Pyrazole urea-based inhibitors of p38 MAP kinase: From lead compound to clinical candidate

Article abstract of DOI:10.1021/jm020057r

We report on a series of N-pyrazole, N′-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5′-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.

Full text of DOI:10.1021/jm020057r

2994
J . Med. Chem. 2002, 45, 2994-3008
P yr a zole Ur ea -Ba sed In h ibitor s of p 38 MAP Kin a se: F r om Lea d Com p ou n d to
Clin ica l Ca n d id a te
J ohn Regan,*,† Steffen Breitfelder,† Pier Cirillo,† Thomas Gilmore,† Anne G. Graham,‡ Eugene Hickey,†
Bernhard Klaus,† J effrey Madwed,§ Monica Moriak,† Neil Moss,† Chris Pargellis,‡ Sue Pav,‡ Alfred Proto,‡
Alan Swinamer,† Liang Tong,† and Carol Torcellini§
Departments of Medicinal Chemistry, Biology, and Pharmacology, Boehringer Ingelheim Pharmaceuticals,
Research and Development Center, 900 Ridgebury Road, Ridgefield, Connecticut 06877
Received February 6, 2002
We report on a series of N-pyrazole, N′-aryl ureas and their mode of binding to p38 mitogen
activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine
5′-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in
part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen
bonding interactions between this class of inhibitors and the protein. We describe the correlation
of the structure-activity relationships and crystallographic structures of these inhibitors with
p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond
at the ATP binding site. This modification affords significant improvements in binding, cellular,
and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the
treatment of inflammatory diseases.
In tr od u ction
In addition to the discovery of this important sig-
nal transduction pathway, pyridinyl imidazole
1
The proinflammatory cytokines tumor necrosis fac-
tor-R (TNF-R) and interleukin-1â (IL-1â) help regulate
the body’s response to infections and cellular stresses.1
However, the pathophysiological consequences resulting
from chronic and excessive production of TNF-R and IL-
1â are believed to underlie the progression of many
inflammatory diseases such as rheumatoid arthritis
(RA),2 Crohn’s disease, inflammatory bowel disease, and
psoriasis.3 Recent data from clinical trials have secured
the continued use of the soluble TNF-R receptor fusion
protein, etanercept, or the chimeric TNF-R antibody,
infliximab, in the treatment of RA4-8 and Crohn’s
disease.9,10 The signal transduction pathway leading to
the production of TNF-R from stimulated inflammatory
cells, while not fully understood, has been shown to be,
in part, regulated by p38 mitogen activated protein
(MAP) kinase.11 p38 MAP kinase belongs to a group of
serine/threonine kinases that includes c-J un NH2-
terminal kinase (J NK) and extracellular-regulated pro-
tein kinase (ERK).12 Upon extracellular stimulation by
a variety of conditions and agents,13 p38 is activated
through bis-phosphorylation on a Thr-Gly-Tyr motif
located in the activation loop. Activation is achieved by
dual-specificity serine/threonine MAPK kinases, MKK3
and MKK6. Once activated, p38 can phosphorylate and
activate other kinases or transcription factors leading
to stabilized mRNA and an increase or decrease in the
expression of certain target genes.14-17
(SB 203580)11 and analogues18-22 have been identified
as potent and selective inhibitors of p38 MAP kinase.
Compound 1 was shown to be an effective orally active
agent in several animal models of acute and chronic
inflammation.23 Recently, an analogue of 1, compound
2 (SB 242235), inhibited endotoxin-induced ex vivo
production of TNF-R and IL-1â in human clinical
trials.24 The interest in p38 as a viable target for drug
intervention has escalated as a result of these early
disclosures. In addition to a plethora of patent applica-
tions on imidazole-based compounds,25-27 several jour-
nal papers have described strategies for the modification
of 1, by either the addition of other substituents to the
imidazole or its replacement with different heterocycles.
These endeavors have produced imidazoles 328 and 4
(RPR200765A),29 a pyrrole analogue of 1,30 oxazole
5,31and pyrrolo[2,3-b]pyridine 6 (RWJ 68354).32 Imid-
azole 7 (RWJ 67657)33 was described to inhibit LPS-
stimulated TNF-R production in human clinical
trials.34 Also, compounds with different structural fea-
tures as compared to 1 have been reported as p38
inhibitors. These include, among others, 8 (VX-745)35
and N,N-diaryl urea 8a 36 as well as pyrazole ketone 9
(RO3201195)37 and pyrimido[4,5-d]pyrimidinone 10.38
Indole amide 11 represents another group of p38 inhibi-
tors.39 A benzophenone class of p38 inhibitors, an
example shown as 12 (EO1428), has recently been
described.40 Diamides (13) are disclosed as p38 inhibi-
tors41 (Chart 1).
* To whom correspondence should be addressesd. Tel.:
(203)798-4768.
Fax:
(203)791-6072.
E-mail:
jregan@
rdg.boehringer-ingelheim.com.
† Department of Medicinal Chemistry.
‡ Department of Biology.
Our focus in cytokine-regulated approaches to inflam-
matory diseases prompted us to evaluate the potential
of p38 MAP kinase as a therapeutic target. Toward this
§ Department of Pharmacology.
10.1021/jm020057r CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/25/2002

Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 2995
Ch a r t 1. Structural Classes of p38 MAP Kinase Inhibitors
Sch em e 1a
Ta ble 1. Substitution at Pyrazole N-2
a
Reagents: (a) Phenylhydrazine, toluene, reflux or aqueous
HCl, ethanol, reflux. (b) 4-Chlorophenyl isocyanate, THF or
CH2Cl2, 25 °C.
14, coupled with its distinction as a new structural type
of inhibitor vs others (e.g., 1-13) prompted us to under-
take a systematic evaluation of its pharmacophores. The
structure-activity relationships (SAR) for this class of
compounds and their correlation to structural data,
which led to the discovery of the clinical candidate BIRB
796,45 are the subjects of this paper.
Ch em istr y
Modifications to the 2-position of the pyrazole nucleus
were prepared as shown in Scheme 1 using 16 as a
representative example of the compounds in Table 1.
The assembly of pyrazole nucleus 15 involved the
condensation of phenylhydrazine and 4,4-dimethyl-3-
oxopentanenitrile in either toluene or aqueous HCl in
ethanol at reflux. Urea formation was accomplished
with 15 and 4-chlorophenyl isocyanate to produce 16.
For examples in Table 1 requiring noncommercially
available aryl hydrazines, the method of Demers46 was
used to convert aryl halides to aryl hydrazines. The
cyclohexylhydrazine that was used in the synthesis of
end, compound 14 was identified from high throughput
screening. Reports on utilizing this compound as a lead
have been disclosed.42-44 While 14 showed only a modest
binding affinity for human p38 MAP kinase (Kd ) 350
nM) (Table 1), our interest in this molecule further
increased upon obtaining a cocrystal structure with
recombinant human p38. The unique binding mode of

2996 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Ta ble 2. Substitution at Pyrazole C-5
Regan et al.
Sch em e 3a
Sch em e 2a
a
Reagents: (a) CNCH2CO2H, n-BuLi, THF, CH2Cl2, -70 °C and
then 25 °C. (b) Phenylhydrazine, toluene, reflux. (c) THF, 25 °C.
(d) LDA, THF, MeI, -78 °C. (e) NaH, CH3CN, THF, 75 °C.
a
Reagents: (a) EDC, CH2Cl2. (b) Tf2O, DTBMP, CH2Cl2, -78
to 0 °C. (c) Tributyl(vinyl)tin, Pd[P(Ph)3]4, LiCl, dioxane, 100 °C.
(d) (i) 9-BBN, THF, reflux. (ii) NaOH, H2O2. (e) J ones reagent. (f)
4-Chloroaniline, DCC, DMAP, CH2Cl2. (g) COCl2, CH2Cl2, aqueous
NaHCO3. (h) CH2Cl2, 25 °C. (i) HCO2H, reflux. (j) BH3-DMS, THF,
25 °C. (k) 4-Chlorophenyl isocyanate, CH2Cl2, 25 °C. (l) 4-Chloro-
phenyl isothiocyanate, CH2Cl2, 25 °C.
target compound 47 was obtained from the sodium
cyanoborohydride-mediated reductive hydrazination of
cyclohexanone with hydrazine.47
Compound 46 was used as a frame of reference for
probing the SAR at the 5-position of pyrazole by re-
placing the t-butyl moiety (Table 2). This effort required
the construction of a diverse set of oxopentanenitrile
subunits. Scheme 2 outlines two general procedures to
prepare oxopentanenitrile derivatives. Briefly, the ad-
dition of the dianion of cyanoacetic acid48 to acid
chlorides (e.g., 17) or the anion of acetonitrile49 to esters
(e.g., 22) supplied the â-keto nitrile components. Fol-
lowing the chemistry described in Scheme 1, pyrazole
formation and urea couplings were completed for target
20 as well as the other compounds in Table 2.
To evaluate the role of the urea linkage to the binding
of p38, the synthesis of several urea analogues was
undertaken. The biological role of each of the urea N-H
groups in 16 was examined by replacement with CH2
(25 and 31) and N-methyl (34 and 37). Amides 25 and
31 were prepared as shown in Scheme 3. EDC-mediated
condensation of aminopyrazole 15 and 4-chlorophenyl-
acetic acid (24) furnished amide 25. Amide 31, however,
required the construction of pyrazole acetic acid 30.
Thus, pyrazolidinone 26 was converted to its O-triflate
derivative 27, which underwent Stille cross coupling
with tributyl(vinyl)tin to give vinyl pyrazole 28. Regio-
selective hydroboration of 28 produced alcohol 29, which
was converted to the desired carboxylic acid 30 with
J ones reagent. Amide bond formation between 30 and
4-chloroaniline with DCC furnished 31. The syntheses
of N-methyl urea analogues 34 and 37 were undertaken
as follows. Exposure of aminopyrazole 15 to phosgene50
produced pyrazole isocyanate 32, which was coupled
with N-methyl-4-chloraniline (33) to provide N-methyl
urea 34. Alternatively, aminopyrazole 15 was heated
with formic acid to produce N-formyl aminopyrazole 35,
which upon reduction with borane51 yielded N-methyl-
aminopyrazole 36. Treatment of 36 with 4-chlorophenyl
isocyanate produced N-methyl urea analogue 37. Thio-
urea 38 served as a basis to understand the part that
the O-atom plays in p38 binding, and its preparation
was accomplished by treatment of aminopyrazole 15
with 4-chlorophenyl isothiocyanate.
The compounds designed to explore the region of the
urea phenyl of 46 are summarized in Table 4. They were
conveniently obtained by the treatment of pyrazole
isocyanate 32 with aniline derivatives or alkylamines.
For example, as shown in Scheme 4, exposure of
isocyanate 32 to 2-aminoindan (39) furnished urea 40.
Other target ureas were prepared according to Scheme
1 wherein amine 15 was coupled to aryl isocyanates.
To access target compounds with groups attached
to the 4-position of the urea naphthalene, the route
shown in Scheme 5 was utilized. Alkylation of N-Boc
naphthol 41, prepared from 4-amino-1-naphthol with
4-(2-chloroethyl)morpholine, gave ether 42. Removal of
the Boc protecting group (43) and urea formation, as
described above with the isocyanate derived from 44,
gave 45.

Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 2997
Sch em e 4a
Ta ble 3. Urea Modifications
a
Reagents: (a) CH2Cl2, 25 °C.
Gly170 (DFG) of the kinase is required for the observed
binding mode of the diaryl urea inhibitor (Figure 2). In
all of the currently known protein Ser/Thr kinase
structures, the residues assume a conformation such
that the Phe side chain is buried in a hydrophobic pocket
in the groove between the two lobes of the kinase (DFG-
in conformation). In the structure of the complex with
compound 14, however, the Phe side chain has moved
by about 10 Å to a new position (DFG-out conformation).
In this position, one face of the Phe side chain and the
urea phenyl ring are involved in hydrophobic interac-
tions whereas the other face is exposed to solvent. This
movement of the Phe side chain reveals a large hydro-
phobic domain in the kinase, and the tert-butyl group
of 14 inserts deep into this pocket (Figure 2). Neither
nitrogen atom on the pyrazole ring participates in
specific hydrogen-bonding interactions with the kinase.
As shown in Figure 3, the urea of 14 establishes a
bidentate hydrogen bond with the conserved side chain
of Glu71.
Ta ble 4. Modification of Urea-Phenyl Ring
Most protein kinase inhibitors use the ATP binding
pocket and inhibit the kinase by directly competing with
the binding of ATP. In contrast, compound 14 does not
compete directly with ATP binding, as it has no struc-
tural overlap with the ATP molecule (Figure 2). How-
ever, our structure shows that the DFG-out conforma-
tion impedes ATP binding, as the side chain of the Phe
residue would be sterically incompatible with the phos-
phate groups of ATP (Figure 2). This is supported by
our observation that compound 14 interferes with the
inactivation of p38 MAP kinase activity by the fluores-
cent ATP analogue 5′-p-fluorosulfonyl benzoyl adenosine
(data not shown). Therefore, the diaryl urea compounds
inhibit p38 MAP kinase by stabilizing a conformation
of the kinase that is incompatible with ATP binding.
The data in Tables 1 and 2 highlight the binding roles
of the groups appended to the pyrazole nucleus of 14s
methyl at N-2 and tert-butyl at C-5. Fortuitously, our
first modification, replacement of the methyl of 14 with
a phenyl group (16), improved binding potency 40-fold
(Table 1) as measured in a fluorescent binding assay.
The crystal structure of 16 and the recombinant human
p38 complex (Figure 4) help rationalize this result. The
phenyl ring at N-2 of the pyrazole participates in
lipophilic interactions with the alkyl portion of the side
chain of the Glu71 residue. In addition, the phenyl ring
may serve as a water shield for the hydrogen bond
network of the urea and the Glu71 carboxylate. The
presence of the phenyl ring causes this Glu residue to
adopt a side chain conformation that results in a
monodentate hydrogen-bonding interaction with the
urea moiety of the inhibitor. This alignment of Glu71
is in contrast to the bidentate interactions in the
complex with 14 (Figure 3).
Resu lts a n d Discu ssion
We recently reported the crystal structure of recom-
binant human p38 MAP kinase in complex with com-
pound 14 at 2.5 Å resolution (Figure 1).45 Interestingly,
the crystal structure reveals that this compound utilizes
binding interactions on the kinase that are spatially
distinct from the adenosine 5′-triphosphate (ATP) pocket.
There is no structural overlap between the atoms of
compound 14 and the ATP (Figure 2). Similarly, there
is only limited spatial overlap between 14 and an iodo
analogue of SB203580 (1a )52 and this occurs in a
lipophilic pocket commonly referred to in the kinase field
as the specificity pocket (Figure 3). A large conforma-
tional change for conserved residues Asp168-Phe169-
Further profiling of this key region in the inhibitor
helped establish the preferred substitution at N-2 of the

2998 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Regan et al.
Sch em e 5a
a
Reagents: (a) Di-tert-butyl dicarbonate, THF, 25 °C. (b) 4-(2-Chloroethyl)morpholine, K2CO3, acetonitrile, heat. (c) HCl, dioxane. (d)
Compound 44, phosgene, THF.
F igu r e 2. Overlap of 14 (blue) and ATP (red). The urea
hydrogen atoms are shown for clarity. Phe169 is shown in red
when occupying the DFG-in conformation (ATP bound) and
in blue in the DFG-out conformation when 14 is bound to p38.
compound (58). This lipophilic binding pocket tolerated
bulkier tert-alkyl groups such as dimethylethyl (60) and
methylcyclohexyl (20). However, the 50-fold loss of
binding observed with dimethylbenzyl analogue 64 may
F igu r e 1. Crystal structure of human p38 MAP kinase and
indicate a size limitation for this domain. A comparison
of cyclohexyl derivatives 62 and 20 further exemplifies
the strong preference for a tertiary group. The relatively
poor activity of compounds 61 and 63 as compared to
60 and 20 suggest that lipophilic substitution at C-5 of
the pyrazole is favored. Taken together, these results
are rationalizable based on the crystal structure of 16
and p38 (Figure 4) that indicate a lipophilic group at
C-5 of the pyrazole has important hydrophobic binding
interactions with the protein in the DFG-out conforma-
tion. The tert-butyl group was incorporated into all
subsequent target molecules since it offered the best
balance of potency and physicochemical properties.
The X-ray crystallographic structure of 16 with p38
reveals a hydrogen bond network consisting of a urea
hydrogen and the carboxylate oxygen of Glu71 and also
the urea oxygen and N-H of Asp168. The data in Table
3 highlight the significance of these interactions on
binding affinity. Replacement of either N-H in the urea
with a methylene group (compounds 25 and 31) or
introduction of N-methyl (34 and 37) results in signifi-
cant loss of activity. Likewise, the thiourea analogue 38
shows a 60-fold decrease in binding potency to p38 as
compared to 16. These findings underscore the crucial
contribution that the urea makes to binding with p38
through extensive hydrogen bonding and, also likely,
to establishing the correct geometric relationships of the
other pharmacophores of the inhibitor.
14 at 2.5 Å resolution.
pyrazole and confirmed our hypothesis regarding bind-
ing interactions at this domain. The diminished potency
of saturated derivative 47 highlighted the necessity for
an aromatic ring to achieve optimal hydrophobic inter-
actions (Table 1). Addition of methyl groups to the 3-
and 4-position of the phenyl ring of 46 provided modest
improvements in binding (49-51). However, 2-methyl
derivative 48 displayed a substantial loss of binding
affinity possibly due to an increase in the torsional angle
favored between the phenyl and the pyrazole rings
beyond the observed angle of 54° for 16 (Figure 4). This
position tolerates bulkier groups as judged by the
binding potency of 2-naphthyl analogue 52. In addi-
tion, heteroatoms (53-57) can be accommodated at this
site. The close proximity of the 3- and 4-positions of
the phenyl ring of 46 to solvent may explain these
results.
In Figure 4, the tert-butyl group at C-5 of pyrazole
16 is embedded deep into a hydrophobic pocket formed
by the reorganization of Phe169 in the DFG-out con-
formation. In an effort to understand the binding role
of the tert-butyl moiety in this class of compounds, we
investigated the size and electronic requirements of this
group. As can be seen in Table 2, removal of one methyl
group lowered potency over 20-fold (cf. 46 vs 59).
Further reduction to a methyl resulted in an inactive

Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 2999
F igu r e 3. Two views of the overlap of 14 and 1a . The urea hydrogen atoms are shown for clarity. The urea phenyl group of 14
occupies the same kinase specificity pocket in p38 as the phenyl ring of 1a . The bindentate hydrogen bond interaction between
the urea hydrogen atoms and the carboxylate oxygens of Glu71 is shown.
can also provide good binding affinity as seen with
bicyclic indan derivatives (40 and 77). Thus, these data
demonstrate that the kinase specificity pocket of p38
favors lipophilic pharamacophores that are not limited
to phenyl rings.
We examined several compounds from this series for
oral activity in a mouse model of LPS-stimulated TNF-R
synthesis. Compound 50 (Table 5) furnished an inter-
esting and important result. The tolyl group on 50
provided a 100-fold increase in plasma concentration in
the mouse vs 46, which lacks this substitution. The
increased plasma levels in combination with a modest
improvement in cellular activity provided our first orally
active compound. Of the 4-methylphenyl derivatives
examined in this model, analogue 78 showed the best
in vivo profile. This compound, with even higher plasma
concentrations than 50, suppressed TNF-R production
by 90% when dosed at 100 mg/kg and also was active
at 30 mg/kg (53% inhibition).
F igu r e 4. X-ray crystallographic structure of human p38 with
16. The urea hydrogen atoms are shown for clarity. The
Unfortunately, we were unable to improve the in vitro
and in vivo activities of the phenyl-based urea inhibitors
hydrophobic effects of the pyrazole phenyl ring and the
beyond that of compound 78. Despite available crystal-
monodendate hydrogen bond of the urea N-H atoms with
lographic data, obvious solutions to achieve additional
Glu71 are seen.
binding interactions were not realized. A breakthrough
As seen in Figure 3, the phenyl ring attached to the
urea of 14 fits into the specificity pocket of p38 in a
manner similar to the pyridinyl imidazole inhibitor 1a .52
The importance of binding in this pocket to potency53
and kinase specificity28 has been described for the
imidazole-based group of inhibitors. Highlights of the
prominent role that the phenyl ring plays in binding
with this series of compounds are shown in Table 4.
Removal of the urea phenyl ring results in complete loss
of binding potency (cf. 46 and 65). Saturation of the
phenyl ring (66) or separation of the ring from the urea
by either one or two carbon atoms (73 and 74) resulted
in decreased binding affinity. Incorporation of polar
groups, through either pyridine (67-69) or aniline
derivatives (70 and 71), lowers potency. However,
lipophilic groups appended to the phenyl nucleus can
improve potency (cf. 72 and 75). Other lipophilic groups
arrived upon establishing a binding assay having more
sensitivity for compounds whose binding activity was
near the limit of the fluorescence assay.45 One observa-
tion from this new assay suggested that 79 was more
potent than initially thought. Table 6 shows selected
examples of the Kd values from the fluorescence assay
vs the exchange curve assay. Remarkably, naphthyl
compound 79 binds 20-fold more tightly to p38 than
phenyl analogue 50 despite similar cellular potencies.
The overlap of the X-ray crystal structures of phenyl
analogue 16 and naphthyl 75 (Figure 5) provides a
possible explanation for this increased binding. The
naphthyl moiety of 75 resides deep in the kinase
specificity pocket and achieves substantial hydrophobic
binding interactions with the protein that are not
possible with the phenyl ring of 16. Thus, it seemed
reasonable that the lack of improvement in cellular

3000 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Ta ble 5. In Vivo Activity of Selected Pyrazole-Phenyl Ureas
Regan et al.
Ta ble 6. Comparison of Phenyl vs Naphthyl Ureas with ATP
Site Binding Pharmacophore
able from the phenyl ring of 16. That is, groups
appended to the 4-position of the naphthalene could
much more readily access the ATP binding region of p38
than the 4-positon of the phenyl ring. Hence, pharma-
cophores attached to the 4-position could have ad-
ditional binding interactions or be used to improve
physicochemical properties. The ethoxy morpholine
group proved to be a very effective group for achieving
both of these goals. This moiety improved binding
potency 10-fold and, more significantly, increased cell
activity over 20-fold. A crystal structure of 45 with
recombinant human p38 (Figure 6a) provided an ex-
planation for the enhanced potency.45 The gauche
conformation of the ethoxy linker of 4554,55 effectively
orients the morpholine group so that the morpholine
oxygen can achieve a strong hydrogen bond with the
N-H of Met109. This hydrogen bond is the same one
used by the adenine base of ATP and the pyridine
nitrogen of the pyridinyl imidazole class of compounds
such as 1a .52 The favorable edge to π hydrophilic
interactions of the phenyl ring of Phe169 and the
naphthalene group of 45 is revealed in Figure 6b and
likely further contributes to binding potency.
To highlight the contribution of the naphthalene
group to 45, we prepared phenyl derivative 80. Despite
the inclusion of the ethoxy morpholine unit in phenyl
analogue 80, it exhibited a dramatic loss in potency vs
45 (Table 6). This result reinforces the importance of
the naphthalene ring in providing better lipophilic
interactions with the specificity pocket and properly
aligning the ethoxy morpholine unit for productive
binding with the ATP binding region.
In addition to superior in vitro and cellular activities,
compound 45 demonstrated enhanced in vivo potency.
For example, in a mouse model of LPS-stimulated
TNF-R synthesis, a 65% inhibition of TNF-R synthesis
was observed when 45 was dosed orally at 10 mg/kg.
In a 5 week model of established collagen-induced
arthritis using B10.RIII mice, 45 produced a 63%
inhibition of arthritis severity when dosed orally at 30
mg/kg qd.56 Some pharmacokinetic data of 45 in mice
and cynomolgous monkeys are summarized in Table 7.
The selectivity profile against a panel of protein kinases
for 45 was determined and is shown in Table 8.45 On
the basis of these and other data, compound 45 (BIRB
796) was selected for human clinical trials.
F igu r e 5. Overlap of 16 (yellow) and 75 (green) with human
p38 MAP kinase. The urea hydrogen atoms are shown for
clarity. The naphthyl group of 75 fits deeper into the kinase
specificity pocket as compared to the phenyl ring of 16. The
4-position of the naphthalene ring points toward the hinge
region and solvent. Phe169 is removed for clarity.
activity of 79 might be a consequence of its higher
lipophilicity as compared to 50.
The crystal structure of 75, in addition to providing
a rationale to the improved binding affinity of the
naphthalene group, offered an opportunity to explore
modifications from this platform that would be unavail-

Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3001
F igu r e 6. (a) X-ray crystallographic complex of human p38 and 45. Phe169 is removed for clarity. (b) X-ray crystallographic
complex of human p38 and 45 with Phe169.
Ta ble 7. Pharmacokinetic Properties of Compound 45
Ta ble 8. Selectivity Profile of Compound 45
in the selection of 45 (BIRB 796) as a clinical candidate
for the treatment of inflammatory diseases.45,57
Exp er im en ta l Section
All solvents and reagents were obtained from commercial
sources and used without further purification unless indicated
otherwise. Melting points were obtained from a Mel-temp 3.0
or Fisher-J ohns melting point apparatus and are uncorrected.
1H nuclear magnetic resonance (NMR) spectrum were recorded
on either a Bruker AC-F-270 spectrometer or Bruker Avance
DPX 400 spectrometer. Chemical shifts are reported in parts
per million (δ) from the tetramethylsilane resonance in the
Con clu sion
indicated solvent. Mass spectra were obtained from a Finni-
gan-SSQ7000 spectrometer. Samples were generally intro-
duced by particle beam and ionized with NH4Cl. Thin-layer
chromatography (TLC) analytical separations were conducted
with E. Merck silica gel F-254 plates of 0.25 mm thickness
and were visualized with UV or I2. Flash chromatographies
were performed according to the procedure of Still et al. (EM
Science Kieselgel 60, 70-230 mesh). Elemental analysis were
performed at Quantitative Technologies, Inc., Whitehouse, NJ .
We have shown that a series of N-pyrazole, N′-aryl
ureas occupy a binding domain on p38 that is exposed
when the conserved binding loop, consisting in part of
Asp168-Phe169-Gly170, adopts a conformation (DFG-
out) not previously noted in other protein Ser/Thr
kinases. A 40-fold improvement in binding was achieved
by the replacement of the methyl group in the original
screening lead (14) with phenyl. The urea atoms, shown
to be involved in an extensive hydrogen bond network
with Glu71 and Asp168 (Figures 3 and 4), proved critical
for binding activity. A toluene ring attached to the
pyrazole nucleus was necessary to secure high plasma
levels in the mouse. The naphthalene was a preferred
pharmacophore as compared to phenyl to bind in the
kinase specificity pocket. We added an ethoxy morpho-
line pharmacophore, which successfully extended the
binding of the inhibitor to also include a hydrogen-
bonding interaction in the ATP binding region of p38.
This modification afforded significant improvements in
binding affinity, cellular activity, and in vivo reduction
of TNF-R production and arthritis severity that resulted
1-(5-ter t-Bu t yl-2-p h en yl-2H -p yr a zol-3-yl)-3-(4-ch lor o-
p h en yl)u r ea (16). A solution of phenyl hydrazine (0.83 mL,
8.39 mmol) and 4,4-dimethyl-3-oxo-pentanenitrile (1.0 g, 8.0
mmol) in toluene (3 mL) was heated to reflux overnight.
Removal of the volatiles in vacuo provided a residue, which
was purified by silica gel chromatography using 50% ethyl
acetate in hexanes as the eluent. Concentration in vacuo of
the product-rich fractions provided 3-amino-5-tert-butyl-2-
phenyl-2H-pyrazole (15) as a light orange solid (1.53 g, 89%).
A solution of 15 (0.058 g 0.27 mmol) and 4-chlorophenyl
isocyanate (0.038 g, 0.25 mmol) in CH2Cl2 (1 mL) was stirred
overnight at room temperature under inert atmosphere.
Removal of the volatiles in vacuo provided a residue, which
was triturated with 50% dichloromethane in hexanes (2 mL).
The urea (16) was filtered and dried in vacuo to afford 0.078
g (85%) and was then recrystallized from methanol to afford
analytically pure material; mp 202-203 °C. 1H NMR (400

3002 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Regan et al.
MHz, dimethyl sulfoxide (DMSO)-d6): δ 1.27 (s, 9H, tert-butyl),
6.36 (s, 1H, pyrazole), 7.28-7.30 (m, 2H, aromatic), 7.39-7.43
(m, 3H, aromatic), 7.50-7.52 (m, 4H, aromatic), 8.42 (s, 1H,
urea), 9.12 (s, 1H, urea). MS (NH3-CI): m/e 369 (MH+). Anal.
(C20H21ClN4O‚CH3OH) C, H, N.
Tr iflu or om eth a n esu lfon ic Acid 5-ter t-Bu tyl-2-p h en yl-
2H-p yr a zol-3-yl Ester (27). To a solution of 5-tert-butyl-2-
phenyl-2,4-dihydro-pyrazol-3-one (26, 5.12 g, 23.7 mmol) and
2,6-di-tert-butyl-4-methyl pyridine (6.12 g, 29.8 mmol) in
CH2Cl2 (50 mL) was added dropwise trifluoromethanesulfonic
anhydride (4.4 mL, 7.4 g, 26 mmol) at -78 °C. The resulting
solution was warmed to 0 °C, saturated NaHCO3 solution (100
mL) was added, and the mixture was stirred vigorously for 10
min. The layers were separated, and the aqueous layer was
extracted with CH2Cl2 (3×). The combined organic layers were
washed with brine and dried (Na2SO4). Removal of the volatiles
in vacuo provided a residue, which was purified by flash
chromatography eluting unpolar impurities with hexanes and
subsequently eluting the product with hexanes:ethyl acetate
(20:1). Concentration in vacuo of the product-rich fractions
gave 8.19 g (99%) of yellow oil 27. 1H NMR (400 MHz,
CDCl3): δ 1.34 (s, 9H, tert-butyl), 6.19 (s, 1H, pyrazole), 7.35
1-[5-(1-Meth ylcycloh exyl)-2-p h en yl-2H-p yr a zol-3-yl]-3-
p h en yl-u r ea (20). A solution of cyclohexane-1-methyl-1-
carboxylic acid (1.31 g, 9.21 mmol), oxalyl chloride (5.5 mL of
a 2.0 M solution in CH2Cl2, 11.05 mmol), and a drop of
anhydrous dimethylformamide (DMF) in CH2Cl2 (5 mL) was
heated to reflux for 3 h and cooled to ambient temperature to
give 17. In a separate flask to a solution of cyanoacetic acid
(1.57 g, 18.4 mmol, freshly dried with MgSO4) and a catalytic
amount of 2,2′-bipyridine in anhydrous tetrahydrofuran (THF)
(68 mL) at -70 °C under an inert atmosphere was added
dropwise n-butyllithium (15 mL of a 2.5 M solution in hexanes,
37.2 mmol). The mixture was slowly warmed to 0 °C until a
persistent red-colored slurry was obtained. The mixture was
cooled to -70 °C, and 17 in CH2Cl2 was slowly added. The
mixture was slowly warmed to room temperature, stirred for
1 h, and quenched with 2 N aqueous HCl. The aqueous layer
was extracted twice with CHCl3. The combined organic layers
were washed with saturated aqueous NaHCO3 and brine and
dried (MgSO4). Removal of the volatiles in vacuo provided a
residue, which was purified by silica gel chromatography using
ethyl acetate in hexanes as the eluent. Concentration in vacuo
of the product-rich fractions provided 1.26 g of 18. A mixture
of 18 (0.80 g, 4.8 mmol) and phenylhydrazine (0.48 mL, 4.8
mmol) in dry toluene (6 mL) was heated to reflux overnight.
Removal of the volatiles in vacuo provided a residue, which
was purified by silica gel chromatography. Concentration in
vacuo of the product-rich fractions provided 1.10 g (90%) of
19. A mixture of 19 (0.288 g, 1.13 mmol) and phenyl isocyanate
(0.12 mL, 1.1 mmol) in anhydrous THF (4 mL) was stirred
overnight under inert atmosphere. Removal of the volatiles
in vacuo provided a residue, which was purified repeatedly
by silica gel chromatography. Concentration in vacuo of the
product-rich fractions provided 0.066 g of 20 as a foam, which
softens at 86-88 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.95
(s, 3H, methyl), 1.43-1.50 (m, 8H, cyclohexyl), 1.98-2.00 (m,
2H, cyclohexyl), 6.36 (s, 1H, pyrazole), 6.95-6.99 (m, 1H,
aromatic), 7.24-7.30 (m, 2H, aromatic), 7.40-7.46 (m, 3H,
aromatic), 7.53-7.57 (m, 4H, aromatic), 8.42 (s, 1H, urea), 9.03
(s, 1H, urea). MS (+ES): m/e 374 (M+). Anal. (C23H26N4O) C,
H, N.
3
3
3
3
(dd, J 1 ) J 2 ) 7.4 Hz, 1H, phenyl), 7.4 (dd, J 1 ) J 2 ) 7.8
Hz, 2H, phenyl), 7.54 (d, 7.96 Hz, 2H, phenyl).
3-ter t-Bu tyl-1-p h en yl-5-vin yl-1H-p yr a zole (28). A solu-
tion of 27 (3.74 g, 10.7 mmol) in dioxane (90 mL) in a sealable
tube was degassed under vacuum and charged with nitrogen.
LiCl (2.91 g, 68.8 mmol) was added, and the mixture was
degassed and charged with nitrogen again. Pd(PPh3)4 (0.491
g, 0.425 mmol) was added, and the mixture was degassed and
charged with nitrogen again. Tributyl(vinyl) tin (4.0 mL, 4.3
g, 14 mmol) was added, and the mixture was degassed and
charged with nitrogen again. The tube was sealed, and the
mixture was heated to 100 °C overnight. After it was cooled
to room temperature, the volatiles were removed in vacuo and
the residue was purified by flash chromatography eluting
unpolar impurities with hexanes and subsequently eluting the
product with hexanes in ethyl acetate (20:1). Concentration
in vacuo of the product-rich fractions gave a residue, which
was dissolved in ethyl acetate (100 mL) and stirred vigorously
with saturated KF solution (30 mL). The organic layer was
dried (Na2SO4). Removal of the volatiles in vacuo provided a
residue, which was purified by flash chromatography using
hexanes in ethyl acetate (20:1) as the eluent. Concentration
in vacuo of the product-rich fractions gave 1.81 g (74%) of 28
as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 1.37 (s, 9H, tert-
3
2
butyl), 5.24 (dd, J (Z) ) 11.1, J ) 1.1 Hz, 1H, vinyl), 5.6 (dd,
3J (E) ) 17.5 Hz, 2J ) 1.1 Hz, 1H, vinyl), 6.4 (s, 1H, pyrazole),
6.51 (dd, 3J (E) ) 17.5, 3J (Z) ) 11.1 Hz, 1H, vinyl), 7.28-7.35
(m, 2H, phenyl), 7.39-7.45 (m, 3H, phenyl).
3-(4-Meth yltetr a h yd r op yr a n -4-yl)-3-oxo-p r op ion itr ile
(23). To a solution of LDA (prepared from n-butyllithium (3.1
mL of a 2.5 M solution in hexanes, 7.78 mmol) and N,N-di-
iso-propylamine (1.09 mL, 7.78 mmol) in THF (2 mL) at -78
°C) was added dropwise pyran 2158 (1.12 g, 7.78 mmol) in THF
(3 mL). The mixture was stirred for 15 min, warmed to 0 °C,
stirred for 30 min, and cooled to -78 °C. Methyl iodide (0.48
mL, 7.78 mmol) was added, and the mixture was stirred
overnight while warming to room temperature. Ethyl acetate
and aqueous HCl were added, and the organic layer was
washed with brine and dried (MgSO4). Removal of the volatiles
in vacuo provided a residue, which was purified by silica gel
chromatography using 25% ethyl acetate in hexanes as the
eluent. Concentration in vacuo of the product-rich fractions
provided 0.345 g (28%) of 22. To a suspension of hexane-
washed NaH (0.090 g of 60% dispersion in mineral oil, 2.2
mmol) in THF (2 mL) at 75 °C was added dropwise 22 (0.32 g,
2.03 mmol) and anhydrous acetonitrile (0.14 mL, 2.63 mmol)
in THF (2 mL). The mixture was stirred for 5 h, cooled to room
temperature, and diluted with ethyl acetate and aqueous HCL.
The organic layer was washed with water and brine and dried
(MgSO4). Removal of the volatiles in vacuo provided 23 (0.244
g, 72%), which was used without further purification.
2-(5-ter t-Bu tyl-2-p h en yl-2H-p yr a zol-3-yl)eth a n ol (29).
To a solution of 28 (0.171 g, 0.756 mmol) in THF (20 mL) was
added 9-BBN (1.9 mL of a 0.5 M solution in THF, 0.90 mmol)
at 0 °C. After it was warmed to room temperature, the mixture
was stirred overnight and heated to reflux for 4 h. Additional
9-BBN (0.8 mL of a 0.5 M solution in THF, 0.4 mmol) was
added, and the mixture was heated to reflux for another 30
min. After it was cooled to room temperature, NaOH 10% (2.0
mL) and H2O2 (1.5 mL) were added and the mixture was
stirred vigorously overnight. The reaction was diluted with
water and Et2O. The aqueous layer was extracted with ethyl
ether (4×). The combined organic layers were washed with
brine and dried (Na2SO4). Removal of the volatiles in vacuo
provided a residue, which was purified by flash chromatogra-
phy using 33% ethyl acetate in hexanes as the eluent.
Concentration in vacuo of the product-rich fractions gave 0.133
1
g (72%) of 29 as a colorless oil. H NMR (400 MHz, CDCl3): δ
1.32 (s, 9H, tert-butyl), 2.72 (t, 3J ) 6.8 Hz, 2H, Ar-CH2-
3
CH2-OH), 3.23 (s (broad), 1H, OH), 3.59 (t, J ) 6.8 Hz, 2H,
Ar-CH2-CH2-OH), 6.07 (s, 1H, pyrazole), 7.26-7.39 (m, 5H,
phenyl).
(5-ter t-Bu tyl-2-p h en yl-2H-p yr a zol-3-yl)a cetic Acid (30).
To a solution of 29 (0.211 g, 0.865 mmol) in acetone (10 mL)
was added dropwise J ones reagent at 0 °C until the orange
color of the reagent persisted (approximately 0.5 mL). The
mixture was stirred for 90 min at 0 °C and quenched with
2-propanol (1 mL). Water and ethyl ether were added, and the
aqueous layer was extracted with ethyl ether (4×). The
combined organic layers were dried (MgSO4). Removal of the
N-(5-ter t-Bu tyl-2-p h en yl-2H-p yr a zol-3-yl)-2-(4-ch lor o-
p h en yl)a ceta m id e (25). mp 158-159 °C. 1H NMR (400 MHz,
CDCl3): δ 1.34 (s, 9H, tert-butyl), 3.69 (s, 2H, CH2), 6.56 (s,
1H, pyrazole), 7.09 (m, 3H, aromatic), 7.18 (s, 1H, NH), 7.25
(m, 4H, aromatic), 7.33 (m, 2H, aromatic). MS (CI): m/e 368
(MH+). Anal. (C21H22ClN3O) C, H, N.

Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3003
volatiles in vacuo provided 0.220 g (98%) of white solid 30. 1H
NMR (400 MHz, CDCl3): δ 1.40 (s, 9H, tert-butyl), 3.58 (s, 2H,
Ar-CH2-COOH), 6.27 (s, 1H, pyrazole), 7.30-7.41 (m, 5H,
phenyl), 9.51 (s (broad), 1H, COOH).
1-(5-ter t-Bu tyl-2-p h en yl-2H-p yr a zol-3-yl)-3-in d a n -2-yl-
u r ea (40). A solution of 32 (0.19 g, 0.81 mmol) and 2-amino-
indan (39, 0.14 g, 1.1 mmol) in CH2Cl2 (5 mL) was stirred at
room temperature overnight. Removal of the volatiles in vacuo
provided a solid, which was recrystallized from hexanes and
2-(5-ter t-Bu tyl-2-p h en yl-2H-p yr a zol-3-yl)-N-(4-ch lor o-
p h en yl)a ceta m id e (31). To a solution of 30 (0.220 g, 0.851
mmol) in CH2Cl2 (10 mL) at 0 °C was added DCC (0.315 g,
1.53 mmol), followed by DMAP (0.041 g (0.34 mmol) and
4-chloroaniline (0.212 g, 1.66 mmol). The mixture was stirred
overnight, while it was warmed to room temperature, and
quenched with the addition of NaHCO3 solution (5 mL). The
mixture was stirred vigorously for 1 h, and water was added.
The organic layer was washed with 10% HCl (10 mL) and dried
(Na2SO4). Removal of the volatiles in vacuo provided a residue,
which was purified by flash chromatography using 25% ethyl
acetate in hexanes as the eluent. Concentration in vacuo of
the product-rich fractions gave 0.133 g (43%) of a white solid.
The material was recrystallized from hexanes/ethyl acetate to
1
ethyl acetate to give 40; wt 0.17 g (56%); mp 222-223 °C. H
NMR (400 MHz, CDCl3): δ 1.32 (s, 9H, tert-butyl), 2.72 (dd,
2H, J ) 4.6, 16.0 Hz, cyclopentyl), 3.26 (dd, 2H, J ) 7.0, 16.0
Hz, cyclopentyl), 4.43-4.60 (m, 1H, cyclopentyl-CH-N), 5.11
(d, 1H, J ) 7.4 Hz, urea), 6.12 (s, 1H, urea), 6.18 (s, 1H,
pyrazole), 7.16-7.22 (m, 4H, aromatic), 7.31-7.38 (m, 1H,
aromatic), 7.41-7.45 (m, 4H, aromatic). MS (NH3-CI): m/e 375
(MH+). Anal. (C23H26N4O‚0.25H2O) C, H, N.
(4-Hyd r oxyn a p h th a len -1-yl)ca r ba m ic Acid ter t-Bu tyl
Ester (41). A solution of 4-amino-1-naphthol (1.88 g, 11.8
mmol) and di-tert-butyl dicarbonate (2.58 g, 11.8 mmol) in
anhydrous THF (25 mL) was stirred overnight at room
temperature, and the volatiles were removed in vacuo. The
residue was purified with flash silica gel chromatography using
33% ethyl acetate in hexanes as the eluent. Concentration in
vacuo of the product-rich fractions provided the solid product
41.
[4-(2-Mor p h olin -4-yl-eth oxy)n a p h th a len -1-yl)ca r ba m -
ic Acid ter t-Bu tyl Ester (42). A mixture of 41 (0.464 g, 1.69
mmol), 4-(2-chloroethyl)morpholine hydrochloride (0.345 g,
1.86 mmol), and powdered potassium carbonate (0.93 g, 6.75
mmol) in acetonitrile (15 mL) was heated at 80 °C for 3 h,
cooled to room temperature, and diluted with ethyl acetate
and water. The organic layer was washed with water and brine
and dried (MgSO4). Removal of the volatiles in vacuo provided
a residue, which was purified by flash silica gel chromatog-
raphy using 12% hexanes in ethyl acetate as the eluent.
Concentration in vacuo of the product-rich fractions provided
0.528 g (84%) of the solid product 42. 1H NMR (270 MHz,
CDCl3): δ 8.25 (dd, 1H), 7.83 (d, 1H), 7.7-7.5 (m, 3H), 6.80
(d, 1H), 6.6 (bs, 1H), 4.30 (t, 2H), 3.75 (m, 4H), 2.92 (t, 2H),
2.61 (m, 4H), 1.55 (s, 9H). MS (CI): m/e 373 (MH+).
1
give 0.093 g (30%) of 31 as a white solid; mp 186 °C. H NMR
(400 MHz, DMSO-d6): δ 1.29 (s, 3H, tert-butyl), 3.81 (s, 2H,
3
Ar-CH2-C(O)NHAr), 6.33 (s, 1H, pyrazole), 7.35 (d, J ) 8.7
3
3
Hz, 2H, 4-chloro-phenyl) overlapping with 7.38 (dd, J 1 ) J 2
) 7.0 Hz, 1H, phenyl), 7.49 (dd, 3J 1 ) 3J 2 ) 7.6 Hz, 2H, phenyl),
7.54 (d, 3J ) 7.6 Hz, 2H, phenyl), 7.59 (d, 3J ) 8.7 Hz, 2H,
4-chloro-phenyl), 10.29 (s, 1H, Ar-CH2-C(O)NHAr). MS
(CI): m/z 368 (M + H)+. Anal. (C21H22ClN3O) C, H, N.
3-(5-tert-Butyl-2-phenyl-2H-pyrazol-3-yl)-1-(4-chlorophen-
yl)-1-m eth yl-u r ea (34). To a solution of 15 (0.52 g, 2.4 mmol)
in CH2Cl2 (25 mL) and aqueous saturated NaHCO3 at 0 °C
was added phosgene (2.5 mL of a 1.9 M solution in toluene,
4.8 mmol). The mixture was rapidly stirred for 10 min and
extracted with CH2Cl2. The combined organic layers were dried
(Na2SO4), and the volatiles were removed in vacuo to provide
pyrazole isocyanate 32, which was used without further
purification. A solution of 32 (0.19 g, 0.80 mmol) and N-methyl-
4-chloroaniline (33, 0.228 g, 1.61 mmol) in CH2Cl2 (3 mL) was
stirred at room temperature overnight. Removal of the vola-
tiles in vacuo provided a residue, which was purified by flash
chromatography using 25% ethyl acetate in hexanes as the
eluent. Concentration in vacuo of the product-rich fractions
gave an oil, which was triturated with petroleum ether to give
0.075 g (24%) of 34; mp 130-131 °C. 1H NMR (400 MHz,
CDCl3): δ 1.32 (s, 9H, tert-butyl), 3.27 (s, 3H, N-Me), 6.41 (s,
1H, urea), 6.49 (s, 1H, pyrazole), 7.08-7.14 (m, 4H, aromatic),
7.27-7.32 (m, 5H, aromatic). Anal. (C21H23ClN4O) C, H, N.
1-Am in o-4-(2-m or p h olin -4-yl-eth oxy)n a p h th a len e Di-
h yd r och lor id e (43). A solution of 42 (0.511 g) and HCl (1
mL of a 4M solution in dioxane) in dioxane (5 mL) was stirred
at room temperature for 20 h, and the volatiles were removed
in vacuo. The residue was used without further purification.
1H NMR (270 MHz, DMSO-d6): δ 8.40 (d, 1Η), 8.05 (d, 1Η),
7.7 (m, 3H), 7.08 (d, 1H), 4.65 (m, 2H), 4.0-3.3 (m, 10H).
1-(5-ter t-Bu t yl-2-p -t olyl-2H -p yr a zol-3-yl)-3-[4-(2-m or -
p h olin -4-yl-eth oxy)n a p h th a len -1-yl]u r ea (45). To a mix-
ture of 44 (prepared as in 15 using 4-methylphenyl hydrazine;
1H NMR (270 MHz, DMSO-d6): δ 7.5-7.4 (m, 4H), 5.6 (s, 1H),
2.33 (s, 3H), 1.25 (s, 9H)) (0.15 g, 0.56 mmol) in CH2Cl2 (15
mL) and saturated aqueous NaHCO3 (15 mL) at 0 °C was
added phosgene (1.2 mL of a 1.9 M solution in toluene, 2.25
mmol). The mixture was stirred rapidly for 15 min, and the
organic layer was dried (MgSO4). Most of the volatiles were
removed in vacuo, and the residue was added to a solution of
aminonaphthalene 43 (0.213 g, 0.620 mmol) and di-iso-
propylethylamine (0.32 mL, 1.86 mmol) in anhydrous THF (10
mL). The mixture was stirred overnight at room temperature
and diluted with water and ethyl acetate. The organic layer
was washed with water and brine and dried (MgSO4). Removal
of the volatiles in vacuo provided a residue, which was purified
by flash silica gel chromatography using ethyl acetate as the
eluent. Concentration in vacuo of the product-rich fractions
provided a solid, which was recrystallized with hexanes and
ethyl acetate and provided 0.065 g (22%) of 45; mp 142-143
1-(5-tert-Butyl-2-phenyl-2H-pyrazol-3-yl)-3-(4-chlorophen-
yl)-1-m eth yl-u r ea (37). A solution of 15 (2.13 g) in formic
acid (7 mL) was heated to reflux for 4 h, cooled to room
temperature, diluted with ethyl acetate, washed with aqueous
NaHCO3 and brine, and dried (Na2SO4). Removal of the
volatiles in vacuo provided a residue, which was purified by
flash chromatography using 20% ethyl acetate in hexanes as
the eluent. Concentration in vacuo of the product-rich fractions
gave 1.0 g (39%) of 35. To a solution of 35 (0.398 g, 1.64 mmol)
in THF (3 mL) at 0 °C was added BH3-DMS (2.0 mL, 4.0 mmol)
dropwise. After the addition was complete, the mixture was
heated to reflux for 1.5 h, cooled to 0 °C, and quenched with
methanol (1 mL). The mixture was stirred for 2.5 h, HCl (0.5
mL of a 4M solution in dioaxane) was added, and the mixture
was heated to reflux for 1 h. After it was cooled to room
temperature, methanol (5 mL) was added and the volatiles
were removed in vacuo. The residue was basified (pH > 12)
with 10% aqueous NaOH and extracted with ether. The
combined extracts were dried (MgSO4). Removal of the vola-
tiles in vacuo provided 0.30 g (80%) of 36. A mixture of 36
(0.15 g, 0.66 mmol) and 4-chlorophenyl isocyanate (0.10 g, 0.68
mmol) in CH2Cl2 (5 mL) was stirred at room temperature for
2 days. Removal of the volatiles in vacuo provided a solid,
which was recrystallized from hexanes and ethyl acetate to
give 37; wt 0.16 g (63%); mp 153-154 °C. 1H NMR (400 MHz,
CDCl3): δ 1.41 (s, 9H, tert-butyl), 3.06 (s, 3H, N-Me), 6.30 (s,
1H, pyrazole), 6.64 (s, 1H, urea), 7.20-7.25 (m, 4H, aromatic),
7.32-7.36 (m, 1H, aromatic), 7.43-7.50 (m, 4H, aromatic).
Anal. (C21H23ClN4O‚0.25H2O) C, H, N.
1
°C. H NMR (270 MHz, CDCl3): δ 8.3 (m, 1H), 7.81 (m, 1H),
7.55 (m, 2H), 7.3 (d, 1H), 6.95 (m, 3H), 6.68 (d, 1H), 6.6 (bs,
1H), 6.48 (bs, 1H), 6.41 (s, 1H), 4,28 (t, 2H), 3.75 (m, 4H), 2.95
(t, 2H), 2.66 (m, 4H), 2.28 (s, 3H), 1.33 (s, 9H). MS (EI): m/e
527 (M+). Anal. (C31H37N5O3) C, H, N.
1-(5-t er t -Bu t yl-2-p h e n yl-2H -p yr a zol-3-yl)-3-p h e n yl-
u r ea (46). This compound was prepared similarly to 16 using
phenyl isocyanate; mp 211 °C. 1H NMR (400 MHz, DMSO-
d6): δ 1.29 (s, 9H, tert-butyl), 6.37 (s, 1H, pyrazole), 6.96-
7.00 (m, 1H, aromatic), 7.24-7.27 (m, 2H, aromatic), 7.38-

3004 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Regan et al.
7.41 (m, 3H, aromatic), 7.50-7.52 (m, 4H, aromatic), 8.39 (s,
1H, urea), 9.00 (s, 1H, urea). MS (NH3-CI): m/e 335 (MH+).
Anal. (C20H22N4O) C, H, N.
the volatiles in vacuo provided a residue, which was crystal-
lized with ethyl acetate and hexanes and furnished the urea
as a white solid (0.096 g, 42%); mp 179-180 °C. 1H NMR (400
MHz, DMSO-d6): δ 1.28 (s, 9H, tert-butyl), 2.38 (s, 3H, Ar-
CH3), 6.39 (s, 1 H, pyrazole C4-H), 6.95 (t, 1H, ArH), 7.22-
7.5 (m, 8H, ArH), 8.35 (s, 1H, urea), 9.07 (s, 1H, urea). MS
(CI): m/e 349 (MH+). Anal. (C21H24N4O) C, H, N.
1-(5-ter t-Bu t yl-2-cycloh exyl-2H -p yr a zol-3-yl)-3-p h en -
yl Ur ea (47). To a solution of anhydrous hydrazine (0.36 mL,
11.6 mmol) in toluene (5 mL) at room temperature was added
cyclohexanone (1.0 mL, 9.6 mmol) dropwise via syringe. After
the addition, the mixture was heated to reflux for 30 min and
cooled to 0 °C and absolute ethanol (5 mL), glacial acetic acid
(15 mL), and sodium cyanoborohydride (0.728 g, 11.6 mmol)
were added. The mixture was warmed to room temperature
and stirred for 3.5 h. The mixture was diluted with 10%
aqueous NaOH solution until a pH of 9-10 was reached
(litmus) and extracted with chloroform. The combined extracts
were dried (MgSO4). Removal of the volatiles in vacuo afforded
an oil and waxy solid (0.94 g) of cyclohexylhydrazine, which
was used without further purifcation. A solution of cyclohexyl-
hydrazine (0.94 g) and 4,4-dimethyl-3-oxo-pentanenitrile (1.21
g, 9.65 mmol) in ethanol (30 mL) was heated at reflux
overnight and cooled to room temperature. Removal of the
volatiles in vacuo provided a residue, which was purified by
silica gel chromatography using 10-20% ethyl acetate in
hexanes as the eluent. Concentration in vacuo of the product-
rich fractions afforded 0.55 g of a mixture containing 4,4-
dimethyl-3-oxo-pentanenitrile and 3-amino-5-tert-butyl-2-
cyclohexyl-2H-pyrazole. This mixture and phenyl isocyanate
(0.17 mL) in 3 mL of anhydrous THF was stirred at room
temperature overnight. The solid was filtered, washed with
CH2Cl2, and dried in vacuo to afford 0.25 g of 47, which was
recrystallized from methanol; mp 236 °C. 1H NMR (400 MHz,
DMSO-d6): δ 1.21 (s, 9H, tert-butyl), 1.20-1.44 (m, 3H,
cyclohexyl), 1.63-1.84 (m, 7H, cyclohexyl), 3.91-3.96 (m, 1H,
cyclohexyl CH-N), 6.03 (s, 1 H, pyrazole C4-H), 6.98 (t, 1H,
J ) 7.3 Hz, phenyl C4-H), 7.28 (dd, 2H, J ) 7.3 Hz, J ) 7.7
Hz, phenyl-C3-H), 7.45 (d, 2H, J ) 7.7 Hz, phenyl C2-H), 8.33
(s, 1H, urea), 8.82 (s, 1H, urea). MS (CI): m/e 341 (MH+). Anal.
(C20H28N4O) C, H, N.
1-[5-ter t-Bu t yl-2-(3,4-d im et h ylp h en yl)-2H -p yr a zol-3-
yl]-3-p h en yl-u r ea (51). This compound was prepared from
3,4-dimethylphenylhydrazine and 4,4-dimethyl-3-oxo-pentane-
1
nitrile as in 16; mp 235-237 °C. H NMR (400 MHz, DMSO-
d6): δ 1.25 (s, 9H, tert-butyl), 2.27 (s, 6H, 2xCH3), 6.33 (s, 1H),
6.94 (t, 3J ) 7.3 Hz, 1H, aromatic), 7.17-7.30 (m, 5H,
aromatic), 7.37 (d, 3J ) 7.6 Hz, 2H, aromatic), 8.30 (s, 1H, urea
N-H), 9.00 (s, 1H, urea N-H). MS (CI): m/e 363 (MH+).
Analysis (C22H26N4O) C, H, N.
1-(5-ter t-Bu t yl-2-n a p h t h a len -2-yl-2H -p yr a zol-3-yl)-3-
p h en yl-u r ea (52). This compound was prepared from 4,4-
dimethyl-3-oxo-pentanenitrile and 2-naphthylhydrazine (from
2-bromonaphthalene as follows: To a solution of 2-bromonaph-
thalene (2.00 g, 9.66 mmol) in THF (10 mL) at -78 °C was
added dropwise s-BuLi (8.17 mL of a 1.3 M solution in
cyclohexane, 10.62 mmol). The green mixture was stirred for
30 min and di-tert-butyl diazodicarboxylate (3.34 g, 14.5 mmol)
was added slowly over 2 min. The reaction was warmed to
room temperature, stirred overnight, quenched with water (10
mL), and extracted with CH2Cl2 (3 × 75 mL). The combined
organic extracts were washed with brine and dried (Na2SO4).
Removal of the volatiles in vacuo provided a residue, which
was triturated with petroleum ether and afforded 1,2-di-(1,1-
dimethylethoxycarbonyl)-1-naphth-2-yl-hydrazine as a pale
orange solid (1.14 g). A mixture of 1,2-di-(1,1-dimethylethoxy-
carbonyl)-1-naphth-2-yl-hydrazine (1.00 g, 2.79 mmol) and HCl
(7.0 mL of a 4 M solution in dioxane) in 2-propanol (15 mL)
was heated at 60 °C for 30 min, cooled to room temperature,
and diluted with ethyl ether. Filtration and drying the solid
in vacuo provided 0.42 g of 2-naphthylhydrazine hydrochloride
as a yellow solid);46 mp 229-230 °C. 1H NMR (400 MHz,
DMSO-d6): δ 1.29 (s, 9H, tert-butyl), 6.43 (s, 1H, pyrazole 4-H),
1-(5-ter t-Bu tyl-2-p yr id in -4-yl-2H-p yr a zol-3-yl)-3-p h en -
yl-u r ea (57). This compound was prepared similarly to 16
using 4-hydrazinopyridine (prepared from 4-bromopyridine as
3
3
3
6.94 (t, J ) 7.3 Hz, 1H, aromatic), 7.23 (dd, J 1 ) J 2 ) 8.0
Hz, 2H, aromatic), 7.37 (d, 3J ) 7.6 Hz, 2H, aromatic), 7.58
(m, 2H, aromatic), 7.67 (dd, 3J ) 8.7 Hz, 4J ) 2.0 Hz, 1H,
aromatic), 8.00 (m, 2H, aromatic), 8.06 (m, 2H, aromatic), 8.50
(s, 1H, urea N-H), 9.00 (s, 1H, urea N-H). MS (CI): m/e 385
(MH+). Anal. (C24H24N4O) C, H, N.
1
in 52);46 mp 178-180 °C. H NMR (400 MHz, CDCl3): δ 1.25
(s, 9H, tert-butyl), 6.45 (s, 1H, pyrazole), 7.08 (m, 1H, aro-
matic), 7.23 (m, 2H, aromatic), 7.31 (m, 2H, aromatic), 7.46
(m, 2H, aromatic), 7.92 (s, 1H, urea), 8.39 (m, 2H, aromatic),
8.47 (s, 1H, urea). MS (CI): m/e 336 (MH+). Anal. (C19H21N5O)
C, H, N.
1-[2-(3-Am in op h en yl)-5-ter t-b u t yl-2H -p yr a zol-3-yl]-3-
p h en yl-u r ea (53). A solution of 3-nitro-phenylhydrazine
hydrochloride (2.0 g, 10.5 mmol), 4,4-dimethyl-3-oxopentane-
nitrile (1.45 g, 11.6 mmol), and concentrated HCl (2 mL) in
ethanol (100 mL) was heated to reflux for 12 h, cooled to room
temperature, basified with 20% aqueous NaOH to pH 12
(litmus), and extracted with ethyl acetate (3 × 20 mL). The
combined extracts were dried (MgSO4). Removal of the vola-
tiles in vacuo afforded 2-(3-nitrophenyl)-5-tert-butyl-2H-pyr-
azol-3-yl-amine as a yellow solid (1.9 g, 70%). A mixture of
this amine (0.40 g, 1.5 mmol) and phenyl isocyanate (0.184
mL, 1.7 mmol) in CH2Cl2 (10 mL) was stirred for 12 h at room
temperature. Removal of the volatiles in vacuo provided a
residue, which was crystallized with ethyl acetate and hexanes
and furnished 1-[5-tert-butyl-2-(3-nitro-phenyl)-2H-pyrazol-3-
yl]-3-phenyl-urea as a white solid (0.54 g, 92%). A mixture of
this urea (0.4 g, 1.0 mmol), 10% Pd/C (0.08 g), and ammonium
formate (0.4 g, 6.0 mmol) in ethanol (20 mL) was heated at
100 °C for 1 h, cooled to room temperture, and filtered through
a plug of Celite. Removal of the volatiles in vacuo provided 53
(0.34 g, 98%); mp 150-151 °C. 1H NMR (400 MHz, DMSO-
d6): δ 1.29 (s, 9H, tert-butyl), 5.41 (s, 2H, Ar-NH2), 6.36 (s, 1
H, pyrazole C4-H), 6.55-6.72 (m, 3H, ArH), 6.99 (t, 1H, ArH),
7.15 (t, 1H, ArH), 7.28 (t, 3H, ArH), 7.43 (d, 2H, ArH), 8.35 (s,
1H, urea), 9.11 (s, 1H, urea). MS (CI): m/e 350 (MH+). Anal.
(C20H23N5O) C, H, N.
1-(5-t er t -Bu t yl-2-o-t olyl-2H -p yr a zol-3-yl)-3-p h e n yl-
u r ea (48). This compound was prepared from the condensation
of o-tolylhydrazine and 4,4-dimethyl-3-oxo-pentanenitrile; see
50; mp 180-182 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.29
(s, 9H, tert-butyl), 1.9 (s, 3H, Ar-CH3), 6.35 (s, 1 H, pyrazole
C4-H), 6.95 (t, 1H, ArH), 7.29 (t, 2H, ArH), 7.35-7.5 (m, 6H,
ArH), 8.18 (s, 1H, urea), 8.93 (s, 1H, urea). MS (CI): m/e 349
(MH+). Anal. (C21H24N4O) C, H; N: calcd, 15.50; found, 16.08.
1-(5-t er t -Bu t yl-2-m -t olyl-2H -p yr a zol-3-yl)-3-p h e n yl-
u r ea (49). This compound was prepared from the condensation
of m-tolylhydrazine and 4,4-dimethyl-3-oxo-pentanenitrile; see
50; mp 120-122 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.28
(s, 9H, tert-butyl), 2.38 (s, 3H, Ar-CH3), 6.38 (s, 1 H, pyrazole
C4-H), 6.95 (t, 1H, ArH), 7.2-7.54 (m, 8H, ArH), 8.38 (s, 1H,
urea), 9.04 (s, 1H, urea). MS (CI): m/e 349 (MH+). Anal.
(C21H24N4O) H, N; C: calcd, 72.39; found, 71.93.
1-(5-t er t -Bu t yl-2-p -t olyl-2H -p yr a zol-3-yl)-3-p h e n yl-
u r ea (50). A solution of p-tolylhydrazine hydrochloride (3 g,
18.9 mmol), 4,4-dimethyl-3-oxopentanenitrile (2.6 g, 20.8
mmol), and concentrated HCl (2 mL) in ethanol (100 mL) was
heated to reflux for 12 h, cooled to room temperature, basified
with 20% aqueous NaOH to pH 12 (litmus), and extracted with
ethyl acetate (3 × 20 mL). The combined organic layers were
dried (MgSO4). Removal of the volatiles in vacuo afforded
5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl-amine (44) as a yellow
solid (3.7 g, 85%). A mixture of the above amine (0.150 g, 0.7
mmol) and phenyl isocyanate (0.07 mL, 0.7 mmol) in CH2Cl2
(10 mL) was stirred for 12 h at room temperature. Removal of
1-[2-(4-Am in op h en yl)-5-ter t-b u t yl-2H -p yr a zol-3-yl]-3-
p h en yl-u r ea (54). mp 199-201 °C. 1H NMR (400 MHz,
DMSO-d6): δ 1.28 (s, 9H, tert-butyl), 5.41 (s, 2H, Ar-NH2), 6.31

Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3005
(s, 1 H, pyrazole C4-H), 6.7 (d, 2H, ArH), 6.98 (t, 1H, ArH),
7.11 (d, 2H, ArH), 7.27 (t, 2H, ArH), 7.42 (d, 2H, ArH) 8.28 (s,
1H, urea), 9.15 (s, 1H, urea). Anal. (C20H23N5O) C, N; H: calcd,
7.12; found, 6.68.
1-(5-Met h yl-2-p h en yl-2H -p yr a zol-3-yl)-3-p h en yl-u r ea
(58). mp 195 °C. 1H NMR (400 MHz, DMSO-d6): δ 2.20 (s,
3H, methyl), 6.29 (s, 1H, pyrazole), 6.95-6.99 (m, 1H, aro-
matic), 7.25-7.29 (m, 2H, aromatic), 7.39-7.44 (m, 3H,
aromatic), 7.51-7.56 (m, 4H, aromatic), 8.42 (s, 1H, urea), 8.98
(s, 1H, urea). MS (PB-NH3-CI): m/e 293 (MH+). Anal.
(C17H16N4O) C, H, N.
1-(5-Isop r op yl-2-p h e n yl-2H -p yr a zol-3-yl)-3-p h e n yl-
u r ea (59). This compound was prepared as in 23 using methyl
isobutyrate; mp 161 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.23
(d, 6H, J ) 6.9 Hz, (CH3)2C-), 2.84-2.94 (m, 1H, Me2CH), 6.34
(s, 1H, pyrazole), 6.95-6.99 (m, 1H, aromatic), 7.24-7.33 (m,
2H, aromatic), 7.39-7.45 (m, 3H, aromatic), 7.51-7.56 (m, 4H,
aromatic), 8.42 (s, 1H, urea), 9.01 (s, 1H, urea). MS (EI): m/e
320 (M+). Anal. (C19H20N4O) C, H, N.
1-[5-(1,1-Dim eth ylp r op yl)-2-p h en yl-2H-p yr a zol-3-yl]-3-
p h en yl-u r ea (60). This compound was prepared as in 20
using 2,2-dimethylbutyric acid; mp 175 °C. 1H NMR (400 MHz,
DMSO-d6): δ 0.79 (t, 3H, propyl C3-H), 1.23 (s, 6H, dimethyl),
1.60 (q, 2H, propyl C2-H), 6.35 (s, 1H, pyrazole), 6.95-6.99
(m, 1H, aromatic), 7.24-7.28 (m, 2H, aromatic), 7.39-7.44 (m,
3H, aromatic), 7.51-7.57 (m, 4H, aromatic), 8.41 (s, 1H, urea),
9.02 (s, 1H, urea). MS (NH3-CI): m/e 349 (MH+). Anal.
(C21H24N4O) C, H, N.
provided a residue, which was purified by silica gel chroma-
tography using 30% ethyl acetate in hexanes as the eluent.
Concentration in vacuo of the product-rich fractions provided
(0.121 g, 23%) of 3-amino-5-(1-methyl-1-phenylethyl)-2-phenyl-
2H-pyrazole as an orange solid. A mixture of this amino-
pyrazole and phenyl isocyanate (50 uL, 0.436 mmol) in
anhydrous THF (2 mL) was stirred at room temperature under
inert atmosphere overnight. Removal of the volatiles in vacuo
provided a residue, which was purified by silica gel chroma-
tography using 1-5% methanol in CH2Cl2 as the eluent.
Concentration in vacuo of the product-rich fractions provided
1
64 as a light tan foam, which softens at 81-83 °C. H NMR
(400 MHz, DMSO-d6): δ 1.67 (s, 6H, dimethyl), 6.25 (s, 1H,
pyrazole), 6.94-6.98 (m, 1H, aromatic), 7.12-7.20 (m, 1H,
aromatic), 7.23-7.32 (m, 4H, aromatic), 7.36-7.39 (m, 4H,
aromatic), 7.42-7.47 (m, 1H, aromatic), 7.55-7.59 (m, 4H,
aromatic), 8.44 (s, 1H, urea), 9.02 (s, 1H, urea). MS (EI): m/e
397 (MH+). Anal. (C25H24N4O) C, H, N.
1-(5-ter t-Bu tyl-2-p h en yl-2H-p yr a zol-3-yl)-3-cycloh exyl-
u r ea (66). This compound was prepared as in 40 using
1
cyclohexylamine; mp 206 °C. H NMR (400 MHz, DMSO-d6):
δ 1.10-1.20 (m, 5H, cyclohexyl), 1.28 (s, 9H, tert-butyl), 1.48-
1.51 (m, 1H, cyclohexyl), 1.61-1.64 (m, 2H, cyclohexyl), 1.75-
1.79 (m, 2H, cyclohexyl), 3.36-3.41 (m, 1H, cyclohexyl-CH-
N), 6.26 (s, 1H, pyrazole), 6.48 (d, 1H, urea), 7.35-7.40 (m,
1H, aromatic), 7.42-7.51 (m, 4H, aromatic), 8.00 (s, 1H, urea).
MS (NH3-CI): m/e 341 (MH+). Anal. (C20H28N4O) C, H, N.
1-(5-t er t -Bu t yl-2-p h e n yl-2H -p yr a zol-3-yl)-3-(2,3-d i-
m eth ylp h en yl)-u r ea (72). This compound was prepared as
in 40 using 2,3-dimethylaniline; mp 210-213 °C. 1H NMR (400
MHz, CDCl3): δ 1.37 (s, 9H, tert-butyl), 2.06 (s, 3H, CH3), 2.26
(s, 3H, CH3), 6.40 (s, 1H, urea), 6.44 (s, 1H, pyrazole), 6.45 (s,
1H, urea), 7.06 (m, 2H, aromatic), 7.18 (m, 1H, aromatic), 7.36
(m, 5H, aromatic). MS (CI): m/e 363 (MH+). Anal. (C22H26N4O)
C, H, N.
1-[5-(2-Met h oxy-1,1-d im et h ylet h yl)-2-p h en yl-2H -p yr -
a zol-3-yl]-3-p h en yl-u r ea (61). This compound was prepared
as in 23 using methyl 2,2-dimethyl-3-methoxypropionate.
Foam, softens 68-72 °C. 1H NMR (400 MHz, DMSO-d6): δ
1.26 (s, 6H, dimethyl), 3.27 (s, 3H, CH3O-), 3.39 (s, 2H, CCH2-
O), 6.38 (s, 1H, pyrazole), 6.95-6.99 (m, 1H, aromatic), 7.25-
7.28 (m, 2H, aromatic), 7.39-7.44 (m, 3H, aromatic), 7.52-
7.57 (m, 4H, aromatic), 8.40 (s, 1H, urea), 9.01 (s, 1H, urea).
MS (NH3-CI): m/e 365 (MH+). Anal. (C21H24N4O2) C, H, N.
1-(5-Cycloh exyl-2-p h en yl-2H -p yr a zol-3-yl)-3-p h en yl-
u r ea (62). mp 182 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.16-
1.20 (m, 1H, cyclohexyl), 1.22-1.47 (m, 4H, cyclohexyl), 1.67-
1.70 (br d, 1H, J ) 12.2 Hz, cyclohexyl), 1.76-1.79 (br d, 2H,
J ) 12.1 Hz, cyclohexyl), 1.92-1.95 (br d, 2H, J ) 10.7 Hz,
cyclohexyl), 2.54-2.60 (m, 1H, cyclohexyl), 6.31 (s, 1H, pyr-
azole), 6.95-6.99 (m, 1H, aromatic), 7.24-7.28 (m, 2H, aro-
matic), 7.39-7.43 (m, 3H, aromatic), 7.51-7.55 (m, 4H,
aromatic), 8.40 (s, 1H, urea), 9.00 (s, 1H, urea). MS (PB-EI):
m/e 360 (M‚). Anal. (C22H24N4O) C, H, N.
1-[5-(1-Meth yl-1-p h en yleth yl)-2-p h en yl-2H-p yr a zol-3-
yl]-3-p h en yl-u r ea (64). Ethyl phenyl acetate (4.0 mL, 25.1
mmol) in 100 mL of anhydrous THF was added dropwise to a
-78 °C solution of lithium hexamethyldisilyl amide (30.1 mL
of a 1.0 M solution in THF). After 30 min, iodomethane (30.1
mmol, 1.9 mL) was added and the mixture was stirred for 30
min, warmed to room temperature, and stirred for 4 h. The
mixture was quenched with a saturated aqueous solution of
NH4Cl and extracted with ethyl acetate. The combined organic
extracts were washed with Na2S2O3 solution and brine and
dried (MgSO4). Removal of the volatiles in vacuo provided a
residue, which was subjected without purification to the same
conditions described above to introduce the second methyl
group. Purification by silica gel chromatography afforded 4.05
g (84%) of ethyl 2-methyl-2-phenylpropionate. A mixture of this
ester (0.370 g, 1.93 mmol) and anhydrous acetonitrile (0.14
mL, 2.70 mmol) in anhydrous toluene (4.0 mL) was added
dropwise to NaH (0.088 g of 60% dispersed in mineral oil, 2.20
mmol) in toluene (3 mL) at reflux. The mixture was heated
for 2 h, cooled to room temperature, carefully quenched with
5 N aqueous HCl, and extracted with CH2Cl2 (3 × 5 mL). The
combined organic extracts were dried (MgSO4). Removal of the
volatiles in vacuo provided a 1:1 mixture of starting ethyl
propionate and desired â-keto-nitrile product. Without any
purification, this material and phenyl hydrazine (0.2 mL, 1.9
mmol) in toluene (12 mL) were heated to reflux overnight and
cooled to room temperature. Removal of the volatiles in vacuo
1-Be n zyl-3-(5-t er t -b u t yl-2-p h e n yl-2H -p yr a zol-3-yl)-
u r ea (73). This compound was prepared as in 40 using
1
benzylamine; mp 190-192 °C. H NMR (400 MHz, CDCl3): δ
1.35 (s, 9H, tert-butyl), 4.40 (d, 2H, J ) 5.8 Hz, benzyl-CH2),
5.19-5.22 (m, 1H, urea), 6.14 (s, 1H, urea), 6.27 (s, 1H,
pyrazole), 7.18-7.19 (m, 1H, aromatic), 7.26-7.37 (m, 4H,
aromatic), 7.42-7.49 (m, 5H, aromatic). MS (NH3-CI): m/e
349 (MH+). Anal. (C21H24N4O) C, H, N.
1-(5-ter t-Bu tyl-2-p h en yl-2H-p yr a zol-3-yl)-3-p h en eth yl-
u r ea (74). This compound was prepared as in 40 using
phenethylamine; mp 144-146 °C. 1H NMR (400 MHz,
CDCl3): δ 1.32 (s, 9H, tert-butyl), 2.81 (t, 2H, J ) 6.7 Hz,
benzylic-CH2), 3.49-3.54 (m, 2H, N-CH2-benzyl), 4.92-4.95
(m, 1H, urea), 6.03 (s, 1H, pyrazole), 7.17-7.18 (m, 2H,
aromatic), 7.23-7.38 (m, 4H, aromatic), 7.44-7.47 (m, 4H,
aromatic). MS (NH3-CI): m/e 363 (MH+). Anal. (C22H26N4O)
C, H, N.
1-(5-ter t-Bu tyl-2-p h en yl-2H-p yr a zol-3-yl)-3-in d a n -1-yl-
u r ea (77). This compound was prepared as in 40 using racemic
1-aminoindan; mp 171-173 °C. 1H NMR (400 MHz, CDCl3):
δ 1.34 (s, 9H, tert-butyl), 1.63-1.73 (m, 1H, cyclopentyl), 2.53-
2.60 (m, 1H, cyclopentyl), 2.79-2.96 (2 m, 2H, cyclopentyl),
5.03 (d, 1H, J ) 8.4 Hz, urea), 5.33 (dd, 1H, J ) 15.8, 8.0 Hz,
cyclopentyl-CH-N), 6.13 (s, 1H, urea), 6.27 (s, 1H, pyrazole),
7.11-7.13 (m, 1H, aromatic), 7.17-7.21 (m, 1H, aromatic),
7.22-7.24 (m, 2H, aromatic), 7.35-7.38 (m, 1H, aromatic),
7.45-7.49 (m, 2H, aromatic), 7.51-7.54 (m, 2H, aromatic). MS
(NH3-CI): m/e 375 (MH+). Anal. (C23H26N4O) C, H, N.
1-(5-ter t-Bu t yl-2-p -t olyl-2H -p yr a zol-3-yl)-3-(2-flu or o-
p h en yl)-u r ea (78). A mixture of 5-tert-butyl-2-p-tolyl-2H-
pyrazol-3-yl-amine (44, 0.10 g, 0.4 mmol) and 2-fluorophenyl
isocyanate (0.053 mL, 0.5 mmol) in CH2Cl2 (10 mL) was stirred
for 12 h at room temperature. Removal of the volatiles in vacuo
provided a residue, which was crystallized with ethyl acetate
and hexanes and furnished the urea as a white solid (0.093 g,
58%); mp 103-104 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.29
(s, 9H, tert-butyl), 2.39 (s, 3H, Ar-CH3), 6.41 (s, 1 H, pyrazole

3006 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Regan et al.
C4-H), 6.95-7.55 (m, 7H, ArH), 8.84 (s, 1H, urea), 8.98 (s,
parameter logistic model was used of the form: percent
inhibition ) Imax × concN/concN + IC50N. The calculated
IC50 value is the concentration of the test compound that
caused a 50% decrease in the maximal inhibition of p38
activity as measured by TNF-R production.
1H, urea). MS (CI): m/e 367 (MH+). Anal. (C21H23FN4O) C, H,
N.
Biologica l Meth od s
P la te Assa y for Estim a tin g Bin d in g Affin ity (Kd )
of Com p ou n d s for p 38 MAP Kin a se. F lu or escen ce
Bin d in g Assa y. The binding affinities for inhibitors of
human recombinant p38 MAP kinase59 were determined
using a simple fluorescent binding assay. The assay is
based upon competition between a fluorescent probe
SK&F 86002 and any inhibitor of choice. Inhibitors were
assayed at two concentrations, typically 1 and 0.1 µM.
The actual concentrations of compounds were verified
by an analytical high-performance liquid chromatogra-
phy (HPLC). Compounds were diluted into binding
buffer (20 mM Bis-TRIS Propane, pH 7.0, 2 mM EDTA,
0.01% sodium azide, and 0.15% octylglucoside) and
placed in a 96 well fluorescent microtiter plate. Fluoro-
probe was added followed by the addition of p38 MAP
kinase. The plate was incubated at room temperature
for 60 min. Plates are read in a fluorescent microtiter
plate reader using an excitation wavelength of 332 nm
and an emission wavelength of 420 nm. The assay was
run in duplicate; all 48 data points (3 × 8 data points
in duplicate) were fit simultaneously to a simple equi-
librium binding equation, which results in an estimation
of binding affinity or Kd. In all cases, standard errors
and P values were within statistically acceptable limits.
A standard compound was included with each set of
experiments and its Kd varied by (25%.
THP -1 Cell Assa y for In h ibition of LP S-In d u ced
TNF -r P r od u ct ion . Cell Cu lt u r e a n d Com p ou n d
P r ep a r a tion . THP-1 cells (ATCC TIB 202, American
Type Culture Collection, Rockville, MD) were main-
tained at 37 °C, 5% CO2 in 10% fetal bovine serum
(FBS)/RPMI 1640 media as previously described.45 The
day of the assay, cells and reagents were diluted in 3%
FBS/RPMI 1640 media. Test compounds in DMSO were
diluted into 3% FBS/RPMI 1640 media and centrifuged
at room temperature for 10 min at 12 000g to precipitate
any undissolved compound. The actual concentration of
dissolved compound was determined by an HPLC
method. Supernatant was diluted serially in 3% FBS/
RPMI 1640 media containing 0.4% DMSO for all
subsequent dilutions (0.2% DMSO final).
In Vivo LP S Ch a llen ge Assa y. Female Balb/c mice,
weighing approximately 20 gm, were used. Mice were
administered 1, test compound, and vehicle in cremo-
phor (po) approximately 30 min prior to LPS/D-gal
administration. The volume of oral gavage was 0.15 mL.
Then, mice were administered LPS (E. coli LPS 0111:
B4, 1.0 µg/mouse) plus D-gal (50 mg/kg) intravenously
in 0.2 mL of pyrogen-free saline. One hour after LPS/
D-gal, each mouse was anesthetized, bled by cardiac
puncture, and collected for serum TNF-R and compound
levels. Blood samples were centrifuged at 2500 rpm for
10-15 min, the serum was decanted, and samples were
stored frozen at -70 °C until transfer either for TNF-R
determination or to Drug Metabolism and Pharmaco-
kinetics for plasma concentration analysis by HPLC.
The concentration of TNF-R in the serum was measured
by a commercially available ELISA kit (R&D Systems,
Minneapolis, MN). ELISA was performed according to
the manufacturers assay procedure. All samples were
assayed in duplicate.
X-r a y Cr ysta llogr a p h y. Crystals of human p38MAP
kinase in complex with inhibitors were prepared by the
hanging drop vapor diffusion method.59 The crystrals
belonged to the space group P2(1)2(1)2(1) and are
isomorphous to those reported for 45.45 X-ray diffraction
data were collected at 100 K, and the structure refine-
ment was carried out with the X-Plor program. An
example is shown below for compound 75. Space group,
P2(1)2(1)2(1); cell parameters (a,b,c), 65.4, 75.0, 78.7;
maximum resolution, 2A; number of observations,
277 841; number of reflections, 26 717; completeness, 97;
R
merge, 5.0%; R/free R factor, 21.4/29.1; rms deviation
in bond lengths, 0.010; rms deviation in bond angles,
1.4.
Ack n ow led gm en t. We thank Lei Zhu for experi-
mental assistance and Ed Harris for mass spectral
analysis. Peter Kinkade is acknowledged for sample
concentration determinations for the cellular assays.
The Drug Metabolism and Pharmacokinetic Depart-
ment is thanked for determination of plasma concentra-
tions and mouse and monkey PK data.
THP -1 Cell Assa y. Confluent THP-1 cells (2 × 106
cells/mL, final concentration) were added to culture
plates containing test compound or DMSO vehicle. The
cell mixture was allowed to preincubate for 30 min at
37 °C, 5% CO2, prior to stimulation with LPS (Sigma
L-2630, from Escherichia coli serotype 0111.B4; 1 ug/
mL final). Blanks (unstimulated) received H2O vehicle.
Overnight incubation (18-24 h) proceeded as described
above. The assay was terminated by centrifuging the
plates for 5 min at room temperature at 400g; super-
natants were transferred to clean culture plates and
stored at -80 °C until analyzed for human TNF-R by a
commercially available enzyme-linked immunosorbent
assay (ELISA) kit (Biosource #KHC3012, Camarillo,
CA).
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