Discovery of S-[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(2,3- dihydroxypropoxy)phenyl]-methanone (RO3201195), an orally bioavailable and highly selective inhibitor of p38 Map kinase

Article abstract of DOI:10.1021/jm050736c

A novel class of highly selective inhibitors of p38 MAP kinase was discovered from high throughput screening. The synthesis and optimization of a series of 5-amino-N-phenyl-1H-pyrazol-4-yl-3-phenylmethanones is described. An X-ray crystal structure of this series bound in the ATP binding pocket of unphosphorylated p38a established the presence of a unique hydrogen bond between the exocyclic amine of the inhibitor and threonine 106 which likely contributes to the selectivity for p38. The crystallographic information was used to optimize the potency and physicochemical properties of the series. The incorporation of the 2,3-dihydroxypropoxy moiety on the pyrazole scaffold resulted in a compound with excellent drug-like properties including high oral bioavailability. These efforts identified 63 (RO3201195) as an orally bioavailable and highly selective inhibitor of p38 which was selected for advancement into Phase I clinical trials.

Full text of DOI:10.1021/jm050736c

1562
J. Med. Chem. 2006, 49, 1562-1575
Discovery of S-[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(2,3-dihydroxypropoxy)phenyl]-
methanone (RO3201195), an Orally Bioavailable and Highly Selective Inhibitor of p38 Map
Kinase
David M. Goldstein,*,† Tom Alfredson,† Jay Bertrand,‡ Michelle F. Browner,† Ken Clifford,† Stacie A. Dalrymple,#
James Dunn,† Jose Freire-Moar,| Seth Harris,† Sharada S. Labadie,† JoAnn La Fargue,† Jean Marc Lapierre, Susan Larrabee,†
Fujun Li,† Eva Papp,† Daniel McWeeney,† Chakk Ramesha,† Rick Roberts,† David Rotstein,† Bong San Pablo,#
Eric B. Sjogren,† On-Yee So,† Francisco X. Talamas,† Will Tao,† Alejandra Trejo,† Armando Villasenor,† Mary Welch,†
Teresa Welch,† Paul Weller,† Phyllis E. Whiteley,§ Kelly Young,† and Sheila Zipfel#
Roche Palo Alto LLC, 3431 HillView AVenue, R6-123, Palo Alto, California 94304, Pharmacia Italy, Pfizer Group, Milan 20014, Italy,
Celera Genomics, 180 Kimball Way, South San Francisco, California 94080, Forest Research Institute, Harborside Financial Center,
Jersey City, New Jersey 07311, Arqule, Inc., 19 Presidential Way Woburn, Massachusetts 01801, and Perlegen Sciences, Inc.,
2021 Stierlin Court, Mountain View, California 94043
ReceiVed July 28, 2005
A novel class of highly selective inhibitors of p38 MAP kinase was discovered from high throughput
screening. The synthesis and optimization of a series of 5-amino-N-phenyl-1H-pyrazol-4-yl-3-phenylmetha-
nones is described. An X-ray crystal structure of this series bound in the ATP binding pocket of
unphosphorylated p38R established the presence of a unique hydrogen bond between the exocyclic amine
of the inhibitor and threonine 106 which likely contributes to the selectivity for p38. The crystallographic
information was used to optimize the potency and physicochemical properties of the series. The incorporation
of the 2,3-dihydroxypropoxy moiety on the pyrazole scaffold resulted in a compound with excellent drug-
like properties including high oral bioavailability. These efforts identified 63 (RO3201195) as an orally
bioavailable and highly selective inhibitor of p38 which was selected for advancement into Phase I clinical
trials.
Introduction
previously reported on the design and synthesis of 4-azaindoles
as potent and selective inhibitors of p38.6 In this manuscript,
we describe our efforts to optimize a lead identified through
high throughput screening (HTS), 4-benzoyl-5-aminopyrazole.
Several benzoyl containing amino pyrazoles were identified in
our HTS campaign, including compound 1 with an IC50 ) 2.3
µM. Early crystallographic efforts resulted in cocrystal structures
of the pyrazole series bound to unphosphorylated p38 MAP
kinase. These X-ray structures revealed a novel interaction in
the ATP binding site. In particular, the amine forms a hydrogen
bond with the side chain hydroxyl of threonine 106, a residue
not prevalent in most human kinases, which we hypothesized
might impart a high degree of selectivity to the series. The
structure also identified portions of the lead molecule which
could be readily substituted to optimize the physicochemical
properties of the series without loss of intrinsic potency. This
paper highlights our strategy utilizing structural data to drive
lead optimization and to ultimately identify 63 (RO3201195),
a highly selective and orally bioavailable inhibitor of p38.
Continued interest in development of small molecule inhibi-
tors of p38 MAP kinase is based on the central role this enzyme
plays in inflammatory cell signaling. It is known that activation
of p38 leads to increased production of pro-inflammatory
cytokines such as TNF-R and IL-1â.1 Human clinical trials with
biologic agents which neutralize TNF (etanercept, infliximab
and adalimumab) or IL-1 (anakinra) have validated the concept
that modulation of these cytokines leads to dramatic improve-
ment in inflammatory diseases such as RA, psoriasis and
inflammatory bowel disease.2 While these biologic agents have
revolutionized the treatment of some inflammatory disorders,
their high cost and inconvenient dosing regimens creates the
need for safe and effective orally active inhibitors of TNF and/
or IL-1. Several excellent reviews describing the potential
importance of p38 inhibitors as novel therapeutics in inflam-
matory disorders have been published.3 To date, however, only
a small number of inhibitors of p38 have been reported to have
advanced into phase II clinical trials. Only Vertex has reported
a correlation between inhibition of p38 and therapeutic benefit
in RA patients.4 A number of other agents have established a
correlation between inhibition of p38 and inhibition of TNF
and/or IL1 in phase I studies in healthy volunteers.5
Chemistry
Two synthetic routes to the (5-amino-1-phenyl-1H-pyrazol-
4-yl)phenylmethanone scaffold were developed (Schemes 1 and
2). The principle approach began with the corresponding methyl
benzoate 6. Addition of the lithium anion derived from
acetonitrile provided cyano ketone 7 in good yield. Ketone 7
reacted with N-phenylformamidine to form enamine 8 in high
yields. Treatment of 8 with the corresponding hydrazine
proceeded with high regioselectivity and exclusively cyclized
onto the nitrile to form pyrazole compounds 1 and 9-21.
Alternatively, the pyrazoles could be assembled according
to the route highlighted in Scheme 2. This approach allowed
the benzoyl ring to be added at a later stage of the synthesis.
Herein, we describe one part of our overall strategy to identify
multiple novel scaffolds as inhibitors of p38 MAP kinase. We
* To whom correspondence should be addressed. Phone (650) 855-5413.
Fax: (650) 852-1311. E-mail: David-M.Goldstein@roche.com.
† Roche Palo Alto LLC.
‡ Pharmacia Italy.
# Celera Genomics.
| Forest Research Institute.
Arqule, Inc.
§ Perlegen Sciences, Inc.
10.1021/jm050736c CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/09/2006

SelectiVe Inhibitor of p38 Map Kinase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1563
Scheme 1. Synthesis of (5-Amino-1-phenyl-1H-pyrazol-4-yl)-
Condensation of ethyl (ethoxymethylene)cyanoacetate with
4-fluorophenyl hydrazine 22 in ethanol formed pyrazole car-
boxylate esters directly. Saponification of the ester with lithium
hydroxide in methanol provided 5-amino-4-carboxypyrazole 23.
Activation of the acid by conversion to the 2-thiopyridyl ester
afforded intermediate 24 which was directly reacted with
substituted phenylmagnesium bromides in THF7 to give ketones
25 and 26. Subsequent deprotection of the silyl ether with TBAF
gave 27 which was then converted to the mesylate 28 under
standard conditions. Treatment of 28 with sodium azide and
potassium carbonate in DMF,8 followed by Staudinger reduction
with triphenylphosphine, provided analogue 29.9 Alternatively,
nucleophilic displacement of the mesylate 28 with 1-methylpip-
erazine under basic conditions in DMF gave compound 30.
phenylmethanones (Method A)a
Elaboration of the aroyl ring using various coupling technolo-
gies is described in Schemes 3 and 4. Coupling of aryl halide
16 or 19 with alkynes under Sonogashira coupling conditions10
gave derivatives 31-38. Alternatively, Heck reactions with
halide 16 provided olefins 41-44 in good yield. Hydrogenations
of the unsaturated side chains using catalytic palladium on
carbon afforded derivatives 45-48. Finally, Stille coupling11
with iodide 18 and vinyltributyltin gave styrene 49. Dihydroxy-
lation of 49 with osmium tetroxide12 gave the desired diol 50
in good yields.
Alternatively, Suzuki coupling of aryl halides could be used
to access compounds 51-58 as described in Scheme 4. Coupling
of pyridylboronic acids with halides 16, 17 or 19 produced
compounds 51-55. The N-oxide analogue 56 was produced via
oxidation of 55 with mCPBA in dichloromethane. Preparation
of analogues 57 and 58 started from iodo derivative 18. Suzuki
coupling between (2-bromoallyloxymethyl)benzene and iodide
18 via in situ boronate formation13 provided intermediate 57 in
a Reagents: (a) CH3CN, LDA, THF; (b) C6H5N:CHNHC6H5, xylenes,
reflux; (c) appropriately substituted phenylhydrazine, ethanol.
Scheme 2. Synthesis of [5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(substituted)phenyl]methanones (Method B)a
a Reagents: (a) ethyl (ethoxymethylene)cyanoacetate, ethanol, reflux; (b) LiOH, methanol, reflux; (c) 2,2′-dipyridinyl disulfide, Ph3P, acetonitrile; (d)
RPhMgBr or RPhLi, THF; (e) TBAF, THF, room temp; (f) CH3SO2Cl, pyridine, room temp; (g) 1-methylpiperazine, K2CO3, DMF, 70 °C; (h) NaN3,
K2CO3, DMF, room temp; (i) Ph3P, THF.

1564 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Goldstein et al.
Scheme 3. Synthesis of [5-Amino-1-(4-fluorophenyl)-
1H-pyrazol-4-yl]-[3-(substituted)phenyl]methanonesa
Scheme 4. Synthesis of [5-Amino-1-(4-fluorophenyl)-
1H-pyrazol-4-yl]-[3-(substituted)phenyl]methanonesa
a Reagents: (a) 2-, 3- or 4-pyridylboronic acid, (Ph3P)4Pd, K3PO4,
dioxane, 85 °C; (b) (2-bromoallyloxymethyl)benzene, KOAc, PdCl2(DPPF),
diboron pinacol ester, DMF, 80 °C; (c) N-methylmorpholine N-oxide,
osmium tetroxide, t-BuOH, water; (d) Pd/C, H2, EtOH.
More polar residues such as nitro and methoxy were less active
than hydrogen. In parallel, attempts to replace the exocyclic
amine resulted in loss of activity. Likewise, conversion of the
ketone to an oxime also resulted in inactive compounds.
X-ray Crystallography. To accelerate optimization of the
pyrazole lead series, significant effort was focused on generation
of X-ray crystal structures of pyrazole analogues bound to p38R
enzyme. The X-ray crystal structures of p38 MAP kinase with
bound pyrazole compounds revealed a unique binding motif
previously not reported for inhibitors of p38 kinase (Figure 1).
This class of inhibitors form a hydrogen bond between the
benzoyl oxygen and the main chain NH of methionine 109 (1.8
Å), a key interaction observed with all ATP competitive p38
inhibitors (Figure 1b). A novel hydrogen bond was observed
between the NH2 group of the aminopyrazole and the side chain
alcohol of threonine 106. A hydrogen bond between a p38
inhibitor and threonine 106 has not been reported previously.
With only approximately 20% of the human kinome having a
threonine at this position, we hypothesized that an inhibitor
series with this unique interaction could confer improved
selectivity versus other kinases which do not contain a similarly
located threonine. The N-phenyl ring of this inhibitor class is
positioned in a hydrophobic pocket toward the back of the
binding site, which is partially defined by threonine 106.
Lead Optimization Strategy. Compounds derived from the
pyrazole series were extremely insoluble and had poor oral
bioavailability. It was clear from the initial X-ray structures that
substitution of the benzoyl ring at either the meta or para position
would be well tolerated by the protein (Figure 1a). We
anticipated that substitutions at this position would be required
to achieve the desired physical properties for the series. Our
basic strategy was to improve the potency of the lead compounds
based on data from in in vitro assays and then to optimize the
pharmacokinetic properties. We put particular emphasis on the
LPS-induced cytokine production assay in undiluted human
whole blood (HWB) as a key driver for compound selection as
this assay provided surrogate efficacy in a physiologically
a Reagents: (a) Zn(CN)2, (Ph3P)4Pd, DMF, 60 °C or alkyne, (Ph3P)2PdCl2,
CuI, diisopropylamine, 70 °C; (b) triethylamine, (Ph3P)2PdCl2, DMF, 100
°C; (c) Pd/C, H2, EtOAc, EtOH, room temp; (d) vinyltributyltin, (Ph3P)4Pd,
DMF, 100 °C; (e) N-methylmorpholine N-oxide, osmium tetroxide, t-BuOH.
acceptable yield. Subsequent oxidation with osmium tetroxide
followed by catalytic hydrogenation to remove the benzyl
protecting group furnished compound 58.
Preparation of oxygen linked derivatives is shown in Scheme
5. Deprotection of the methoxy group of 20 with boron
tribromide provided phenol 59 which served as an intermediate
for the preparation of analogues 61-63. Analogue 61 was
prepared by Mitsunobu coupling of phenol 58 with bromoet-
hanol followed by subsequent halide displacement with piperi-
dine. Alternatively, alkylation of phenol 59 with (R)- or (S)-
O-isopropylideneglycerol tosylate followed by deprotection of
the acetal with p-toluenesulfonic acid gave chiral alcohols 62
and 63.
Results and Discussion
Initial Lead Identification. HTS was performed using a
radioactive filtration binding format using [33P] ATP (250 µM)
and myelin basic protein (150 µM) as the substrate. Compounds
were initially tested at 40 µM, and those which displayed >40%
inhibition of the enzyme activity were retested in the same
format at multiple concentrations. Several benzoyl amino
pyrazoles were identified in our HTS campaign, including
compound 1, which was confirmed to have an IC50 ) 2.3 µM.
To our knowledge, this was the first example of this scaffold
to be identified as an inhibitor of a protein kinase. Preliminary
SAR studies revealed that only small hydrophobic substituents
on the N-phenylpyrazole ring were tolerated (Table 1). It was
found that substitution at the para position of the phenyl ring
(compare ortho (13) vs meta (12) vs para (11) fluorine
derivatives and ortho (15)/para (14) methoxy) was preferred.

SelectiVe Inhibitor of p38 Map Kinase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1565
Scheme 5. Synthesis of [5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-(3-hydroxy-substituted-phenyl)methanonesa
a Reagents: (a) BBr3, CH2Cl2, room temp; (b) 2-bromoethanol, Ph3P, diethyl azodicarboxylate, toluene, 0 °C room temp; (c) piperidine, ethanol, reflux;
(d) (R)-O-isopropylidene glycerol tosylate or (S)-O-isopropylidene glycerol tosylate, K2CO3, DMF, 80 °C; (e) p-toluenesulfonic acid monohydrate, water,
50 °C.
Table 1. In Vitro Activity of 2-15 versus Compound 1
P38R
,
THP-1 TNF-R,
IC50 ( ASE (µM)
R
R1
R2
R3
R4
R5
IC50a ( ASE (µM)
1
H
H
H
H
H
H
H
NH2
CN
O
O
O
H
2.30 ( 0.6
>100
0.57 ( 0.05
2
H
H
H
3
H
H
OH
CH3
H
>100
4
H
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
N-OCH3
N-OCH3
51 ( 12
>100
>18
>18
0.51 ( 0.09
>56
0.10 ( 0.04
1.59 ( 0.47
0.56 ( 0.02
0.64 ( 0.06
0.53 ( 0.12
5
H
H
H
9
CH3
H
H
H
O
O
O
O
O
O
O
H
1.08 ( 0.16
64.2 ( 30.6
1.10 ( 0.11
6.7 ( 0.96
2.86 ( 0.21
7.06 ( 0.44
8.38 ( 2.5
10
11
12
13
14
15
NO2
H
H
H
H
F
H
H
F
H
H
F
H
H
H
H
H
OCH3
H
H
OCH3
H
H
a IC50s were calculated from the dose-response curves. Positive control: 4-[5-(4-Fluorophenyl)-4-pyridin-4-yl-1H-imidazol-2-yl]phenol, compound 64
(SB202190),20 IC50 of 0.03 ( 0.003 (µM).
relevant environment in the presence of serum albumin and other
proteins. Initial focus was to install either polar or ionizable
functional groups to either the para or the meta position of the
aryl ring either directly or via a linker group of different lengths.
It became apparent after synthesis of a number of analogues
that the meta position offered significantly greater tolerability
to a wide range of substituents than the corresponding para-
substituted analogues (examples include meta-substituted com-
pounds 31, 37 and 39 vs para analogues 32, 38 and 40). While
it was not immediately evident from our X-ray structures why
there was a preference for meta substitution, we focused our
optimization on a broader range of substituents at the meta
position. The installation of ionizable function, as expected, did
lead to dramatic improvements in intrinsic solubility; an increase
in solubility from 1 to 5 µg/mL in the lead molecule to >6
mg/mL and 5-10 mg/mL was obtained with analogues 25 and
36 respectively. This prompted us to explore other basic
containing groups (Table 2). While these derivatives had
desirable solubilities, they also exhibited greater inhibition in
cellular assays than expected based on their intrinsic in vitro
enzyme potency. We postulated that these molecules may have
other nonspecific effects resulting in cytotoxicy. In addition,
compounds such as 21 suffered from problems such as poor
metabolic stability and/or a complex metabolic profile specific
to the amine-bearing side chains. We ultimately chose to look
for alternate ways to improve the physicochemical properties
including installation of less-basic amines which would be
uncharged at physiologic pH. To this end, another series of
pyridyl analogues were synthesized and the results are shown
in Table 3. Within this series, several analogues were identified
that had enhanced potency with a similar correlation between
the isolated enzyme and the cellular assays as observed with
the more basic amine derivatives. While these compounds
showed good metabolic stability (only the N-oxide metabolite
of the pyridyl ring was identified, data not shown), they
unfortunately exhibited toxicity upon chronic administration in
rodents.
On the basis of these findings, alternate nonionizable solu-
bilizing functional groups became the primary focus of the lead
optimization strategy (Table 4). A variety of polar functional

1566 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Table 2. Basic Amines as Solubilizing Groups
Goldstein et al.
P38R
,
THP-1 TNF-R,
IC50 ( ASE (µM)
aqueous solubility
X
IC50a ( ASE (µM)
(mg/mL)
21
25
29
30
31
32
33
34
35
36
37
38
39
40
61
3-(2-morpholin-4-ylethoxy)
4-methylpiperazin-1-yl
2-aminoethyl
2-(4-methylpiperazin-1-yl)ethyl
3-cyano
1.27 ( 0.5
2.00 ( 0.3
2.44 ( 0.8
1.64 ( 0.3
4.76 ( 1.4
8.38 ( 3.5
4.04 ( 0.67
2.28 ( 0.7
2.65 ( 0.3
1.32 ( 0.18
1.72 ( 0.3
3.13 ( 0.8
1.74 ( 0.4
22.3 ( 4.4
1.84 ( 0.6
0.37 ( 0.02
1.21 ( 0.19
0.52 ( 0.05
0.36 ( 0.12
2.78 ( 0.9
>30
0.21 ( 0.03
0.15 ( 0.01
0.78 ( 0.18
0.06 ( 0.01
0.13 ( 0.01
11.22 ( 2.4
1.04 ( 0.09
>200
>6
>10
1
4-cyano
3-(3-hydroxyprop-1-ynyl)
3-dimethylaminoprop-1-ynyl
3-(4-methylpiperazin-1-yl
3-piperidin-1-ylprop-1-ynyl
3-(3-morpholin-4-ylprop-1-ynyl)
4-(3-morpholin-4-ylprop-1-ynyl)
3-(3-morpholin-4-ylpropyl)
4-(3-morpholin-4-ylpropyl)
2-piperidin-1-ylethoxy
5-10
1.41 ( 0.19
a IC50s were calculated from the dose-response curves. Positive control: Compound 64. IC50 of 0.03 ( 0.003 (µM).
Table 3. Pyridyl Substitution
P38R
,
THP-1 TNF-R,
IC50 ( ASE (µM)
aqueous solubility,
X
R
IC50a ( ASE (µM)
µg/mL
51
52
53
54
55
56
3-pyridin-3-yl
4-pyridin-3-yl
3-pyridin-4-yl
3-pyridin-4-yl
3-pyridin-3-yl
3-(1-oxypyridin-3-yl
H
H
H
F
F
F
0.78 ( 0.25
0.98 ( 0.2
1.28 ( 0.3
0.31 ( 0.04
0.18 ( 0.03
0.34 ( 0.04
0.047 ( 0.005
0.17 ( 0.03
>10
18.1
0.03 ( 0.003
0.04 ( 0.005
0.04 ( 0.01
1.4
10.7
5.8
a IC50s were calculated from the dose-response curves. Positive control: Compound 64. IC50 of 0.03 ( 0.003 (µM).
Table 4. Nonbasic Functional Groups as Solubilizing Agents
P38R
,
THP-1 TNF-R,
IC50 ( ASE (µM)
HWB IL-1,
IC50 ( ASE (µM)
aqueous solubility,
X
IC50a ( ASE (µM)
µg/mL
27
45
46
47
48
50
58
62
63
2-hydroxyethyl
2-ethanesulfonic acid amide
2-methanesulfonylethyl
2-(1-hydroxycyclopentyl)ethyl
3-hydroxy-3-methylbutyl
1,2-dihydroxyethyl
1,2-dihydroxy-1-hydroxymethylethyl
2,3-dihydroxypropoxy
0.85 ( 0.5
1.25 ( 0.1
0.79 ( 0.25
0.23 ( 0.07
0.59 ( 0.18
3.5 ( 0.8
9.54 ( 4.4
1.20 ( 0.4
0.7 ( 0.1
0.19 ( 0.03
0.51 ( 0.02
0.52 ( 0.03
0.13 ( 0.007
6.59 ( 0.18
1.30 ( 0.12
>10
0.69 ( 0.13
3.60 ( 0.6
0.22 ( 0.03
4.64 ( 0.4
0.92 ( 0.13
1.20 ( 0.36
5.6
121
0.7
ca. 40
>300
0.2 ( 0.04
0.25 ( 0.01
0.76 ( 0.4
0.57 ( 0.04
36.2
35.4
2,3-dihydroxypropoxy
a IC50s were calculated from the dose-response curves. Positive control: Compound 64. IC50 of 0.03 ( 0.003 (µM).
derivatives were made including amides, sulfonamides, ethers,
alcohols and diols. While several functionalities maintained
excellent potency against p38, only a few derivatives retained
both intrinsic potency as well as good inhibition of LPS-induced
Il-1â production in undiluted human whole blood. The com-
pounds which had the best balance between desirable physical
properties and potency in both in vitro and acute in vivo assays
were compounds 27 and 46 and diols 62 and 63. As there was
no precedence for the use of diols as solubilizing groups for
kinase inhibitors, there was some concern that these diols would
be prone to rapid clearance in vivo. We chose to evaluate 27
and 46 and both enantiomers 62 and 63 in vivo to evaluate their
pharmacokinetic properties. Definitive PK studies ultimately
resulted in the selection of diol 63 (the (S) enantiomer) due to
its high bioavailability and lower clearance rates relative to the
(R) enantiomer and analogues 27 and 46 across multiple species
Pharmacokinetics and Metabolism of 63. A summary of
the pharmacokinetic parameters for the S isomer 63 is detailed
in Table 5. In animals, systemic clearance of 63 was low to
moderate, while volume of distribution (Vdss) was medium to
high. The absorption was rapid and complete. Oral bioavail-
ability was 62%, 57% and 88% in rat, monkey and dog,

SelectiVe Inhibitor of p38 Map Kinase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1567
Table 5. Single Dose Plasma Pharmacokinetic Parameters of 63
Following Dosing in Animals
species, strain
rat,
monkey,
dog,
Hanover-Wistar
cyno-molgus
beagle
dose (mg/kg)
gender
sample size (iv, po)
po Tmax (h)
po Cmax (µg/mL)
po T1/2 (h)
po AUC Inf (µg‚h/mL)
po F%
iv Cl (L/h/kg)
iv Vdss (L/kg)
% protein bindingc
10
10
10
males
60,a 15b
3
4.48
1.64
19.50
61.70
0.32
1.27
12.10
male
4, 3b
1.08
2.02
2.41
7.56
56.80
0.76
21.40
11.20
female
4, 3b
0.83
4.21
1.57
13.30
87.50
0.67
2.55
21.30
a iv values based on mean plasma concentrations; plasma from three
rats per sample time. b Values based on mean parameter; serial plasma
samples obtained from each animal. c The in vitro binding of [14C]-63 to
plasma protein at 37 °C was studied by ultrafiltration methods.
inhibition detected in the unfractionated whole blood, whereas
TNFR was more potently inhibited in the mononuclear cell
fraction. To evaluate 63 in vivo, the compound was tested in a
number of acute inflammatory models. In the first study, 63
was dosed orally and was evaluated for its ability to inhibit the
production of TNFR and IL-6 after IP injection of LPS into
Hanover-Wistar and Sprague-Dawley rats. In this model, 63
was administered to rats 0.5 h prior to challenge with 50 µg/kg
LPS. Serum was collected 1.5 h after LPS injection for analysis
of cytokines and concentrations of drug. Compound 63 dem-
onstrated significant dose-dependent inhibition of serum TNFR
and IL-6. The estimated effective dose (ED) for TNFR inhibition
by 63 is reported in Table 6. In addition to inhibition of TNFR
production, compound 63 also regulates TNFR signaling. In
another rat model, 63 was administered to rats 0.5 h prior to
challenge with TNFR. Serum was collected 2 h after IP injection
of TNFR for analysis of cytokines and concentrations of drug.
Compound 63 inhibited TNFR-induced IL-6 at an ED50 ( ASE
of 1.5 ( 0.5 mg/kg. Furthermore, compound 63 was evaluated
in a chronic disease model, rat adjuvant arthritis (AI), by
subcutaneous delivery using osmotic minipumps. Lewis rats
were treated with vehicle or compound 63 delivered at a rate
of 0.5 mg/hr from day 1 to 14. Compound 63 inhibited the
adjuvant-induced increase in hind paw weight by 58% (p <
0.01) compared to vehicle-treated animals. In addition, the mean
clinical score exhibited by compound 63 treated rats was 6 ((
1) compared to 10 (( 0.7) out of a possible 19 in vehicle-treated
rats (p < 0.05). Plasma concentrations of compound 63 were
7.6 ( 0.8 and 7.5 ( 0.6 µM (mean ( SEM) on days 3 and 14,
respectively.
Figure 1. X-ray crystal structure of compound 63 bound in the ATP
binding site of unphosphorylated p38 MAP kinase. A. Ribbon diagram
of the p38 MAP kinase with the N-terminal domain colored blue and
the C-terminal domain in green. Substitutions at the meta or para
positions of the benzoyl ring are well tolerated because of their location
outside of the surface enclosed binding site. The enzyme is represented
by a transparent representation of the enzyme surface. B. Close-up of
the inhibitor binding to p38 MAP kinase. Hydrogen bond interactions
between the pyrazole and protein atoms are indicated, including the
novel interaction with side chain of threonine 106. This interaction may
be critical for the observed kinase selectivity of this class of inhibitors.
respectively. Compound 63 was moderately bound (79-88%)
to plasma in all species studied. In vitro, 63 is a poor substrate
for cytochrome P-450 and had a low affinity for the major
isoforms. In Inhibition studies with human cytochrome P-450
enzymes, 63 modestly inhibited only the 2C9 isoform with an
IC50 of 11 µM (IC50 values for human cytochromes 1A2, 2C19,
2D6 and 3A4 were greater than 25 µM).
Pharmacological Evaluation of 63. The complete in vitro
and in vivo pharmacology for diol 63 is summarized in Table
6. Compound 63 decreased the production of secreted cytokines
such as TNFR in a human monocytic cell line, THP1, after
stimulation with LPS. Compound 63 showed a concentration-
dependent inhibition of TNFR production with an estimated IC50
of 0.25 µM. The inhibitory effect of 63 on TNFR and IL-1â
production also was tested in a mononuclear cell fraction isolated
from HWB. Inhibitor 63 displayed a similar concentration-
dependent inhibition for both IL-1â and TNFR. The concentra-
tion-dependent inhibition of IL-1â was comparable to the
Kinase Selectivity Profiling. Protein kinases are considered
“higher” risk drug discovery targets in part due to the difficulty
to both measure and achieve kinase selectivity. We have
continued to improve our ability to assess the kinase selectivity
and to correlate the profiles of our kinase inhibitors with their
in vitro and in vivo pharmacology. As part of these efforts, we
evaluated the in vitro kinase selectivity profile of 63 against
105 kinases using the Ambit Biosciences platform.14 Of the 105
kinases tested, 63 inhibited only six other kinases with inhibition
>50% at 10 µM concentration. For each of these kinases, the
Kd was determined as is reported in Table 7. Compound 63 is
highly selective for p38 and inhibits only two other kinases at
comparable concentrations to its p38 activity. Compound 63
binds to both p38R and p38â isoforms but does not bind to
either the p38 γ or p38 δ isoforms. Compound 63 binds to both

1568 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Table 6. Summary of Biological Efficacy of Analogue 63
Goldstein et al.
in vitro
HWBa
in vivo
TNFRc
IL-6
THP-1a
mononuclearb
TNFR IL-1â
LPSc
LPSc
IL-6
AI
TNFR
TNFR
IL-1â
TNFR
paw weight
[7.5 ( 0.6]
IC50 (µM) or [plasma] (µM) 0.25 ( 0.01 1.97 ( 0.1 0.57 ( 0.04 0.4 ( 0.1 0.5 ( 0.1
ED50 (mg/kg)
% inhibition (vs vehicle)
0.2 ( 0.04 0.3 ( 0.05 1.5 ( 0.5
58, p < 0.01
a The data are expressed as the combined mean IC ( SEM. b The data are expressed as the mean IC ( ASE. c The data are expressed as the mean ED
( SEM.
Table 7. Kinases Inhibited by Compound 63 from a Panel of 105
Kinases Tested at 10 µM
reaction progress was determined by thin-layer chromatography
(TLC) using Analtec 250-µm silica gel plates. Visualization was
done with UV light (254 nm) or iodine. Yields are of purified
compounds and were not optimized. 1H NMR measurements were
recorded at 75.40 MHz using a Bruker AMX 300 instrument in
DMSO with tetramethylsilane as the internal standard. Melting
points recorded were uncorrected. Elemental analyses were within
(0.4% except for compound 50 for which hydrogen analysis
exhibits (0.41% error. Purity of compounds 9-15 and 19 was
determined using a Micromass Platform LC, single quadruple mass
spectrometer with a electrospray (ES) probe using a mobile phase
of acetonitrile/water with 0.1% TFA.
kinase
kinase assay Kda (µM)
EPHA5
GAK
JNK2
JNK3
LCK
6.6
0.59
5.0
6.9
3.8
PDGFRâ
0.61
0.20
0.05
P38R
P38â
a The Kds were determined using the Ambit Bioscience platform.14
(5-Amino-1-phenyl-1H-pyrazol-4-yl)phenylmethanone (1). A
solution of benzoylacetonitrile (10 g, 68.9 mmol) and N,N-
diphenylformamidine (13.5 g, 68.9 mmol) in xylenes (50 mL) was
heated at reflux under argon. After 2 h, the reaction mixture was
cooled to room temperature and diluted with ether to give
2-benzoyl-3-phenylaminoacrylonitrile (14.0 g) as a solid.
A mixture of 2-benzoyl-3-phenylaminoacrylonitrile (14.0 g, 56.4
mmol) and phenylhydrazine (5.6 mL, 56.4 mmol) in ethanol (10
mL) was heated at reflux. After 3 h, the reaction mixture was cooled
to room temperature and the solid was filtered and washed with
ethanol to give (5-amino-1-phenyl-1H-pyrazol-4-yl)phenylmetha-
none(4.75 g). 1H NMR (DMSO-d6) δ 7.19 (s, 2H), 7.42-7.60 (m,
8H), 7.77-7.80 (m, 2H), 7.81 (s, 1H). Ms (EI/CI) m/z: (M + H)
262. Anal. (C16H13N3O) C, H, N.
the phosphorylated and unphosphorylated form of p38R (data
not shown) as commonly reported for ATP competitor inhibitors
of p38.
On the basis of all of these results, compound 63 was selected
as a candidate for Phase I clinical trials as an oral agent that
will suppress TNF and related cytokine production, with the
potential to offer increased safety and efficacy compared to the
current standard therapies for RA at appreciably less cost than
that of biological anticytokine therapy.
Conclusion
A novel class of p38 inhibitors (5-amino-1-phenyl-1H-
pyrazol-4-yl)phenylmethanones was identified by HTS. Initial
SAR studies and the cocrystal structure of 25 with unphospho-
rylated p38 guided our efforts to improve both in vitro and in
vivo potency as well as the physical properties of our initial
lead. After surveying a wide variety of different solubilizing
groups, only nonbasic solubilizing groups, particularly 2(S)
glycerol monoether 63, resulted in a balance of good potency
and favorable drug delivery characteristics with a desirable
metabolic profile. To our knowledge, compound 63 represents
the first use of a diol moiety as a solubilizing group for a kinase
inhibitor selected for clinical study. Our data suggests that diols
can be effectively used to improve the physical properties of
otherwise insoluble kinase scaffolds. The use of the glycerol
mono ether moiety on the pyrazole scaffold resulted in a
molecule with excellent drug-like properties and high oral
bioavailability while maintaining intrinsic potency against p38.
Compound 63 selectively inhibits p38 MAP kinase, an enzyme
in the intracellular signaling pathway for the generation of TNFR
and IL-1â. The compound reduced the production of TNFR from
human monocytes, whole blood, and a monocytic cell line in
vitro as well as reduced the production of downstream cytokine
IL-6 in vivo. Upon the basis of the range of efficacy in the
biological models, compound 63 was selected as a clinical
candidate for the treatment of inflammatory diseases including
rheumatoid arthritis.
4-Benzoyl-2-phenyl-2H-pyrazole-3-carbonitrile (2). To a mix-
ture of anhydrous copper bromide (2.3 g, 10.3 mmol) and tert-
butyl nitrite (1.54 mL, 12.9 mmol) in anhydrous acetonitrile (5 mL)
at 65 °C was added 5-amino-1-phenyl-1H-pyrazol-4-yl)phenyl-
methanone (2.3 g, 8.6 mmol) as a solid. The reaction mixture was
stirred for 24 h, cooled to room temperature, poured into 2 N HCl
(100 mL) and extracted into ethyl acetate, the organic phase was
washed with brine, dried over sodium sulfate and concentrated in
vacuo and the residue was purified by flash chromatography, eluting
with 10-40% ethyl acetate/hexane to afford (5-bromo-1-phenyl-
1H-pyrazol-4-yl)phenylmethanone (1.25 g).
A mixture of (5-bromo-1-phenyl-1H-pyrazol-4-yl)phenylmetha-
none (900 mg, 2.75 mmol) and CuCN (370 mg, 4.13 mmol) in
NMP (100 mL) was refluxed for 2 h and then cooled to room
temperature, and an excess of ethylenediamine and ethyl acetate
was added. This resulting mixture was filtered through Celite. The
filtrate was washed with water and brine and then dried over sodium
sulfate. Concentration in vacuo and purification by flash chroma-
tography with 10-40% ethyl acetate/hexane afforded 4-benzoyl-
2-phenyl-2H-pyrazole-3-carbonitrile (460 mg). 1H NMR (DMSO-
d6) δ 7.61-7.78 (m, 6H), 7.82-7.85 (m, 2H), 7.95-7.98 (m, 2H),
8.45 (s, 1H). Ms (EI/CI) m/z: (M + H) 273. Anal. (C17H11N3O)
C, H, N.
(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)phenylmetha-
none (3). A suspension of 1-phenyl-3-methyl-5-pyrazolone (95.9
g, 28.7 mmol) in dioxane (30 mL) was heated and stirred with an
overhead stirrer. When the mixture was homogeneous, calcium
hydroxide (4.0 g, 54.0 mmol) was added portion wise followed by
dropwise addition of benzoyl chloride (3.3 mL, 28.4 mmol). The
mixture was heated at reflux under nitrogen for 30 min, cooled to
room temperature and then poured into 2 N HCl (100 mL). The
brown solid formed was filtered and purified by flash chromatog-
raphy eluting with 10-25% ethyl acetate/hexane. 1H NMR (DMSO-
Experimental Section
Reagents and solvents were obtained from commercial suppliers
and were used without further purification. Flash chromatography
was performed with Merck silica gel 60 (230-400 mesh), and

SelectiVe Inhibitor of p38 Map Kinase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1569
d6) δ 2.50 (s, 3H), 7.29-7.34 (m, 1H), 7.46-7.52 (m, 4H), 7.54-
7.60 (m, 1H), 7.69-7.76 (m, 4H). Ms (EI/CI) m/z: (M + H) 278.
Anal. (C17H14N2O2) C, H, N.
5-Amino-1-(4-fluorophenyl)-4-[3-iodobenzoyl]pyrazole (18).
n-Butyllithium (30.5 mL, 76 mmol, 2.5 M solution in hexane) was
added dropwise to a cooled (0 °C) solution of diisopropylamine
(10.6 mL, 76 mmol) in 10 mL dry tetrahydrofuran. Once addition
was complete, the solution was kept at 0 °C for 10 min and was
then cooled to -50 °C. This cold LDA solution was then added to
a -50 °C solution of acetonitrile (2.37 mL, 45.3 mmol) and ethyl
iodobenzoate (10.0 g, 36.2 mmol) in dry tetrahydrofuran (18 mL).
Once addition was complete, the reaction was stirred at -50 °C
for 3 h and was subsequently warmed to 0 °C. Saturated acqueous
ammonium chloride solution was added (20 mL), and the reaction
mixture was allowed to warm to room temperature. The product
was extracted into ether and washed with 1 N hydrochloride acid
(50 mL). The organics were washed with brine (50 mL), dried over
magnesium sulfate and then concentrated in vacuo to give a red
oil. The oil was purified through a small plug of silica gel using
3:1-2:1 hexanes/ethyl acetate as eluent. Concentration of the
column fractions in vacuo gave 2-(3-iodobenzoyl)acetonitrile (8.3
g) as a yellow oil.
(5-Amino-1-phenyl-1H-pyrazol-4-yl)phenylmethanone O-Meth-
yloxime (4 and 5). (5-Amino-1-phenyl-1H-pyrazol-4-yl)phenyl-
methanone (250 mg, 0.95 mmol) in methanol (5 mL) with
methoxylamine hydrochloride (95 mg, 1.14 mmol) was treated with
pyridine (0.36 mL). The mixture was refluxed for 5 days and then
concentrated in vacuo, and the residue was partitioned between
water (10 mL) and ethyl acetate (10 mL), separated and extracted
with ethyl acetate (2 × 10 mL). The combined organic layers were
washed with brine (10 mL) and then dried over sodium sulfate,
concentrated in vacuo and purified by preparative thin-layer
chromatography eluting with 3:1 hexane/ethyl acetate to afford 4
1
(84 mg). H NMR (DMSO-d6) δ 3.88 (s, 3H), 5.47 (br, 2H), 7.05
(s, 1H), 7.35-7.56 (m, 8H), 7.57-7.64 (m, 2H). MS (EI/CI) m/z:
(M + H) 294. Anal. (C17H16N4O) C, H, N and 5 (140 mg) 1H NMR
(DMSO-d6) δ 4.09 (s, 3H), 4.55 (br, 2H), 7.36-7.46 (m, 5H), 7.47-
7.67 (m, 6H). MS (EI/CI) m/z: (M + H) 294. Anal. (C17H16N4O)
C, H, N.
A mixture of 2-(3-iodobenzoyl)acetonitrile (36.2 g, 133.5 mmol)
and N,N-diphenylformamidine (26.2 g, 133.5 mmol) in toluene (200
mL) was heated at reflux under a nitrogen atmosphere. After 8 h,
the reaction mixture was cooled to room temperature and diluted
with ether (200 mL) to give 2-(3-iodobenzoyl)-3-phenylaminoacry-
lonitrile (31.2 g) as a solid.
(5-Amino-1-O-tolyl-1H-pyrazol-4-yl)phenylmethanone (9). 1H
NMR (DMSO-d6) δ 2.11 (s, 3H), 6.89 (s, 2H), 7.33-7.62 (m, 7H),
7.77-7.80 (m, 3H). MS (EI/CI) m/z: (M + H) 278.
[5-Amino-1-(3-nitrophenyl)-1H-pyrazol-4-yl]phenylmetha-
none (10). 1H NMR (DMSO-d6) δ 7.42 (s, 2H), 7.53-7.61(m, 3H),
7.77-7.89 (m, 4H), 8.07 (d, J ) 8.03 Hz, 1H), 8.28 (d, J ) 8.27,
1H), 8.38-8.39 (m, 1H). MS (EI/CI) m/z: (M + H) 309
[5-Amino-1-(4-methoxyphenyl)-1H-pyrazol-4-yl]phenylmetha-
none (14). 1H NMR (DMSO-d6) δ 7.02 (s, 2H), 7.10 (d, J ) 8.94
Hz, 2H), 7.47 (d, J ) 8.96 Hz, 2H), 7.50-7.58 (m, 3H), 7.76-
7.78 (m, 3H). MS (EI/CI) m/z: (M + H) 294.
A mixture of 4-fluorophenylhydrazine (26.6 g, 211 mmol) and
2-(3-iodobenzoyl)-3-phenylaminoacrylonitrile (79 g, 211 mmol) in
ethanol (400 mL) was heated at reflux under a nitrogen atmosphere.
After 30 min, the reaction mixture was cooled to room temperature
and then diluted with hexane to give 5-amino-1-(4-fluorophenyl)-
1
4-(3-iodobenzoyl)pyrazole (75.1 g) as a solid. H NMR (DMSO-
d6) δ 7.19 (s, 2H), 7.32-7.44 (m, 3H), 7.59-7.63 (m, 2H), 7.77
(s, 1H), 7.79 (d, J ) 7.99 Hz, 1H), 7.96 (d, J ) 7.78 Hz, 1H), 8.02
(s, 1H). MS (EI/CI) m/z: (M + H) 407. Anal. (C16H11F2I N3O) C,
H, N.
[5-Amino-1-(2-methoxyphenyl)-1H-pyrazol-4-yl]phenylmetha-
none (15). 1H NMR (DMSO-d6) δ 3.83 (s, 3H), 6.85 (s, 2H), 7.10
(t, J ) 7.60 Hz, 1H), 7.26 (d, J ) 7.38 Hz, 1H), 7.36 (dd, J ) 6.09
Hz, J ) 1.70 Hz, 1H), 7.48-7.59 (m, 4H), 7.74 (s, 1H), 7.75-
7.79 (m, 2H). MS (EI/CI) m/z: (M + H) 294.
5-Amino-1-(4-fluorophenyl)-4-[3-(2-morpholin-4-ylethoxy)-
benzoyl]pyrazole Hydrochloride (21). A mixture of methyl
3-hydroxybenzoate (8.0 g, 56 mmol) and 4-(2-chloroethyl)mor-
pholine hydrochloride (15.7 g, 84 mmol) and potassium carbonate
(11.5 g, 83 mmol) in toluene (50 mL) was heated at reflux. After
4 days, the reaction mixture was cooled to room temperature and
diluted with ethyl acetate. The organic layer was washed with water
and then extracted with dilute hydrochloric acid. The acidic layer
was separated, basified with 5 N sodium hydroxide, and the product
was extracted into ethyl acetate. The organics were removed in
vacuo, and the residue was purified by flash chromatography
(elution gradient 3% acetone/methylene chloride) to give methyl
3-(2-morpholin-4-ylethoxy)benzoate (9.0 g) as an oil.
Lithium diisopropylamide (18.8 mL, 37 mmol, 2.0 M solution
in heptane/tetrahydrofuran/ethylbenzene) was added dropwise to a
solution of acetonitrile (1.58 g, 37 mmol) in dry tetrahydrofuran
(50 mL) at -78 °C. After stirring the reaction mixture for 30 min,
a solution of methyl 3-(2-morpholin-4-ylethoxy)benzoate in dry
tetrahydrofuran (50 mL) was added dropwise over 10 min. After
15 min, water was added and the reaction mixture was allowed to
warm to room temperature. The aqueous layer was separated and
neutralized with dilute hydrochloric acid to pH 7. The product was
extracted into ethyl acetate, washed with water and brine and dried
over magnesium sulfate. The organics were removed in vacuo to
give 2-[3-(2-morpholin-4-ylethoxy)phenyl]acetonitrile (5.0 g) as an
oil which was used in the next step without further purification.
A mixture of 2-[3-(2-morpholin-4-ylethoxy)phenyl]acetonitrile
(5.0 g) and N,N-diphenylformamidine (5.0 g, 25.5 mmol) in xylene
(150 mL) was heated at 100 °C under a nitrogen atmosphere. After
3 h, the reaction mixture was cooled to room temperature and
diluted with hexane to give 2-[3-(2-morpholin-4-ylethoxy)benzoyl]-
3-phenylaminoacrylonitrile (5.0 g) as a solid.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-(3-bromophen-
yl)methanone (16). n-Butyllithium (214 mL, 340 mmol, 1.6 M
solution in hexane) was added dropwise to a solution of acetonitrile
(23.8 mL, 460 mmol) in dry tetrahydrofuran (1000 mL) at -78
°C. After stirring the reaction mixture for 20 min, a solution of
4-bromobenzoyl chloride in dry tetrahydrofuran (50 mL) was added
dropwise over 20 min. After 1 h, saturated aqueous ammonium
chloride solution was added (200 mL) and the reaction mixture
was allowed to warm to room temperature. The product was
extracted into ether and washed with 1 N hydrochloric acid (400
mL). The organics were removed in vacuo and the residue was
redissolved in ethyl acetate. Ammonium hydroxide was added to
give a solid that was filtered, redissolved in ethyl acetate and washed
with 2 N hydrochloric acid. The organic layer was washed with
brine, dried over sodium sulfate and concentrated in vacuo to give
2-(3-bromobenzoyl)acetonitrile (16.6 g) as a solid.
A mixture of 2-(3-bromobenzoyl)acetonitrile (16.5 g, 73.6 mmol)
and N,N-diphenylformamidine (14.5 g, 73.6 mmol) in xylene (100
mL) was heated at reflux under a nitrogen atmosphere. After 3 h,
the reaction mixture was cooled to room temperature and diluted
with ether to give 2-(3-bromobenzoyl)-3-phenylaminoacrylonitrile
(17.9 g) as a solid.
A mixture of 4-fluorophenylhydrazine (4.25 g, 33.7 mmol) and
2-(3-bromobenzoyl)-3-phenylaminoacrylonitrile (10.0 g, 30.7 mmol)
in ethanol (100 mL) was heated at reflux under a nitrogen
atmosphere. After 4 h, the reaction mixture was cooled to room
temperature, diluted with hexane to give 5-amino-4-(3-bromoben-
zoyl)-1-(4-fluorophenyl)pyrazole (9.7 g) as a solid. 1H NMR
(DMSO-d6) δ 7.18 (s, 2H), 7.41 (t, J ) 8.83 Hz, 2H), 7.50 (t, J )
7.83 Hz, 1H), 7.59-7.63 (m, 2H), 7.77-7.10 (m, 3H), 7.84-7.86
(m, 1H). MS (EI/CI) m/z: (M + H) 360.
[5-Amino-1-(2,4-difluorophenyl)-1H-pyrazol-4-yl]-(3-bromophen-
A mixture of 4-fluorohydrazine (1.0 g, 6.8 mmol) and 2-[3-(2-
morpholin-4-ylethoxy)benzoyl]-3-phenylaminoacrylonitrile (2.0 g,
5.3 mmol) in ethanol (30 mL) was heated at reflux under a nitrogen
atmosphere. After 6 h, the reaction mixture was cooled to room
1
yl)methanone (17). H NMR (DMSO-d6) δ 7.27-7.33 (m, 3H),
7.48-7.69 (m, 3H), 7.77-7.86 (m, 4H). MS (EI/CI) m/z: (M +
H) 378. Anal. (C16H10BrF2N3O) C, H, N.

1570 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Goldstein et al.
temperature and diluted with water. The product was extracted into
ethyl acetate, and the organic layer was washed with brine, dried
over sodium sulfate and concentrated in vacuo. Purification by flash
chromatography (elution gradient: CH2Cl2-3% MeOH/CH2Cl2)
gave 5-amino-1-(4-fluorophenyl)-4-[3-(2-morpholin-4-ylethoxy)-
benzoyl]pyrazole which was converted to the hydrochloride salt
butyldimethylsiloxyethyl)bromobenzene (5.0 g, 15.9 mmol), and
the reaction mixture was heated at reflux. After 3 h, the reaction
mixture was cooled to room temperature, 5-amino-1-(4-fluorophe-
nyl)-4-(2-pyridylthiocarboxy)pyrazole (2.37 g, 7.6 mmol) was added
and the stirring was continued for 16 h. The reaction mixture was
concentrated in vacuo. The residue was dissolved in ethyl acetate,
washed with saturated aqueous ammonium chloride solution and
brine and dried over sodium sulfate. The organics were removed
in vacuo, and the residue was purified by flash chromatography
(elution gradient: 10-30% ethyl acetate/hexane) to give 5-amino-
1-(4-fluorophenyl)-4-[3-(2-tert-butyldimethylsiloxyethyl)benzoyl]-
pyrazole (1.20 g).
To a solution of 5-amino-1-(4-fluorophenyl)-4-[3-(2-tert-bu-
tyldimethylsiloxyethyl)benzoyl]pyrazole (1.2 g, 3.0 mmol) in
tetrahydrofuran (25 mL) was added tetrabutylammonium fluoride
(3.6 mL, 3.6 mmol, 1 M solution in tetrahydrofuran). After 1 h,
the reaction mixture was poured into brine, and the product was
extracted into ethyl acetate. The organic layer was dried over sodium
sulfate, filtered and concentrated in vacuo. The residue was purified
by flash chromatography (elution gradient: 40-100% ethyl acetate/
hexane) to give 5-amino-1-(4-fluorophenyl)-4-[3-(2-hydroxyethyl)-
benzoyl]pyrazole (0.8 g). 1H NMR (DMSO-d6) δ 2.82 (t, J ) 6.69
Hz, 2H), 3.67 (dd, J ) 5.27 Hz, J ) 11.94 Hz, 2H), 4.71 (t, J )
5.17 Hz, 1H), 7.16 (s, 2H), 7.37-7.45 (m, 4H), 7.57-7.64 (m,
4H), 7.81 (s, 1H). MS (EI/CI) m/z: (M + H) 326. Anal. (C18H16-
FN3O2) C, H, N.
5-amino-4-[3-(2-aminoethyl)benzoyl]-1-(4-fluorophenyl)pyra-
zole Hydrochloride (29). To a solution of 5-amino-1-(4-fluorophen-
yl)-4-[3-(2-hydroxyethyl)benzoyl]pyrazole 27 (0.8 g, 2.5 mmol) in
pyridine (10 mL) was added methanesulfonyl chloride (0.29 mL,
3.7 mmol). After 2 h, the reaction mixture was poured into 2 N
hydrochloric acid (40 mL), and the product was extracted into ethyl
acetate. The organic layer was washed with brine, dried over sodium
sulfate, filtered and concentrated in vacuo. The residue was purified
by flash chromatography (elution gradient: 40-100% ethyl acetate/
hexane) to give methanesulfonic acid 2-{3-[5-amino-1-(4-fluo-
rophenyl)-1H-pyrazole-4-carbonyl]phenyl}ethyl ester (0.87 g).
A mixture of 5-amino-1-(4-fluorophenyl)-4-[3-(2-methanesulfo-
nyloxyethyl)benzoyl]pyrazole 28 (0.40 g, 0.99 mmol), sodium azide
(0.19 mL, 2.97 mmol) and potassium carbonate (0.41 g, 2.97 mmol)
in dimethylformamide (15 mL) was stirred at room temperature.
After 16 h, the reaction mixture was poured into brine and the
product was extracted into ethyl acetate. The organic layer was
dried over sodium sulfate, filtered and concentrated in vacuo. The
residue was purified by flash chromatography (elution gradient:
20-50% ethyl acetate/hexane) to 5-amino-1-(4-fluorophenyl)-4-
[3-(2-azidoethyl)benzoyl]pyrazole (0.32 g).
To a solution of 5-amino-1-(4-fluorophenyl)-4-[3-(2-azidoethyl)-
benzoyl]pyrazole (0.31 g, 0.9 mmol) in tetrahydrofuran (15 mL)
was added triphenylphosphine (3.55 g, 1.36 mmol). After 48 h,
the reaction mixture concentrated in vacuo. The residue was
dissolved in 2 N sodium hydroxide, and the product was extracted
into ethyl acetate. The organic layer was dried over sodium sulfate,
filtered and concentrated in vacuo. The product was converted to
its hydrochloride salt and recrystallized from a mixture of methanol-
ethyl acetate to give 5-amino-4-[3-(2-aminoethyl)benzoyl]-1-(4-
fluorophenyl)pyrazole hydrochloride salt (0.22 g). 1H NMR (DMSO-
d6) δ 2.50-3.02 (m, 2H), 3.09-3.35 (m, 2H), 7.28 (s, 2H), 7.42
(t, J ) 8.76 Hz, 2H), 7.50 (d, J ) 5.50 Hz, 2H), 7.59-7.67 (m,
4H), 7.85 (s, 1H), 8.10 (br, 2H). MS (EI/CI) m/z: (M + H) 361.
Anal. (C18H17FN4O2) C, H, N.
1
(0.7 g). H NMR (DMSO-d6) δ 3.12-3.30 (m, 2H), 3.47-3.63
(m, 4H), 3.79-3.91 (m, 2H), 3.93-4.02 (m, 2H), 4.50-4.56 (m,
2H), 7.19 (s, 2H), 7.21-7.26 (m, 1H), 7.32 (s, 1H), 7.37-7.52
(m, 4H), 7.58-7.65 (m, 2H), 7.83 (s, 1H). MS (EI/CI) m/z: (M +
H) 465. Anal. (C22H23FN4O3) C, H, N.
5-Amino-1-(4-fluorophenyl)-1H-pyrazole-4-carbothioic Acid
S-Pyridin-2-yl Ester (24). A mixture of ethyl (ethoxymethylene)-
cyanoacetate (26 mL, 154 mmol) and 4-fluorophenyl hydrazine
(19.4 g, 154 mmol) in ethanol (125 mL) was heated at reflux. After
16 h, the reaction mixture was cooled to room temperature. The
solid was filtered and dried to give 5-amino-4-ethylcarboxy-1-(4-
fluorophenyl)pyrazole (28 g) which was suspended in a mixture
of 1 N lithium hydroxide (100 mL) and methanol (250 mL). The
reaction mixture was heated at reflux. After 16 h, the reaction
mixture was filtered through a sinter funnel and the filtrate was
acidified with 2 N hydrochloric acid (65 mL). The solid was filtered
and dried to give 5-amino-4-carboxy-1-(4-fluorophenyl)pyrazole (21
g).
A mixture of 5-amino-4-carboxy-1-(4-fluorophenyl)pyrazole (15
g, 68 mmol), 2,2′-dipyridinyl disulfide (14.9 g, 68 mmol) and
triphenylphosphine (17.8 g, 68 mmol) in acetonitrile (2 L) was
stirred at room temperature. After 16 h, the product was filtered
and dried to give 5-amino-1-(4-fluorophenyl)-4-(2-pyridylthiocar-
boxy)pyrazole (14 g).
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(4-meth-
ylpiperazin-1-yl)phenyl]methanone (25). Into an oven dried flask
containing magnesium turnings (1.1 g, 45.2 mmol) and tetrahy-
drofuran (50 mL) was added 1-(3-bromo-phenyl)-4-methylpipera-
zine (11.6 g, 45.5 mmol), and the reaction mixture was heated at
reflux. 5-Amino-1-(4-fluorophenyl)-1H-pyrazole-4-carbothioic acid
S-pyridin-2-yl ester 23 (6.82 g, 12.7 mmol), partially suspended in
tetrahydrofuran (75 mL), was added and the mixture was stirred
for 18 h at room temperature. The reaction was diluted with ethyl
acetate (50 mL) and washed with aqueous ammonium chloride
solution and brine and dried over sodium sulfate. The organics were
removed in vacuo and the residue was purified by flash chroma-
tography (elution gradient: 3% methanol/dichloromethane) to give
[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(4-methyl-piper-
azin-1-yl)phenyl]methanone (3.5 g) which was converted to the
1
hydrochloride salt. H NMR (DMSO-d6) δ 2.82 (d, J ) 4.69 Hz,
3H), 3.14-3.17 (m, 2H), 3.49-3.52 (m, 1H), 3.90-3.94 (m, 1H),
7.23-7.29 (m, 3H), 7.38-7.44 (m, 3H), 7.59-7.63 (m, 2 H), 7.79
(s, 1H). MS (EI/CI) m/z: (M + H) 443. Anal. (C21H22FN5O‚HCl)
C, H, N..
5-Amino-1-(4-fluorophenyl)- 4-[3-(2-hydroxyethyl)benzoyl]-
pyrazole (27). To a solution of 3-bromophenylacetic acid (10 g,
46.5 mmol) in tetrahydrofuran (100 mL) at 0 °C was added diborane
(70 mL, 1.0 M solution in tetrahydrofuran). The reaction mixture
was allowed to warm to room temperature. After 16 h, the reaction
mixture was cooled to 0 °C and water was added dropwise (50
mL). The organic layer was separated, washed with brine, dried
over sodium sulfate and concentrated in vacuo. The residue was
purified by flash chromatography (elution gradient: 40-60% ethyl
acetate/hexane) to give 3-(2-hydroxyethyl)bromobenzene (9.0 g).
To a solution of 3-(2-hydroxyethyl)bromobenzene (4.0 g, 20
mmol) in methylene chloride (100 mL) at 0 °C was added a solution
of tert-butyldimethylsilyl chloride (3.6 g, 24 mmol), (dimethylami-
no)pyridine (0.61 g, 5 mmol) and triethylamine (3.6 mL, 25.9
mmol). After 1 h, the reaction mixture was washed with brine and
saturated aqueous ammonium chloride solution, dried over sodium
sulfate and concentrated in vacuo. The residue was purified by flash
chromatography (elution gradient: 0-10% hexane/ethyl acetate)
to give 3-(2-tert-butyl-dimethylsiloxyethyl)bromobenzene (6.0 g).
Into an oven dried flask containing magnesium turnings (0.386
g, 15.9 mmol) and tetrahydrofuran (10 mL) was added 3-(2-tert-
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-{3-[2-(4-meth-
yl-piperazin-1-yl)ethyl]phenyl}methanone (30). A mixture of
5-amino-1-(4-fluorophenyl)-4-[3-(2-methanesulfonyloxyethyl)ben-
zoyl]pyrazole 28 (0.22 g, 0.55 mmol), N-methylpiperazine (0.18
mL, 1.64 mmol) and potassium carbonate (0.22 g, 1.64 mmol) in
dimethylformamide (10 mL) was heated at 70 °C. After 4 h, the
reaction mixture was cooled to room temperature and poured into
water and the product was extracted into ethyl acetate. The organic
layer was washed with brine, dried over sodium sulfate, filtered
and concentrated in vacuo. The residue was purified by flash

SelectiVe Inhibitor of p38 Map Kinase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1571
chromatography (elution gradient: ethyl acetate-20% methanol/
ethyl acetate) to afford 5-amino-1-(4-fluorophenyl)-4-{3-[4-meth-
ylpiperazin-1-yl)ethyl)benzoyl]pyrazole which was converted to the
) 8.31 Hz, 2H), 7.82 (s, 1H). MS (EI/CI) m/z: (M + H) 404.
Anal. (C23H21FN4O2) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(3-morpho-
1
1
hydrochloride salt. H NMR (DMSO-d6) δ 2.14 (s, 3H), 2.32-
lin-4-ylpropyl)phenyl]methanone (39). H NMR (DMSO-d6) δ
2.57 (m, 10H), 2.83 (t, J ) 7.48 Hz, 2H), 7.16 (s, 2H), 7.38-7.44
(m, 4H), 7.56-7.64 (m, 4H), 7.82 (s, 1H). MS (EI/CI) m/z: (M +
H) 499. Anal. (C23H26FN5O2‚2HCl‚0.75H2O) C, H, N.
2.09 (m, 2H), 2.76 (t, J ) 7.68 Hz, 2H), 3.02-3.09 (m, 4 H), 3.77-
3.95 (m, 4H), 7.16 (br, 2H), 7.39-7.49 (m, 2H), 7.59-7.63 (m,
2H), 7.81 (s, 2H). MS (EI/CI) m/z: (M + H) 408. Anal. (C23H25-
FN4O2‚HCl) C, H, N.
3-[5-Amino-1-(4-fluorophenyl)-1H-pyrazole-4-carbonyl]-ben-
zonitrile (31). A mixture of 5-amino-4-(3-bromobenzoyl)-1-(4-
fluorophenyl)pyrazole 19 (2 g, 5.5 mmol), zinc cyanide (652 mg,
5.5 mmol) and tetrakis(triphenylphosphine)palladium (0) (64 mg,
0.55 mmol) in DMF (2 mL). was heated at 60 °C under argon.
After 16 h, the mixture was cooled to room temperature, diluted
with ethyl acetate and washed with 2 N NH4OH. The insoluble
material was removed by filtration, and the filtrate was dried over
sodium sulfate, concentrated and recrystallized from ethyl acetate/
hexane to afford 3-[5-amino-1-(4-fluorophenyl)-1H-pyrazole-4-
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[4-(3-morpho-
1
lin-4-ylpropyl)phenyl]methanone (40). H NMR (DMSO-d6) δ
2.09 (m, 2H), 2.74 (t, J ) 7.66 Hz, 2H), 3.02-3.09 (m, 4 H), 3.40-
3.50 (m, 4H), 3.83-3.96 (m, 4H), 7.15 (br, 2H), 7.38-7.44 (m, 4
H), 7.59-7.64 (m, 2H), 7.74 (d, J ) 8.11 Hz, 2H) 7.81 (s, 1H).
MS (EI/CI) m/z: (M + H) 408. Anal. (C23H25FN4O2‚HCl) C, H,
N.
5-Amino-4-[3-(2-aminosulfonylethyl) benzoyl]-1-(4-fluorophen-
yl)pyrazole (45). A mixture of 5-amino-4-(3-bromobenzoyl)-1-(4-
fluorophenyl)pyrazole (1.5 g, 4.14 mmol), vinylsulfonamide (1.33
g, 12.4 mmol), bis(triphenylphosphine)palladium chloride (0.3 g,
0.42 mmol) and triethylamine (6 mL, 43 mmol) in dimethylfor-
mamide (18 mL) was heated at 100 °C under argon. After 16 h,
the reaction mixture was cooled to room temperature and poured
into 1 N hydrochloric acid. The product was extracted into ethyl
acetate, washed with brine, dried over sodium sulfate and filtered.
The organic layer was removed in vacuo, and the residue was
purified by flash chromatography (elution gradient: 40-80% ethyl
acetate/hexane) to give 5-amino-4-[3-(2-aminosulfonylethenyl)-
benzoyl]-1-(4-fluorophenyl)pyrazole which was recrystallized from
a mixture of methanol/ethyl acetate/hexane to give 0.78 g of the
desired product.
1
carbonyl]benzonitrile (720 mg). H NMR (DMSO-d6) δ 7.22 (s,
2H), 7.38-7.44 (m, 2H), 7.59-7.63 (m, 2H), 7.74 (t, J ) 7.76
Hz, 1H), 7.86 (s, 1H), 8.04-8.09 (m, 2H), 8.14-8.15 (m, 1H).
MS (EI/CI) m/z: (M + H) 306. Anal. (C17H11FN4O‚ 0.25 H2O) C,
H, N.
4-[5-Amino-1-(4-fluorophenyl)-1H-pyrazole-4-carbonyl]ben-
1
zonitrile (32). H NMR (DMSO-d6) δ 7.24 (br, 2H), 7.38-7.44
(m, 2H), 7.54-7.64 (m, 2H), 7.86 (s, 1H), 7.91 (d, J ) 8.35 Hz,
2H), 8.00 (d, J ) 8.52 Hz, 2H), 8.14-8.15 (m, 1H). MS (EI/CI)
m/z: (M + H) 306. Anal. (C17H11FN4O‚0.1H2O) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(3-hydroxy-
prop-1-ynyl)phenyl]methanone (33). A mixture of 5-amino-4-(3-
bromobenzoyl)-1-(4-fluorophenyl)pyrazole 19 (1.5 g, 4.2 mmol),
propargyl alcohol (0.5 mL, 8.33 mmol), bis(triphenylphosphine)-
palladium(II) chloride (146 mg, 0.21 mmol) and copper iodide (79
mg, 0.42 mmol) was heated at 70 °C under argon. After 16 h, the
mixture was cooled to room temperature, diluted with ethyl acetate
and washed with brine. The insoluble material was removed by
filtration, and the filtrate was dried over sodium sulfate, concentrated
and purified by flash chromatography eluting with 40%-100%
ethyl acetate/hexane. The product was recrystallized from ethyl
acetate/hexane to afford [5-amino-1-(4-fluorophenyl)-1H-pyrazol-
4-yl]-[3-(3-hydroxyprop-1-ynyl)phenyl]methanone (420 mg). 1H
NMR (DMSO-d6) δ 4.33 (d, J ) 5.97 Hz, 2H), 5.37 (t, J ) 5.94
Hz, 1H), 7.17 (br, 2H), 7.38-7.44 (m, 2H), 7.50-7.65 (m, 4H),
7.72-7.79 (m, 3H). MS (EI/CI) m/z: (M + H) 335. Anal. (C19H14-
FN3O2 C19H14FN3O2‚0.5H2O) C, H, N.
A mixture of 5-amino-4-[3-(2-aminosulfonylethenyl)benzoyl]-
1-(4-fluorophenyl)pyrazole (2.1 g, 5.43 mmol) and palladium
hydroxide (0.6 g) in methanol (150 mL) was shaken in a Parr
apparatus under hydrogen atmosphere at 50 psi. After 4 days, the
reaction mixture was filtered through Celite, and the filtrate was
concentrated. The residue was purified by flash chromatography
(elution gradient: 40-100% ethyl acetate/hexane) to give a crude
product which was recrystallized from methanol/ethyl acetate/
hexane to give 5-amino-4-[3-(2-aminosulfonylethyl)benzoyl]-1-(4-
1
fluorophenyl)pyrazole (0.95 g) as a solid. H NMR (DMSO-d6) δ
3.09-3.15 (m, 2H), 3.32-3.37 (m, 2H), 6.90 (s, 2H), 7.17 (s, 2H),
7.38-7.50 (m, 4H), 7.59-7.65 (m, 4H), 7.85 (s, 1H). MS (EI/CI)
m/z: (M + H) 388. Anal. (C18H17FN4O3S) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(2-methane-
1
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(3-(dimethyl-
sulfonylethyl)phenyl]methanone (46). H NMR (CDCl3) δ 2.89
1
amino)prop-1-ynyl)phenyl]methanone (34). H NMR (DMSO-
(s, 3H), 3.24-3.39 (m, 4H), 6.05 (s, 2H), 7.22-7.28 (m, 2H), 7.42-
7.58 (m, 4H), 7.68-7.74 (m, 2H), 7.76 (s, 1H). MS (EI/CI) m/z:
(M + H) 388. Anal. (C19H18FN3O3S‚ 0.45 H2O) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-{3-[2-(1-hydroxy-
d6) δ: 2.87 (s, 6 H), 4.34 (s, 2 H), 7.21 (s, 2 H), 7.42 (t, J ) 8.82
Hz, 2 H), 7.58-7.64 (m, 3 H), 7.75-7.87 (m, 4 H). MS (EI/CI)
m/z: (M + H) 362. Anal. (C21H19FN4O‚HCl) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-{3-[3-(4-meth-
yl-piperazin-1-yl)prop-1-ynyl]phenyl}methanone (35). 1H NMR
(DMSO-d6) δ 2.85 (s, 3H), 3.32 (br, 4H), 3.64 (br, 4H), 4.21 (s,
2H), 7.41 (t, J ) 8.83 Hz, 2H), 7.59-7.76 (m, 3H), 7.77-7.84
(m, 4H). MS (EI/CI) m/z: (M + H) 417. Anal. (C24H24FN5O‚2HCl)
C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(3-piperidin-
1-ylprop-1-ynyl)phenyl]methanone (36). 1H NMR (DMSO-d6) δ
1.82 (br, 6H), 3.02 (br, 2H), 3.45 (br, 2H), 4.31 (s, 2H), 7.20 (s,
2H), 7.41 (t, J ) 8.82 Hz, 2H), 7.57-7.64 (m, 3H), 7.74-7.86
(m, 4H). MS (EI/CI) m/z: (M + H) 439. Anal. (C24H23FN4O‚HCl)
C, H, N.
1
cyclopentyl)ethyl]phenyl}methanone (47). H NMR (CDCl3) δ
1.59-1.96 (m, 10 H), 2.83-2.88 (m, 2H), 6.07 (s, 2H), 7.20-
7.27 (m, 2H), 7.40 (d, J ) 5.01 Hz, 2H), 7.52-7.58 (m, 2H), 7.60-
7.65 (m, 2H), 7.78 (s, 1H). MS (EI/CI) m/z: (M + H) 394. Anal.
(C23H24FN3O2) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(3-hydroxy-
3-methyl-butyl)phenyl]methanone (48). 1H NMR (CDCl3) δ 1.16
(s, 6 H), 1.66-1.72 (m, 2H), 2.70-2.76 (m, 2H), 4.30 (s, 1 H),
7.16 (s, 2H), 7.38-7.44 (m, 4H), 7.56-7.65 (m, 4H), 7.91 (s, 1H).
MS (EI/CI) m/z: (M + H) 368. Anal. (C21H22FN3O2) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]- [3-(1,2-dihy-
droxy-ethyl)phenyl]methanone (50). To a solution of 5-amino-
1-(4-fluorophenyl)-4-[3-iodobenzoyl]pyrazole (15.0 g, 36.85 mmol)
in DMF (150 mL) were added vinyltributyltin (12.85 g, 40.54
mmol) and tetrakis(triphenylphosphine)palladium(0) (2.13 g, 1.84
mmol). The reaction was degassed with argon and then warmed to
100 °C under argon. After 18 h the reaction was cooled to room
temperature and then poured into water (500 mL). The product was
extracted into 1:1 ether/ethyl acetate, and the combined organic
extracts were washed with water and brine and dried over
magnesium sulfate. Concentration in vacuo gave an oil which was
purified by flash column chromatography using 5:1-4:1 hexane/
5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(3-morpho-
1
lin-4-ylprop-1-ynyl)phenyl]methanone (37). H NMR (DMSO-
d6) δ 3.22 (br, 2H), 3.41 (br, 2H), 3.92 (br, 2H), 4.37 (s, 2H), 7.20
(s, 2H), 7.38-7.44 (m, 2H), 7.58-7.63 (m, 3H), 7.74-7.87 (m,
4H). MS (EI/CI) m/z: (M + H) 404. Anal. (C23H21FN4O2‚HCl) C,
H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[4-(3-morpho-
1
lin-4-ylprop-1-ynyl)phenyl]methanone (38). H NMR (DMSO-
d6) δ 3.49-2.56 (m, 4H), 3.57 (s, 2H), 3.63 (t, J ) 4.64 Hz, 4H),
7.18 (br, 2H), 7.38-7.44 (m, 2H), 7.57-7.63 (m, 2H), 7.77 (d, J

1572 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Goldstein et al.
ethyl acetate to remove the byproducts and 3:1 to 2:1 hexane/ethyl
acetate to elute [5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-(3-
vinylphenyl)methanone (10.06 g).
aqueous Na2SO3 and extracted into dichloromethane, dried over
sodium sulfate, concentrated in vacuo and purified by flash
chromatography eluting with 100% ethyl acetate and then 3%
methanol/ethyl acetate to give [5-amino-1-(2,4-difluorophenyl)-1H-
pyrazol-4-yl]-[3-(1-oxypyridin-3-yl)phenyl]methanone which was
recrystallized from methanol (1.3 g). 1H NMR (DMSO-d6) δ 7.27-
7.34 (m, 1H), 7.50-7.56 (m, 5 H), 7.84 (d, J ) 6.43 Hz, 1H),
7.93-7.97 (m, 2H), 8.02 (s, 1H), 8.26 (d, J ) 4.68 Hz, 1H), 8.68
(s, 1H). MS (EI/CI) m/z: (M + H) 392. Anal. (C21H14F2N4O2‚
0.2H2O) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(1,2-dihy-
droxy-1-hydroxymethylethyl)phenyl]methanone (58). To a solu-
tion of 5-amino-1-(4-fluorophenyl)-4-(3-iodobenzoyl)pyrazole (1.0
g, 2.46 mmol) in DMF (10 mL) were added potassium carbonate
anhydrous (733 mg, 7.37 mmol), diboron pinacol ester (0.686 g,
2.70 mmol) and dichloro[1,1′-bis(diphenylphosphino)ferrocene]-
palladium(II) dichloromethane adduct (0.20 g, 0.246 mmol). The
solution was degassed with argon and warmed to 80 °C for 2 h.
The reaction was then cooled to room temperature and dichloro-
[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane
adduct (200 mg, 0.246 mmol) was added along with (2-bromoal-
lyloxymethyl)benzene (0.613 g, 2.70 mmol) and sodium carbonate
(1.3 g, 12.3 mmol) in water (6 mL). The reaction was again
degassed and warmed back to 80 °C. After 18 h, the reaction was
cooled to room temperature and was poured into water (100 mL),
and the product was extracted into ethyl acetate. The combined
ethyl acetate extracts were washed with brine and dried over
magnesium sulfate. Concentration in vacuo gave a brown oil which
was purified by flash column chromatography using 4:1-2:1
hexane/ethyl acetate to yield [5-amino-1-(4-fluorophenyl)-1H-
pyrazol-4-yl]-[3-(1-benzyloxymethylvinyl)phenyl]methanone (380
mg).
To a solution of [5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-
[3-(1-benzyloxymethylvinyl)phenyl]methanone 57 (0.38 g, 0.889
mmol) in tert-butyl alcohol were added N-methylmorpholine
N-oxide (0.114 g, 0.978 mmol) and water (3 mL). To this solution
was then added osmium tetroxide (0.56 mL, 0.04 mmol). The
reaction was stirred at room temperature for 8 h. The reaction was
poured into ethyl acetate (25 mL) and washed with brine. The
organic layer was separated and dried over magnesium sulfate.
Concentration in vacuo gave a brown oil which was purified by
flash column chromatography using 1:1 hexane/ethyl acetate to
remove impurities and 4:1 ethyl acetate/hexane to elute [5-amino-
1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(2-benzyloxy-1-hydroxy-1-
hydroxymethylethyl)phenyl]methanone (73 mg).
To a suspension of [5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-
yl]-(3-vinylphenyl)methanone (8.0 g, 24.92 mmol) in tert-butyl
alcohol (90 mL) was added N-methylmorpholine N-oxide (3.2 g,
27.41 mmol) in water (90 mL). To this slurry at room temperature
was then added osmium tetroxide (9.375 mL, 0.747 mmol, 2.5%
in tert-butyl alcohol). After 18 h, the reaction was complete and
homogeneous. The product was extracted into ethyl acetate, and
the combined organic extracts were washed with water and brine
and dried over magnesium sulfate. Concentration in vacuo gave a
brown oil which was purified by flash column chromatography
using 1:1 hexane/ethyl acetate as eluent. The product eluted with
100% ethyl acetate to afford [5-amino-1-(4-fluorophenyl)-1H-
pyrazol-4-yl]-[3-(1,2-dihydroxyethyl)phenyl]methanone (4.96 g). 1H
NMR (DMSO-d6) δ 3.44-3.99 (m, 2H), 4.62-4.68 (m, 1H), 4.80
(t, J ) 5.78 Hz, 1H), 5.40 (d, J ) 4.33 Hz, 1H), 7.16 (s, 2H),
7.38-7.65 (m, 7H), 7.77 (s, 1H), 7.81 (s, 1H). MS (EI/CI) m/z:
(M + H) 342 Anal. (C18H16FN3O3) C, H, N.
Synthesis of 5-Amino-1-(4-fluorophenyl)-4-[3-(pyridin-3-yl)-
benzoyl]pyrazole (51). A mixture of 5-amino-4-(3-bromobenzoyl)-
1-(4-fluorophenyl)pyrazole (0.9 g, 2.5 mmol), pyridine-3-boronic
acid, 1,3-propanediol cyclic ester (0.5 g, 3 mmol), potassium
phosphate (0.8 g, 3.73 mmol) and tetrakis(triphenylphosphine)-
palladium (0.3 g, 0.25 mmol) in dioxane (20 mL) was heated at 85
°C under argon. After 16 h, the reaction mixture was cooled to
room temperature and poured into brine. The product was extracted
into ethyl acetate, dried over sodium sulfate and filtered. The organic
layer was removed in vacuo, and the residue was purified by flash
chromatography (elution gradient: 40-80% ethyl acetate/hexane)
to give 5-amino-1-(4-fluorophenyl)-4-[3-(pyridin-3-yl)benzoyl]pyra-
zole (0.50 g) which was recrystallized from ethyl acetate. 1H NMR
(DMSO-d6) δ 7.25 (br, 2H), 7.39-7.45 (m, 2H), 7.60-7.65 (m,
2H), 7.75 (t, J ) 7.71 Hz, 1H), 7.90 (d, J ) 7.75 Hz, 1H), 7.95 (s,
1H), 8.08-8.10 (m, 2H), 8.19 (s, 1H), 8.86-8.91 (m, 2H), 9.33
(s, 1H). MS (EI/CI) m/z: (M + H) 359. Anal. (C21H15FN4O‚HCl)
C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-(4-pyridin-3-
1
ylphenyl)methanone (52). H NMR (DMSO-d6) δ 7.25 (br, 2H),
7.39-7.45 (m, 2H), 7.60-7.65 (m, 2H), 7.86 (s, 1H), 7.95 (d, J )
6.51 Hz, 2H), 8.03-8.09 (m, 3H), 8.82 (d, J ) 4.68 Hz, 1H), 8.90
(d, J ) 5.48 Hz, 1H), 9.29 (d, J ) 2.05 Hz, 1H). MS (EI/CI) m/z:
(M + H) 359. Anal. (C21H15FN4O‚HCl) C, H, N.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-(3-pyridin-4-
1
ylphenyl)methanone (53). H NMR (DMSO-d6) δ 7.25 (br, 2H),
To a solution of [5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-
[3-(2-benzyloxy-1-hydroxy-1-hydroxymethylethyl)phenyl]metha-
none (320 mg, 0.694 mmol) in ethanol (25 mL) was added 10%
Pd/C (100 mg). The reaction was stirred at room temperature under
hydrogen. After 72 h, the palladium was removed by filtration
through Celite. The filtrate was concentrated to an oil and purified
by flash chromatography using 2:1 ethyl acetate/hexane-100%
ethyl acetate as eluent to yield [5-amino-1-(4-fluorophenyl)-1H-
pyrazol-4-yl]-[3-(1,2-dihydroxy-1-hydroxymethylethyl)phenyl]meth-
7.39-7.45 (m, 2H), 7.60-7.65 (m, 2H), 7.79 (t, J ) 7.74 Hz, 1H),
7.92 (s, 1H), 7.99 (d, J ) 7.79 Hz, 1 H), 8.21 (d, J ) 7.78 Hz, 1
H), 8.28 (s, 1H), 8.45 (d, J ) 6.73 Hz, 2H), 8.98 (d, J ) 6.71 Hz,
2H). MS (EI/CI) m/z: (M + H) 359. Anal. (C21H15FN4O‚HCl‚
0.15H2O) C, H, N.
[5-Amino-1-(2,4-difluorophenyl)-1H-pyrazol-4-yl]-(3-pyridin-
1
4-ylphenyl)methanone (54). H NMR (DMSO-d6) δ 7.28-7.34
(m, 3H), 7.56-7.66 (m, 2H), 7.79 (t, J ) 7.74 Hz, 1H), 7.92 (s,
1H), 7.99 (d, J ) 7.79 Hz, 1H), 8.21 (d, J ) 7.78 Hz, 1H), 8.28 (s,
1H), 8.45 (d, J ) 6.73 Hz, 2H), 8.98 (d, J ) 6.71 Hz, 2H). MS
(EI/CI) m/z: (M + H) 377. Anal. (C21H14F2N4O‚HCl‚0.25 H2O)
C, H, N.
1
anone (152 mg). H NMR (DMSO-d6) δ 3.78 (d, J ) 11.4 Hz,
2H), 3.94 (d, J ) 11.4 Hz, 2H), 6.31 (s, 2H), 7.21-7.26 (m, 2H),
7.45-7.58 (m, 3H), 7.68-7.73 (m, 2H), 7.80 (s, 1H), 7.98 (s, 1H).
MS (EI/CI) m/z: (M + H) 372. Anal. (C19H18FN3O4‚ 0.7 H2O) C,
H, N.
[5-Amino-1-(2,4-difluorophenyl)-1H-pyrazol-4-yl]-(3-pyridin-
1
3-ylphenyl)methanone (55). H NMR (DMSO-d6) δ 7.29-7.34
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(2-piperidin-
1-yl-ethoxy)phenyl]methanone (61). [5-Amino-1-(4-fluorophenyl)-
1H-pyrazol-4-yl]-(3-hydroxyphenyl)methanone (1.5 g, 5.05 mmol)
was combined with toluene (50 mL). 2-Bromoethanol (1.79 mL,
25.23 mmol) was added, and the mixture was cooled to 0 °C.
Triphenylphosphine (5.425 g, 20.69 mmol) and diethyl azodicar-
boxylate (3.26 mL, 20.69 mmol) were then added. The reaction
was allowed to warm to room temperature. After stirring for 16 h,
the reaction was quenched with a saturated aqueous ammonium
chloride solution, extracted with ethyl acetate, dried over magnesium
sulfate, filtered and concentrated in vacuo. The product was purified
by column chromatography on silica gel using 40:1 CH2Cl2/MeOH
(m, 1H), 7.52-7.69 (m, 2H), 7.78 (t, J ) 7.73 Hz, 1H), 7.92 (s, 1
H), 7.94 (s, 1H), 8.06-8.14 (m, 2H), 8.17 (s, 1H), 8.88-8.91 (m,
2H), 9.30 (s, 1H). MS (EI/CI) m/z: (M + H) 377. Anal.
(C21H14F2N4O‚HCl) C, H, N.
[5-Amino-1-(2,4-difluorophenyl)-1H-pyrazol-4-yl]-[3-(1-oxy-
pyridin-3-yl)phenyl]methanone (56). To a suspension of [5-amino-
1-(2,4-difluorophenyl)-1H-pyrazol-4-yl]-(3-pyridin-3-ylphenyl)-
methanone 56 (4.4 g, 11.7 mmol) in dichloromethane (100 mL) at
room temperature under nitrogen was added 3-chloroperoxybenzoic
acid (7.8 g, 45.2 mmol). The mixture turned reddish-brown and
became homogeneous. After 18 h the mixture was poured into 10%

SelectiVe Inhibitor of p38 Map Kinase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1573
and then stirred with ether for 20 min, filtered and dried to give
0.785 g of [5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-(3-bro-
mophenyl)methanone 60.
generously allowed region. Details of the data collection and
statistics and structure refinement are reported along with the
structure coordinates, which have been deposited in the Protein Data
Bank.17 Pictures of the p38 MAP kinase structure (Figure 1) were
made using PyMOL.18
Biological Methods. p38 MAP Kinase Inhibition. HTS Assay.
HTS was performed using a Zymark robotic system and a 96-well,
radioactive filtration binding format. The enzyme was preincubated
with test compounds (40 µM) for 10 min. at 30 °C, following which
the reaction was initiated by adding [33P] ATP (250 µM) and myelin
basic protein (150 µM). Following an additional incubation for 20
min at 30 °C the reaction was terminated, and the radiolabeled
product was captured on nitrocellulose filtration plate, washed and
counted for radioactivity. Compounds which displayed >40%
inhibition of the enzyme activity were retested at multiple concen-
trations.
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]- (3-bromophe-
nyl)methanone 60 (0.6 g, 1.48 mmol) was combined with piperidine
(1.47 mL, 14.8 mmol) and ethanol (10 mL) and heated at reflux
for 16 h. The reaction mixture was concentrated in vacuo. The
resulting residue was partitioned between a saturated aqueous
sodium hydrogen carbonate solution and ethyl acetate. The organic
extracts were dried over magnesium sulfate, filtered, concentrated
under vacuum and purified by column chromatography on silica
gel using 16:1 CH2Cl2/MeOH. Dissolving the product in ethyl
acetate then adding hydrochloric acid (1.0 M, 1.0 equivalent) formed
the hydrochloric salt which was filtered and dried to give 0.413 g
of [5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(2-piperidin-
1
1-yl-ethoxy)phenyl]methanone. H NMR (DMSO-d6) δ 1.39 (m,
In Vitro Assay. Inhibition of human recombinant active p38R
[5 nM] was tested by measuring the incorporation of 33P from
γ-[33P] ATP [50 µM, 1 µCi] into myelin basic protein (MBP) (35
µM]. The assay was performed in a 96-well microtiter plate. In 40
µL reaction volume, 26 µL diluted p38R (5 nM) in assay dilution
buffer (ADB) (20 mM MOPS pH 7.4, 40 mM MgCl2, 1 mM DTT,
25 mM â-glycerol phosphate, 5 mM EGTA, 1 mM sodium
orthovanadate) were preincubated at room temperature for 10 min
with 4 µL test compounds (0-100 µM), dissolved in dimethyl
sulfoxide (DMSO) (10%). The kinase reaction was initiated by the
addition of 10 µL assay mix, containing ADB and γ-[33P] ATP
(50 µM, 1 µCi) and MBP (35 µM). After 30 min incubation at 30
°C, the reactions were terminated by transferring 25 µL reaction
samples onto a prewetted phosphocellulose filter plate (Millipore)
containing 200 µL 0.75% phosphoric acid (H3PO4). The plate
membranes were washed three times with 200 µL 0.75% H3PO4
for the removal of the free radionucleotide. 50 µL Microscint-20
scintillant (Packard) was added to each well; the plate was sealed
with a plastic film and counted in a Packard Topcount microplate
scintillation counter. The 50% inhibitory value was calculated by
fitting the data to the equation:
1H), 1.66-1.98 (m, 5 H), 3.00 (m, 2H), 3.49 (m, 4H), 4.49 (t, J )
4.92 Hz, 2H), 7.17-7.23 (m, 3H), 7.29 (m, 1 H), 7.38-7.51 (m,
4H), 7.57-7.62 (m, 2H), 7.81 (m, 1H). MS (EI/CI) m/z: (M + H)
409. Anal. (C23H25FN4O2) C, H, N.
5-Amino-1-(4-fluorophenyl)-4-[3-{2(R), 3-dihydroxypropoxy}
benzoyl]pyrazole (62). To a solution of 5-amino-4-(3-hydroxy-
benzoyl)-1-(4-fluorophenyl)pyrazole (8.0 g, 26.9 mmol) in 30 mL
dry dimethylformamide was added D-R,â-isopropylideneglycerol
γ-tosylate (11.3 g, 39.5 mmol) followed by anhydrous potassium
carbonate (11.13 g, 80.7 mmol). The reaction was warmed to 80
°C under argon. After 24 h, the reaction was cooled to room
temperature and diluted with distilled water (100 mL), and the
product was extracted into ethyl acetate. The combined organics
were washed with brine, dried over magnesium sulfate and then
concentrated in vacuo to a brown oil. The oil was purified by flash
column chromatography on silica gel using 2:1-1:1 hexanes/ethyl
acetate as eluent. Concentration of the column fractions in vacuo
gave 9.32 g of the desired acetal.
To a solution of the acetal formed above (17.42 g, 42 mmol) in
methanol (80 mL) were added distilled water (20 mL) and
p-toluenesulfonic acid monohydrate (500 mg). The solution was
warmed to 50 °C under an argon atmosphere. After 6 h, the reaction
mixture was cooled to room temperature and concentrated in vacuo
to a yellow oil. The oil was redissolved in ethyl acetate and 5%
sodium bicarbonate solution (50 mL). The organic layer was
separated, dried over magnesium sulfate and concentrated in vacuo,
and the desired diol precipitated out of solution to give 13 g of a
white solid. The solid had a small amount of impurity and was
thus suspended in 150 mL 1:1 hexanes/ethyl acetate and stirred
for 1 h. Filtration of the undissolved solid gave pure diol (12.03
fractional activity ) 1/[I]/IC50 + 1
Induction of TNF Biosynthesis. THP1 cells were suspended
in culture medium [RPMI (Gibco-BRL, Gailthersburg, MD)
containing 15% fetal bovine serum, 0.02 mM 2-mercaptoethanol],
at a concentration of 2.5 × 106 cells/mL and then plated in a 96-
well plate (0.2 mL aliquots in each well). Test compounds were
dissolved in DMSO and then diluted with the culture medium such
that the final DMSO concentration was 5%. Twenty-five microliter
aliquots of test solution or medium with DMSO (control) were
added to each well. The cells were incubated for 30 min, at 37 °C.
LPS (Sigma, St. Louis, MO) was added to the wells at a final
concentration of 0.5 µg/mL, and cells were incubated for an
additional 2 h. At the end of the incubation period, culture
supernatants were collected and the amount of TNF-R present was
determined by a specific trapping ELISA assay using two anti-
TNF-R antibodies (2TNF-H22 and 2TNF-H34) as described.19
Inhibition of TNFR and IL-1â Biosynthesis in LPS-Stimu-
lated Peripheral Blood Mononuclear Cells. Blood was collected
from healthy volunteers (drug-free for two weeks) into vacutainers
(Becton Dickinson, Mountainview, CA) containing 19 units/mL
sodium heparin. Peripheral blood mononuclear cells (PBMC) were
separated using a Histopaque-1077 gradient (Sigma; H-8889).
Briefly, whole blood was diluted 1:1 in Dulbecco’s phosphate-
buffered saline (Gibco; #14190-136) and layered onto the histo-
paque solution followed by centrifugation at 400g for 20 min at
room temperature. Cells were collected and washed once in cold
PBS followed by red cell lysis with lysing media (Sigma #R-7757,
Lot #46H2373). A final cell suspension was prepared at 2 × 106
per mL in RPMI 1640 (Gibco BRL, #11875-085), 10% FBS, 2
mM l-glutamine, 5.5 × 10-5 M â-mercaptoethanol. Dilutions of
test compounds were predispensed in 25 µL aliquots (before
addition of cells) into round-bottom 96-well plates (U bottom TC
1
g). H NMR (DMSO-d6) δ 3.47 (t, J ) 5.69 Hz, 2H), 3.78-3.87
(m, 1H), 3.92-3.97 (m, 1H), 4.07-4.12 (m, 1H), 4.72 (t, J ) 5.71
Hz, 1H), 5.02 (d, J ) 5.13 Hz, 1H), 7.14-7.18 (m, 3H), 7.25-
7.26 (m, 1H), 7.32-7.47 (m, 4H), 7.59-7.66 (m, 2H), 7.81 (s,
1H). MS (EI/CI) m/z: (M + H) 372. Anal. (C19H18FN3O4‚0.25H2O)
C, H, N.
5-Amino-1-(4-fluorophenyl)-4-[3-{2(S),3-dihydroxypropoxy}-
1
benzoyl]pyrazole (63). H NMR (DMSO-d6) δ 3.47 (t, J ) 5.68
Hz, 2H), 3.78-3.85 (m, 1H), 3.92-3.97 (m, 1H), 4.07-4.12 (m,
1H), 4.72 (t, J ) 5.71 Hz, 1H), 5.02 (d, J ) 5.12 Hz, 1H), 7.14-
7.18 (m, 3H), 7.25-7.26 (m, 1H), 7.32-7.47 (m, 4H), 7.59-7.66
(m, 2H), 7.81 (s, 1H). MS (EI/CI) m/z: (M + H) 372. Anal. (C19H18-
FN3O4) C, H, N.
Crystallization and Structure Determination. Crystallization
and structure determination of p38 MAP kinase with compound
55 was performed as reported previously.4 Crystals were obtained
in the space group P212121 with cell dimensions of a ) 45.3 Å, b
) 86.5 Å, c ) 124.1 Å. X-ray diffraction data were collected at
the Advance Light Source, Lawrence Berkeley National Laboratory,
and data between 36.6 and 1. 75 Å (41 760 unique reflections) were
reduced using HKL2000.15 The structure was refined using CNS16
to a final Rfactor ) 20.1 and Rfree ) 24.1, including 293 water
molecules in the final structure. Overall the protein structure has
good geometry, with 89.7% of the residues in the core region of a
Ramachandran plot, 10% in the allowed region and 0.3% in the

1574 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Goldstein et al.
plate; Costar #3799). The starting concentration was 100 µM in
5% DMSO and 6 half log serial dilutions were made. After the
addition of 200 µL cell suspension and 25 µL of 5 µg/mL
lipopolysaccharide (LPS, E. coli; 0127; B8; Cat. No. L3129, Sigma
Chemical Co., St. Louis, MO) in medium, the final DMSO
concentration was 0.5%. Aliquots of test compounds were diluted
an additional 10-fold, and the final LPS concentration was 500 ng/
mL. PBMC suspension (200 uL per sample) was preincubated with
the diluted compound for 30 min at 37 °C followed by addition of
25 µL of LPS dissolved in RPMI (Gibco BRL, #11875-085) to
produce a final LPS concentration of 0.5 µg/mL. Negative control
wells received RPMI alone. Plates were incubated for 2 or 5 h at
37 °C in a 5% CO2 atmosphere for determination of TNFR or IL-
1â, respectively, followed by centrifugation at 150g for 10 min.
Supernatants were collected from each sample and stored in
polypropylene plates at 4 °C. TNF-R and IL-1â were determined
by Elisa (Antibody Solutions, Palo Alto, CA) following the
manufacturer’s instructions. Optical density of each sample was
read at 450 nm with a reference of 650 nm. Cytokine concentrations
were determined from a standard curve using Molecular Devices
SoftMax Pro. The percent inhibition was calculated for each
concentration tested and an IC50 curve was constructed using Xlfit
software.
Systemic LPS and TNF-R Challenge. All animal procedures
were approved by the Institutional Animal Care and Use Committee
of Roche Palo Alto. Female Hanover-Wistar rats (100-130 g)
were purchased from Charles River Laboratories (Hollister, CA).
Animals were randomly segregated into groups (8 per group) and
treated with LPS (Sigma, St Louis, MO; 50 µg/kg intraperitoneally
in 0.9% saline) or TNF-R (R&D Systems, Minneapolis, MN; 5
µg/kg intraperitoneally in 0.9% saline). Animals were treated with
vehicle or compound 63 by oral gavage 30 min prior to LPS or
TNF-R challenge. Animals were euthanized 90 min after the
challenge by CO2 inhalation and serum harvested. TNF-R and IL-6
levels were measured in serum samples by ELISA (Biosource
International, Camarillo, CA) according to the manufacturer’s
instructions.
Rat Adjuvant Arthritis (AI). Female Lewis rats (120-150 g)
from Charles River Laboratories were randomized and immunized
with heat killed Mycobacterium butyricum (Difco Laboratories,
Detroit, MI) in light mineral oil (Sigma; 5 mg/ml) by intradermal
injection (0.1 mL) into the tail on day 0. Vehicle (PEG 400) and
compound 63 were subcutaneously delivered from day one onward
by osmotic minipump (Alza; Mountain View, CA). Two minipumps
were implanted to each rat and replaced on day eight. Plasma
samples were collected on days three and fourteen. Animals were
euthanized on day fourteen and hind paw weights recorded in
addition to clinical scores.
Rat Phamacokinetic Study. Female Wistar/Han (CRL:WI) Rats
(Charles River, Hollister, CA) weighing between 180 and 220 g
were used. Animals were allowed free access to a standard
laboratory chow and tap water and were housed in a constant
temperature-humidity environment. Three rats per dose regime were
administered either single 10-mg/kg iv bolus doses (50% cyclo-
dextran/water) or single 10-mg/kg oral suspension doses prepared
in aqueous vehicle containing 0.9% NaCl, 0.5% sodium carboxym-
ethyl cellulose, 0.4% polysorbate 80 and 0.9% benzyl alcohol.
Blood was collected from each rat anesthetized with CO2:O2 (60:
40) via the orbital sinus or cardiac puncture at 1, 3, 6, 8 and 24 h
after dosing. Plasma levels of test compounds were assayed by a
LC/MS method. In this method, an aliquot of plasma was treated
by mixing with acetonitrile to precipitate protein, centrifuged to
clarify the supernatant and then further diluted with formate buffer
(50 mM) and injected onto an HPLC. Test compounds were
separated from endogenous interfering substances and subsequently
eluted from the HPLC column for mass spectrometric quantification.
Microsomal Incubations. Nicotine adenine dinucleotide phos-
phate (NADPH)-dependent transformation was studied with con-
centrations of 100 µM of test compound. Incubations were
conducted at 37 °C and contained 2 mg/mL of liver microsomal
protein in pH 7.4, 40 mM phosphate buffer containing 3 mM
magnesium chloride and 1.0 mM NADPH. Aliquots of the
incubations were taken at specific time points over a 30 min
incubation period. To stop the reaction the timed aliquots were
added to an equal volume of acetonitrile containing an internal
standard (IS). The IS-acetonitrile solution denatured and precipitated
the microsomal protein. The protein was separated by centrifugation,
and an aliquot of the resultant supernatants were assayed by a
HPLC/UV method. Incubations prepared without the addition of
NADPH acted as controls.
Acknowledgment. The Advanced Light Source is supported
by the Director, Office of Science, Office of Basic Energy
Sciences, Materials Sciences Division, of the U.S. Department
of Energy under Contract No. DE-AC03-76SF00098 at Lawrence
Berkeley National Laboratory.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org
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