Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase

Article abstract of DOI:10.1021/jm9805236

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-α (TNF-α) release and an animal model of rheumatoid arthritis. The SAR

Full text of DOI:10.1021/jm9805236

2
180
J . Med. Chem. 1999, 42, 2180-2190
Design a n d Syn th esis of P oten t, Selective, a n d Or a lly Bioa va ila ble
Tetr a su bstitu ted Im id a zole In h ibitor s of p 38 Mitogen -Activa ted P r otein Kin a se
Nigel J . Liverton,* J ohn W. Butcher, Christopher F. Claiborne, David A. Claremon, Brian E. Libby,
Kevin T. Nguyen, Steven M. Pitzenberger, Harold G. Selnick, Garry R. Smith, Andrew Tebben, J oseph P. Vacca,
†
§
†
†
‡
Sandor L. Varga, Lily Agarwal, Kim Dancheck, Amy J . Forsyth, Daniel S. Fletcher, Betsy Frantz,
‡
‡
†
‡
§
†
William A. Hanlon, Coral F. Harper, Scott J . Hofsess, Matthew Kostura, J iunn Lin, Sylvie Luell,
Edward A. O’Neill, Chad J . Orevillo, Margaret Pang, J aney Parsons, Anna Rolando, Yousif Sahly,
Denise M. Visco, and Stephen J . O’Keefe
‡
†
‡
‡
‡
§
†
‡
Departments of Medicinal Chemistry and Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486,
and Departments of Inflammation Research and Pharmacology, Merck Research Laboratories, Rahway, New J ersey 07065
Received September 16, 1998
Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein)
kinase are described which have activity in both cell-based assays of tumor necrosis factor-R
(TNF-R) release and an animal model of rheumatoid arthritis. The SAR leading to the
development of selectivity against c-Raf and J NK2R1 kinases is presented, with key features
being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl
ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation
of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous
solubility. In general, oral bioavailability of this class of compounds was found to be poor unless
the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38
MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits
lipopolysaccharide-stimulated release of TNF-R from human blood with an IC50 2.2 nM, shows
good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement
in measures of disease progression in a rat adjuvant-induced arthritis model.
In tr od u ction
Our goal was to develop selective small-molecule
inhibitors of p38 MAP kinase that could be tested as a
therapy for inflammatory conditions resulting from
excess cytokine production. One obstacle to overcome
is selectivity for p38 kinase inhibition over the vast
number of other cellular kinases. The prototypical
inhibitor of p38 kinase, 1, which has shown activity in
a number of animal models of rheumatoid arthritis14
demonstrated limited selectivity in our kinase enzyme
Tumor necrosis factor-R (TNF-R) and interleukin-1
IL-1) are proinflammatory cytokines produced in
1
(
response to infection and other cellular stresses. Al-
though TNF-R in appropriate amounts plays an impor-
tant role in the host immune response, excess levels are
thought to underlie a number of serious inflammatory
2
,3
diseases. Recent clinical data, obtained with either
4
-7
12
chimeric TNF-R antibodies
or soluble TNF-R recep-
assays (Table 1), in contrast to reported data. Specif-
8
tor in the treatment of rheumatoid arthritis and
Crohn’s disease, has confirmed the key role of TNF-R
in these inflammatory conditions. While the complete
biochemical pathway for the production of TNF-R and
IL-1 in response to inflammatory stimuli has yet to be
elucidated, p38 MAP (mitogen-activated protein) kinase
ically, significant inhibitions of c-Raf and J NK2R1
kinases were observed. In addition 1 was weak as an
inhibitor of TNF-R production in human monocytes (IC50
84 ( 11 nM, n ) 32) and in human whole blood (IC50
1
000 ( 175 nM, n ) 2). The key objectives for our effort
were to increase selectivity, particularly with respect
to c-Raf and J NK2R1, and to reduce the IC50 for
inhibition of TNF-R in human whole blood, thereby
minimizing required drug levels in a therapeutic setting.
From a physical chemistry perspective, we sought to
improve aqueous solubility of these inhibitors (1: 0.0019
mg/mL at pH 7.4, 0.043 mg/mL at pH 5).
9
has been shown to play a role in this process. p38 MAP
kinase is one member of a group of serine-threonine
kinases that are activated via dual phosphorylation of
a TXY motif (TGY in the case of p38, TEY in ERK2,
and TPY in J NK).10 This phosphorylation is carried out
by dual specificity kinases (MKK3 and MKK6 in the
case of p38) in response to extracellular stimuli such
as osmotic shock, endotoxin (lipopolysaccharide, LPS),
UV light, or cytokines.12 Previous work has demon-
strated that small-molecule inhibitors of p38 MAP
kinase such as SB203580 (1)13 can reduce TNF-R levels.
1
1
In this paper we describe our investigations into the
structure-activity relationship (SAR) of a series of
imidazole inhibitors of p38 MAP kinase and the evolu-
tion of the aminopyridine derivative 48 as an extremely
potent and selective inhibitor of p38 MAP kinase. 48
also demonstrates good oral bioavailability in two spe-
cies, reduces LPS-stimulated production of TNF-R in
human whole blood at low-nanomolar concentrations,
and is active in an animal model of rheumatoid arthritis.
The approach taken was to carry out separate optimiza-
*
To whom correspondence should be addressed.
Department of Drug Metabolism.
Department of Inflammation Research.
Department of Pharmacology.
§
‡
†
1
0.1021/jm9805236 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/21/1999

Imidazole Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2181
Ta ble 1. Kinase Selectivity of Key Compoundsa
Sch em e 2a
IC50 (n)
kinase
p38b
1
39
48
39 ( 11 (70)
330 ( 155 (7)
0.11 ( 0.046 (21) 0.19 ( 0.08 (29)
>1000 (2)
675 (1)
1550 (1)
>20000 (1)
d
c-Raf
810 ( 100 (2)
1170 ( 790 (2)
2200 ( 1030 (3)
>10000 (1)
>10000 (1)
>10000 (1)
>1500 (1)
c
J NK2R1 290 ( 110 (4)
a
J NK2R2 1900 ( 28 (2)
d
Lck
3450 ( 2000 (2)
d
EGF
96000 ( 8000 (2) >10000 (1)
61000 ( 6000 (2) >10000 (1)
d
a
MEK
PKA
PKC
Reagents: (a) PhC(dNH)NH , DMF, 40 °C; (b) NaH, THF, 0
2
d
50000 (1)
75000 ( 35000 (2) >6000 (1)
>24000 (1)
°C then methoxymethyl chloride; (c) n-BuLi, THF, -78 °C then
d
>1500 (1)
Me SnCl; (d) 3-CF PhI, DMF, (Ph P) Pd, 80 °C; (e) 6 M HCl, reflux.
3
3
3
4
a
IC50 values for inhibition of kinases are in nM ((SD (n)).
b
c
Reference 32. Determined as in ref 13, replacing p38 with
J NK2R2 or J NK2R1 and using 2 µM ATP. Carried out as
described in the Experimental Section.
the less hindered isomer 12. Deprotonation at the
-position with n-butyllithium and quenching with
d
5
trimethyltin chloride afforded the stannane 13 which
Sch em e 1a
underwent smooth palladium-catalyzed coupling17 with
3
-iodo(trifluoromethyl)benzene to give 14 and deprotec-
tion with aqueous HCl to afford 3a .
A related stannane coupling-based route was used for
the preparation of 5-(4-pyrimidinyl)-2-phenylimidazole
derivatives 19-23 (Scheme 3). Palladium-catalyzed
coupling of the stannyl derivative 16 with 4-iodo-2-
1
8
(
methylthio)pyrimidine and subsequent acid hydroly-
sis of 17 afforded 18. Reductive removal of the meth-
ylthio substituent was then accomplished with Raney
nickel to give the unsubstituted derivative 19. Introduc-
tion of amino substituents was carried out by first
oxidizing the sulfide 18 to the corresponding sulfone 20,
for which Oxone was found to be the most convenient
reagent, followed by displacement with the desired
amine. The parent amino-substituted compound 23 was
then prepared by acid hydrolysis of the 4-methoxybenz-
ylamino derivative 22.
a
Reagents: (a) LDA/THF then XPhCON(CH3)OCH3; (b) R2CHO,
While a preparation of compounds combining the
Cu(OAc)2, NH4OAc, AcOH, reflux; (c) HCl, EtOAc, MeOH.
2
-piperidine and 5-pyrimidinyl substituents could be
envisaged using chemistry related to that in Scheme 3,
the necessary intermediate corresponding to 16 would
require several synthetic steps to prepare. An alterna-
tive route which avoided tin chemistry and proved
readily applicable to large-scale synthesis was developed
(Scheme 4). Deprotonation of the methyl group of
tion of each of the imidazole substituents independently
and then establish their additivity.
Ch em istr y
2
-Phenylimidazole derivatives were prepared by two
different routes. In the first (Scheme 1), the anion of
-[(tert-butyldimethysilyloxy)methyl]pyridine was con-
1
9
4-methyl-2-(methylthio)pyrimidine was readily carried
out with LDA at -78 °C and the resulting anion
quenched with the Weinreb amide 24 of 3-(trifluorom-
ethyl)benzoic acid to provide the ketone 25. R-Oximi-
nation was carried out in high yield by treatment with
4
1
5
densed with an appropriate Weinreb amide, and the
resulting protected benzoins 2 and 4 were condensed
with ammonium acetate and the desired aldehyde in
acetic acid to give 3d , 3g, 3h , 3k , 3m , 3p , and 3q (Table
2
0
sodium nitrite in an acetic acid, THF, water solvent
system to afford a mixture of oxime isomers 26. Both
isomers participated in the key cyclization reaction
when treated with CBZ piperidine-4-carboxaldehyde21
and ammonium acetate in acetic acid at reflux, to afford
the hydroxyimidazole 27. Several methods are available
for the reduction of hydroxyimidazoles, including
2
) and 5-8 (Table 4). HCl deprotection of 7 and 8
afforded piperidines 9 and 10.
In a second approach which allows for ready variation
of the 4-aryl substituent, a stannane coupling reaction
was used as the key step (Scheme 2). 2-Phenyl-4-(4-
pyridyl)imidazole (11) was prepared from 4-(bromoacetyl)-
pyridine hydrobromide16 and excess benzamidine in
DMF. Protection of the imidazole as the methoxymethyl
ether afforded a 3:1 mixture of regioisomers favoring
22
23
P(OMe)3, Raney nickel-catalyzed hydrogenation, and
2
4
aqueous titanium trichloride. Hydrogenation would be
incompatible with the CBZ functionality and further

2
182 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Liverton et al.
Sch em e 3a
a
Reagents: (a) n-BuLi, THF, -78 °C then Bu3SnCl; (b) 4-iodo-2-(methylthio)pyrimidine, DMF, (Ph3P)4Pd, 80 °C; (c) 6 M HCl, reflux;
(
d) Raney Ni, EtOH, H2O, reflux; (e) Oxone, MeOH, H2O; (f) RNH2, neat, 100 °C.
Sch em e 4a
pared by treatment of the 4-methoxybenzyl derivative
3
0 with aqueous HCl at reflux.
Initial attempts to prepare the corresponding N-
methylimidazole derivatives focused on methylation of
sulfone 29 using iodomethane and cesium carbonate in
DMF. This provided the undesired regioisomer 35 with
less than 5% of the desired product 34 (Table 3). The
sulfide 28 under the same conditions yielded 25% of the
desired isomer 36, and this ratio was not altered by
using methyl triflate as the electrophile. In contrast
treatment of 28 with DMF dimethyl acetal in toluene
at reflux formed predominantly the desired isomer 36.
Oxidation of 36 with Oxone, displacement of the sulfone
3
4 with amine, and deprotection afforded methylated
analogues 39 and 41 (Scheme 5). In a similar manner,
the regioisomer (42) of 39 was prepared from the other
methylation product 37.
The analogue 48, in which the pyrimidine ring is
replaced by pyridine, was prepared by an analogous
route from 2-fluoro-4-methylpyridine (Scheme 6) with
two significant differences. In the key cyclization step,
only one of the oxime isomers was consumed when the
reaction was carried out at 80 °C, and heating for
sufficient time at higher temperatures in an effort to
utilize the other isomer resulted in concomitant conver-
sion of the desired product to the corresponding pyri-
done, presumably by nucleophilic displacement of the
fluoropyridine by acetate and hydrolysis to the pyridone
on workup. While sodium nitrite in aqueous acetic acid
provided a 1:1 mixture of oxime regioisomers, use of tert-
butyl nitrite in ethanol containing HCl gave >90% of
the more reactive isomer. In the second difference,
attempted methylation of the imidazole ring of fluoro-
pyridine gave <25% yield of the desired isomer, and the
methylation was instead carried out on the amine
displacement product 46, which afforded 61% of the
desired regioisomer 47. Hydrogenolysis of the CBZ
group then gave pyridine analogue 48.
a
Reagents: (a) LDA, THF, -78 °C then 3-CF3PhCON(CH3)OCH3
24); (b) NaNO2, AcOH, THF, H2O; (c) CBZ piperidine-4-carbox-
aldehyde, NH4OAc, AcOH, reflux; (d) TiCl3, MeOH, H2O, reflux;
e) Oxone, MeOH, H2O; (f) RNH2, neat, 140 °C; (g) HBr, AcOH or
(
(
H2, Pd/C, i-PrOH; (h) 3 M HCl, reflux.
2
5
complicated by the presence of the (methylthio)pyrimi-
dine. Treatment of the crude cyclization product 27 in
methanol with an aqueous HCl solution of titanium-
III) chloride proved rapid and high yielding. The
-pyrimidinyl position of the product 28 was then
activated toward nucleophilic displacement by oxidation
to the sulfone 29 with Oxone in aqueous methanol.
Displacement was carried out with different amines, if
necessary in a sealed tube for volatile examples, and
the CBZ group deprotected with HBr/AcOH or by
hydrogenolysis to afford 30, 32, and 33. The parent
aminopyrimidine analogue 31 was conveniently pre-
(
2
Biologica l Resu lts a n d Discu ssion
An aryl group at the 4-position was important for p38
kinase inhibition, with the 4-isopropyl analogue 3q

Imidazole Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2183
Ta ble 2. 4-Position Substitution
Sch em e 5a
IC50 (nM)
prep
a
compd
R
p38
c-Raf
method
anal.
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
3-CF3
4-CF3
2-Cl
3-Cl
4-Cl
4-CN
4-OCH3
4-HO
4-CO2CH3
4-Ph
86
200
150
130
48
2600
1400
610
90
56
86
950
390
320
2500
23
3000
210
20
1600
570
B
B
B
A
B
B
A
A
B
B
A
B
A
B
B
A
A
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
970
300
1400
>10000
1000
140
3-HO
3-F
495
550
28
137
3-OCH3
3,4-DiCl
3,5-DiCl
cycloC6H11
isopropyl
>10000
2100
>10000
a
Method A: prepared according to Scheme 1. Method B:
a
Reagents: (a) Oxone, MeOH, H2O; (b) RNH2, neat, 150 °C; (c)
prepared according to Scheme 2.
H2, Pd/C, 2-propanol.
Ta ble 3. Regiochemistry of Methylation
Sch em e 6a
starting material
conditions
product
ratiosa
2
2
2
2
9
8
8
8
Cs2CO3, MeI, DMF
Cs2CO3, MeI, DMF
34, <5% 35, >95%
36, 25% 37, 75%
Cs2CO3, MeOTf, DMF 36, 25% 37, 75%
DMF dimethyl acetal 36, 71%b 37, 29%
a
Ratios determined by HPLC. b Isolated yield after chroma-
tography 65%.
and 3n which showed equal potency versus p38 and
c-Raf.
Examining SAR at the 2-position (Table 4), the
2
2
-unsubstituted compound 5 demonstrated that the
-aryl moiety of 1 does not contribute significantly to
potency or selectivity. Preparation of the cyclohexyl
analogue 6 reinforced this and demonstrated tolerance
for nonplanar groups at this position. Selectivity was
not, however, improved by this modification. Based on
this observation and the desire to generate compounds
with improved aqueous solubility, the 4-piperidinyl
analogue 9 was prepared and found to provide improved
inhibition of p38 MAP kinase combined with enhanced
selectivity. Preparation of the 3-piperidinyl derivative
10 demonstrated that the nitrogen position could be
altered with only minor effects on activity.
a
Reagents: (a) LDA, THF, -78 °C then 3-CF3PhCON(CH3)OCH3
(
25); (b) t-BuONO, EtOH, HCl; (c) CBZ piperidine-4-carboxalde-
hyde, NH4OAc, AcOH, 80 °C; (d) TiCl3, MeOH, H2O; (e) RNH2,
neat, 150 °C; (f) Cs2CO3, MeI, DMF; (g) H2, Pd/C, 2-propanol.
showing significantly reduced activity and the cyclo-
hexyl compound 3p showing no significant inhibitory
activity (Table 2). While a number of small substituents
were tolerated on the 4-phenyl, at the 2-, 3-, and
4
-positions, our key goal at the time was to focus on
In the third independent study of substituents, data
from the 5-(4-pyrimidinyl) series of compounds (19, 21-
23) clearly demonstrated that c-Raf activity could be
significantly reduced by utilizing the pyrimidine ring
and that the inhibition of p38 MAP kinase was tolerant
of a relatively large substituent on the 2-amino group,
pursuing selectivity. As a result we chose to focus on
the 3-trifluoromethyl substituent present in 3a , which
provided a modest 2-fold increase in p38 potency
but was accompanied by 30-fold selectivity over Raf
rather than the somewhat more active compounds 3e

2
184 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Liverton et al.
Ta ble 4. Kinase Inhibition Data
IC50 (nM)
compd
p38
c-Raf
J NK2R1
TNF-R whole blood
1
3
39
180
86
170
70
9
19
700
120
57
38
7.0
7.6
0.75
0.97
1.5
330
380 ( 115 (2)
2600 (1)
1300 ( 280 (2)
135 ( 7 (2)
80 ( 15 (2)
110 ( 60 (2)
5000 ( 2900 (2)
2100 ( 1260 (4)
750 ( 360 (2)
1000 ( 280 (2)
36% @ 1000 (2)
66% @ 1000 (1)
3500 (1)
290
1000
ND
130 ( 0 (2)
910 ( 50 (2)
195 ( 7 (2)
29 ( 1 (2)
3
5
6
9
0
9
1
3
2
1
0
2
3
1
9
8
2
a
>100000 (2)
>10000 (2)
34 ( 1.4 (2)
175 ( 7 (2)
585 ( 275 (2)
>100000 (2)
>100000 (2)
>100000 (2)
>100000 (2)
190 (1)
290 ( 0 (2)
6 ( 0.6 (2)
0.4 (1)
50 ( 23 (4)
4.3 ( 2.9 (12)
2.8 ( 1.5 (4)
24000 ( 6000 (2)
210 (1)
1
1
2
2
2
3
3
3
3
4
3
4
4
140 ( 14 (2)
1420 ( 340 (2)
175 ( 7 (2)
180 ( 0 (2)
98 ( 17 (2)
3300 ( 3600 (2)
735 ( 21 (2)
1750 (1)
295 (1)
300 (1)
675 (1)
1170 ( 790 (2)
37% @ 40000
4500 (1)
48% @ 5000 (1)
>1000 (2)
810 ( 100 (2)
26% @ 5000 (1)
0.11 ( 0.046 (21)
0.19 ( 0.08 (29)
1220
Ta ble 5. Pharmacokinetic Parameters of Selected p38 Kinase
Inhibitors
an observation that would prove useful in modifying the
physical properties of later compounds in order to gain
oral bioavailability.
On the basis of the data from these three separate
studies, a series of compounds (30-33) were prepared
with the optimal features at the 2-, 4-, and 5-positions
of the imidazole. The parent aminopyrimidine derivative
iv t1/2
(min)
Vdsss
(L/kg)
Clpl
(mL/min/kg)
po Cmax
(µM)
F
(%)
compd
3
3
3
1
2
9
7.9 ( 1.3 1.23 ( 0.07
167 ( 3.3
146 ( 46
38.3
ND
ND
0.78
0.39
0
0
64
85
1 ( 0.1
6.5 ( 0.7
294
12.7
48
171
12.3
63
3
1 clearly demonstrated that not only were the incre-
mental increases in p38 inhibition additive, but the
improvements in selectivity were also combined to give
greater than 100-fold selectivity versus both c-Raf and
J NK2R1. Once again the 4-methoxybenzylamino sub-
stituent in 30 was tolerated, and (R)- and (S)-R-
methylbenzylamino analogues 41 and 39, respectively,
were prepared based on the hypothesis that removing
the electron-donating methoxy substituent and increas-
ing steric congestion at the benzylic position would lead
to reduced oxidative metabolic debenzylation in later
in vivo studies. The (S)-enantiomer 32 inhibited p38
MAP kinase with an IC50 of 0.75 nM, without any
concomitant increase in c-Raf or J NK2R1, resulting in
a selectivity ratio of >1000. As demonstrated by the
enantiomer 33, the chirality of the methyl substituent
has no significant effect on activity. Methylation of the
imidazole, however, caused a significant difference in
p38 MAPK kinase inhibition between enantiomers, with
the (S)-enantiomer 39 being the more active p38 MAP
kinase inhibitor (IC50 0.11 nM) and the (R)-enantiomer
kinase inhibition assay and the whole cell data. The
triaryl systems prepared (3a , 5, 19, 21-23) all showed
minimal inhibition of TNF-R production, which may be
a consequence of the anticipated high protein binding
of these largely planar compounds. Breaking up the
planarity without large effects on lipophilicity can have
a dramatic effect as shown by the cyclohexyl analogue
6
which has whole blood activity comparable to the
kinase assay. The piperidine derivatives 9 and 10 show
an approximately 20-fold shift in whole cells. The
pyrimidine analogues 30-33, 39, and 41 showed a
1
-80-fold range of attenuation in the whole cell assay.
P h a r m a cok in etics a n d Dr u g Meta bolism . Initial
screening of several compounds for both intravenous
and oral pharmacokinetics was carried out in the rat
(Table 5). The initial product of combining independent
optimization studies, 31, was evaluated and found to
show a disappointingly short iv half-life and no oral
bioavailability. The drug is cleared extremely rapidly,
and HPLC analysis showed complete excretion of intact
compound in both urine (80%) and bile (20%), possibly
resulting from the low log P value of 0.39. A simple
relationship between log P and oral bioavailability was
ruled out in this series of compounds when it was found
that the much more lipophilic 32 (log P > 3.9) showed
the same profile of short intravenous half-life coupled
with no oral bioavailability. In complete contrast, the
methylated analogue 39 showed a highly desirable
profile with a greatly increased iv t1/2, coupled with good
oral bioavailability. Compound 39 has an intermediate
lipophilicity (log P 2.37), in the range commonly associ-
ated with improved prospects for oral bioavailability.
The pyridine analogue 48 showed a similar pharmaco-
kinetic profile (Figure 1) and lower clearance combined
with a lower volume of distribution.
4
1 being 30-fold less active. The critical importance of
the position of the methyl group on the imidazole was
exemplified by the preparation of the regioisomer of 39
(42) which showed >20 000-fold reduced inhibition of
p38 MAP kinase. The pyridine analogue 48 of pyrimi-
dine 39 proved to have similar activity in the enzyme
inhibition assay.
Cell-Ba sed Assa ys of TNF -r Relea se. To select a
compound for testing in animal models of rheumatoid
arthritis, activity of the compounds in inhibiting the
LPS-induced release of TNF-R from human whole blood
was evaluated (Table 4). The shift of potency in this
assay relative to the p38 kinase inhibition assay pro-
vides information on the varying cell permeability and
protein binding of the compounds. For this reason there
is not a simple relationship between IC50 in the p38

Imidazole Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2185
mg/kg/day. At 60 mg/kg/day, 48 inhibited secondary paw
swelling by 76%. J oint destruction was assessed using
radiographic total scores (RTS). The RTS on day 21 of
primary (PP) and secondary (SP) paws of rats at both
2
6
0 mg/kg/day (RTS-PP 7.2 ( 2.2, RTS-SP 3.3 ( 1.8) and
0 mg/kg/day (RTS-PP 3.2 ( 1.4, RTS-SP 2.1 ( 0.6) or
indomethacin at 1 mg/kg/day (RTS-PP 5.5 ( 2.0, RTS-
SP 4.9 ( 2.0) were significantly less than those from
rats administered vehicle (RTS-PP 13.0 ( 3.0, RTS-SP
1
0.2 ( 2.2).
Compound 48 was also effective in the adjuvant-
induced arthritis model when given therapeutically
beginning on day 14 through day 21. 48 inhibited the
swelling of the secondary paw dose dependently with
an ID50 value of 17.5 mg/kg/day. The primary and
secondary paw RTS of all arthritic rats were signifi-
cantly greater than those of nonadjuvant control rats
on day 21. The RTS of primary and secondary paws of
rats administered 48 at 20 and 60 mg/kg/day (RTS-PP
F igu r e 1. Mean plasma concentrations of 48 after iv (2 mg/
kg, closed circles) or oral (10 mg/kg, open circles) administra-
tion; n ) 3, (SD dosed to male Sprague-Dawley rats.
9
0
.4 ( 1.8 and 8.1 ( 3.0, RTS-SP 6.2 ( 2.5 and 3.4 (
.5, respectively) were significantly less than those from
rats administered vehicle (RTS-PP 13.0 ( 3.0, RTS-SP
0.2 ( 2.2). These results indicate that when 48 is
1
administered at a total daily dose of 60 mg/kg/day b.i.d.
progression of tissue destruction is almost completely
prevented.
Con clu sion
A highly potent and selective inhibitor (39) of p38
MAP kinase has been identified by combining the SAR
data from several independent studies of the imidazole
substituents. Incorporating a methyl group at N-1 of the
imidazole improved selectivity and oral bioavailability.
Comparison with the corresponding aminopyridine ana-
logue (48) demonstrated that the two compounds were
similar in their biological profiles, with the exception
of moderate p450 inhibition shown by 39 in human liver
microsomes. 48 was evaluated and found to show
significant reductions in measures of disease progres-
sion in a functional model of arthritis and represents a
potential candidate to permit the evaluation of a p38
MAP kinase inhibitor as a new therapy for the treat-
ment of inflammatory diseases mediated by TNF-R.
F igu r e 2. Mean plasma concentrations of 48 after iv (1 mg/
kg, closed circles) or oral (2 mg/kg, open circles) adminstration;
n ) 3, (SD dosed to fasted male rhesus monkeys.
One significant and unexpected difference noted
between 39 and 48 was that while neither compound
inhibited rat p450, the pyrimidine analogue 39, but not
pyridine 48, proved to be a modest time-dependent
inhibitor of p450 (CYP3A4) in human liver microsomes.
As a result, 48 emerged as the leading candidate for
further evaluation, and oral bioavailability in rat (Fig-
ure 1) and in fasted male rhesus monkey (Figure 2) was
determined as 85% and 86%, respectively.
In Vivo Effica cy Mod el. On the basis of combination
of p38 kinase inhibition, selectivity, and whole blood,
pharmacokinetic, and p450 inhibition data above, ami-
nopyridine analogue 48 was selected for evaluation in
two in vivo models. In a murine LPS challenge model,
Exp er im en ta l Section
Gen er a l. All reagents and solvents were of commercial
quality and used without further purification unless indicated
otherwise. All reactions were carried out under an inert
1
atmosphere of nitrogen or argon. H NMR spectra were
obtained on a Varian Unity 300 spectrometer or a Varian
Unity-Plus 500 spectrometer. Chemical shifts are reported in
parts per million relative to TMS as internal standard. Optical
rotations were obtained on a Perkin-Elmer 241 polarimeter
using a 1-dm cell. Melting points were determined in open
glass capillaries using a Thomas-Hoover UniMelt melting
point apparatus and are uncorrected. Microanalyses were
carried out on a Perkin-Elmer 2400-II instrument. Log P
values were determined by the traditional shake flask method
partitioning between octanol and pH 7.4 buffer. Relative
concentrations were then determined by HPLC analysis.
4
0
8 had an ED50 for reduction of TNF-R production of
.06 mg/kg (n ) 2) when given intravenously at the
same time as LPS dosing and an ED50 of 0.6 mg/kg
n ) 2) when administered po 6 h prior to dosing with
LPS.
The efficacy of 48 was also determined in rat adjuvant-
induced arthritis26 at total daily doses of 2, 6, 20, and
(
2
-(ter t-Bu tyld im eth ylsila n yloxy)-1-p h en yl-2-p yr id in -4-
yleth a n on e (2). To a stirred solution of lithium diisopropyl-
amide (8.4 mmol) in THF (25 mL) at -78 °C was added a
2
7
solution of 4-[(tert-butyldimethylsilyloxy)methyl]pyridine (1.34
g, 6.0 mmol) in THF (10 mL). After 5 min, a solution of
N-methoxy-N-methylbenzamide (0.89 g, 5.4 mmol) in THF (10
mL) was added; the reaction mixture was allowed to warm to
6
0 mg/kg per os b.i.d. from day 1 (prophylactic regimen).
4
8 produced a dose-dependent inhibition of secondary
paw swelling at day 21 with an ED50 on day 21 of 8.2

2
186 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Liverton et al.
room temperature, poured into saturated aqueous sodium
bicarbonate solution, and extracted with EtOAc. The organic
MgSO
4
, and the solvent was evaporated. Purification by flash
column chromatography eluting with 5-10% MeOH/DCM
1
phase was washed with brine and dried over MgSO
4
and the
afforded 11 as a solid: 6.2 g (80%); mp 212-214 °C; H NMR
solvent evaporated. Purification by flash column chromatog-
6
(DMSO-d ) δ 7.38 (t, J ) 7.3 Hz, 1H), 7.47 (t, J ) 7.8 Hz, 2H),
raphy (25-100% EtOAc-hexane) afforded ketone 2 as an oil:
7.77 (d, J ) 5.9 Hz, 2H), 7.99 (d, J ) 7.6 Hz, 2H), 8.03 (s, 1H),
8.50 (d, J ) 5.6 Hz, 2H). Anal. (C14 ‚0.1H O) C, H, N.
1
H NMR (CDCl
3
) δ 0.10 (s, 6H), 0.90 (s, 9H), 5.64 (s, 1H), 7.3-
H
11
N
3
2
7
.5 (m, 5H), 7.95 (d, J ) 8 Hz, 2H), 8.58 (d, J ) 6.5 Hz, 2H).
,4-Dip h en yl-5-(4-p yr id yl)im id a zole (3). A solution of 2
655 mg, 2.0 mmol), ammonium acetate (1.54 g, 20 mmol),
benzaldehyde (212 mg, 2.0 mmol), and cupric acetate (726 mg,
.0 mmol) in acetic acid (15 mL) was heated to reflux for 1.5
1-(Meth oxym eth yl)-2-ph en yl-4-(4-pyr idyl)im idazole (12).
Sodium hydride (690 mg, 60% dispersion, 28.8 mmol) was
added to a stirred solution of 11 (5.3 g, 24 mmol) in THF (200
mL) at 0 °C, and after 10 min, methoxymethyl chloride (1.95
g, 24 mmol) was added. The reaction mixture was allowed to
warm to room temperature, stirred for 1 h, poured into
2
(
4
h. The cooled reaction mixture was poured into ice/ammonium
hydroxide, stirred for 10 min, and partitioned with EtOAc. The
saturated aqueous NaHCO
The combined extracts were dried over MgSO
3
, and extracted with EtOAc (×3).
organic layer was washed with brine and dried over MgSO
4
4
, the solvent was
and solvent evaporated. Purification by flash column chroma-
tography eluting with EtOAc followed by crystallization from
evaporated, and the residue was purified by flash column
chromatography eluting with 5-10% MeOH/DCM to afford 12
EtOAc/hexane afforded 160 mg (27%) of 3: 1H NMR (CDCl
)
(3.3 g, 57%): 1H NMR (CDCl
3
3
) δ 3.42 (s, 3H), 5.29 (s, 2H), 7.2-
δ 7.4-7.7 (m, 10H), 8.01 (d, J ) 7.4 Hz, 2H), 8.41 (d, J ) 7.4
Hz, 2H). Anal. (C20 ) C, H, N.
-(ter t-Bu tyld im eth ylsila n yloxy)-1-(4-flu or op h en yl)-2-
p yr id in -4-yleth a n on e (4). This compound was prepared as
7.85 (m, 8H), 8.60 (d, J ) 4.6 Hz, 2H).
-(Meth oxym eth yl)-2-p h en yl-4-(4-p yr id yl)-5-(tr im eth -
15 3
H N
1
2
ylsta n n yl)im id a zole (13). n-Butyllithium (4.4 mL, 1.6 M in
hexane, 11 mmol) was added to a cooled (-78 °C) solution of
12 (2.65 g, 10 mmol) in THF (100 mL), and after 5 min,
chlorotrimethyltin (10 mL, 1 M in THF, 10 mmol) was added.
After 5 min, the reaction mixture was poured into saturated
1
described in the synthesis of 2 (75%): H NMR (CDCl
s, 6H), 0.90 (s, 9H), 5.60 (s, 1H), 7.03 (t, J ) 8.55 Hz, 2H),
.45 (d, J ) 5.1 Hz, 2H), 8.0 (m, 2H), 8.59 (m, 2H).
-(4-Flu or oph en yl)-4-(4-pyr idyl)im idazole (5): paraform-
3
) δ 0.11
(
7
5
aqueous NaHCO
phases were dried over MgSO
3
and extracted with EtOAc (×2). The organic
1
aldehyde, yield 0.25 g, 68%; mp 243-245 °C; H NMR (CD
OD) δ 7.19 (brt, 2H), 7.45 (m, 3H), 7.88 (s, 1H), 8.42 (brs, 2H).
Anal. (C14 ‚0.5H O) C, H, N.
-Cycloh exyl-5-(4-flu or op h en yl)-4-(4-p yr id yl)im id a -
zole (6): cyclohexanecarboxaldehyde, yield 0.125 g, 26%; mp
3
-
4
, the solvent was evaporated,
and the residue was purified by flash column chromatography
1
H
13FN
3
2
eluting with EtOAc, to afford 1.2 g (31%) of 13: H NMR
2
3
(CDCl ) δ 0.33 (s, 9H), 3.16 (s, 3H), 5.28 (s, 2H), 7.4-7.7 (m,
7H), 8.60 (d, J ) 5.8 Hz, 2H).
1
2
79-280 °C; H NMR (CD
8.8, 3.4 Hz, 1H), 7.16 (t, J ) 8.0 Hz, 2H), 7.3-7.6 (m, 4H),
.39 (brs, 2H). Anal. (C20 ) C, H, N.
-[4-(N-(ter t-Bu t yloxyca r b on yl)p ip er id in yl)]-4-(4-p y-
r id yl)-5-(4-flu or op h en yl)im id a zole (7): N-(tert-butyloxy-
3
OD) δ 1.3-2.1 (m, 10H), 2.81 (tt, J
1-(Meth oxym eth yl)-2-p h en yl-4-(4-p yr id yl)-5-[3-(tr iflu o-
r om eth yl)p h en yl]im id a zole (14). A mixture of 13 (200 mg,
0.59 mmol), 3-iodobenzotrifluoride (192 mg, 0.71 mmol), and
tetrakis(triphenylphosphine)palladium(0) (68 mg, 0.059 mmol)
in dry DMF was heated to 80 °C for 1 h, cooled to room
temperature, and partitioned between saturated aqueous
)
8
H20FN
3
2
2
8
carbonyl)piperidine-4-carboxaldehyde, 0.28 g, 46%; mp 157-
1
1
2
7
60 °C; H NMR (CD
3
OD) δ 1.48 (s, 9H), 1.7-1.9 (m, 2H), 1.9-
NaHCO
3
(150 mL) and EtOAc (3 × 75 mL). The combined
.05 (m, 2H), 2.82-3.08 (m, 2H), 4.21 (d, J ) 13.2 Hz, 2H),
.10-7.25 (m, 2H), 7.45-7.50 (m, 4H), 8.4 (s, 2H). Anal.
organic phases were dried over MgSO
4
, the solvent was
evaporated, and the residue was purified by flash column
chromatography eluting with EtOAc to afford 180 mg of solid.
(C
24
H
27FN
4
O
2
2
‚0.70H O) C, H, N.
1
2
-[3-(N-(ter t-Bu t yloxyca r b on yl)p ip er id in yl)]-4-(4-p y-
Crystallization from ether gave 40 mg (16%) of 14: H NMR
r id yl)-5-(4-flu or op h en yl)im id a zole (8): N-(tert-butyloxy-
(CDCl
7.46-7.56 (m, 3H), 7.60-7.94 (m, 6H), 8.46 (d, J ) 6.1 Hz,
2H). Anal. (C23 O‚0.15H O) C, H, N.
3
) δ 3.26 (s, 3H), 4.91 (s, 2H), 7.42 (d, J ) 6.1 Hz, 2H),
2
6
1
carbonyl)piperidine-3-carboxaldehyde, mp 193-194 °C; H
NMR (CDCl ) δ 1.46 (s, 9H), 1.45-1.70 (m, 1H), 1.75-2.00 (m,
H), 2.08-2.20 (m, 1H), 2.70-3.00 (m, 1H), 3.12-3.22 (m, 1H),
d, J ) 13.4 Hz, 1H), 4.26 (d, J ) 10.7 Hz, 1H), 7.10-7.25 (m,
H
18
F
3
N
3
2
3
2
(
2-P h en yl-4-(4-p yr id yl)-5-[3-(t r iflu or om et h yl)p h en yl]-
im id a zole (3a ). A mixture of 14 (140 mg, 0.327 mmol) and 6
M HCl (10 mL) was heated to reflux for 2 h. The cooled mixture
2
0
H), 7.40-7.55 (m, 4 H), 8.40 (s, 2H). Anal. (C24
.20H O) C, H, N.
-(4-P ip er id in yl)-4-(4-p yr id yl)-5-(4-flu or op h en yl)im i-
4 2
H27FN O ‚
2
was poured into saturated aqueous NaHCO
M NaOH (50 mL) and partitioned with EtOAc (3 × 100 mL),
The combined extracts were dried over MgSO , and solvent
3
(150 mL) and 1
2
d a zole (9). Hydrogen chloride was passed into a cooled (0 °C)
solution of 7 (140 mg, 0.33 mmol) in EtOAc (5 mL) and MeOH
4
was evaporated. Crystallization of the crude product from
1
(1 mL) for 30 min, and the volatiles were evaporated. The
EtOAc afforded 67 mg (54%) of 3a : mp 228-230 °C; H NMR
residue was partitioned between saturated aqueous NaHCO
3
3
(CD OD) δ 7.4-7.58 (m, 5H), 7.64 (t, J ) 7.6 Hz, 1H), 7.72 (d,
and DCM (×2). The combined extracts were dried over MgSO
4
,
J ) 7.2 Hz, 1H), 7.77 (d, J ) 7.6 Hz, 1H), 7.86 (s, 1H), 8.03 (d,
and the solvent was evaporated to give a solid which was
14 3 3
J ) 6.8 Hz, 2H), 8.48 (d, J ) 5.4 Hz, 2H). Anal. (C21H F N )
triturated with EtOAc and filtered to afford 30 mg (28%) of 9:
C, H, N.
1
mp 267-270 °C; H NMR (CD
3
OD) δ 1.83 (qd, J ) 12.6, 3.3
2,5-Dip h en yl-1-(m et h oxym et h yl)-5-(t r ib u t ylst a n n yl)-
im id a zole (16). n-Butyllithium (870 µL, 2.18 mmol) was
added to a cooled (-78 °C) solution of 2,5-diphenyl-1-(meth-
oxymethyl)imidazole29 (523 mg, 1.98 mmol) in THF (10 mL).
After 15 min, tributyltin chloride (590 µL, 2.18 mmol) was
added; the reaction mixture was stirred for 20 min, poured
Hz, 2H), 1.95-2.05 (m, 2H), 2.75 (brd, J ) 10.6 Hz, 2H), 2.97
tt, J ) 12.3, 3.7 Hz, 1H), 3.1-3.3 (m, 2H), 7.18 (t, J ) 9.0 Hz,
H), 7.4-7.5 (m, 4H), 8.40 (d, J ) 6.2 Hz, 2H). Anal.
F‚0.40H O) C, H, N.
-(3-P ip er id in yl)-4-(4-p yr id yl)-5-(4-flu or op h en yl)im i-
d a zole (10). This was prepared from 8 as described in the
(
2
(C
19
H
19
N
4
2
2
into saturated aqueous NaHCO
The organic phase was dried over Na
3
, and extracted with EtOAc.
SO , the solvent evapo-
1
synthesis of 9: mp 267-270 °C; H NMR (CD
3
OD) δ 1.75-
.90 (m, 2H), 1.95-2.05 (m, 2H), 2.68-2.80 (m, 2H), 2.92-
.04 (m, 2H), 3.12-3.22 (m, 2H), 7.18 (t, J ) 8.8 Hz, 2H), 7.41-
.50 (m, 4H), 8.40 (d, J ) 6.2, 2H). Anal. (C19 F‚0.40H O)
2
4
1
3
7
rated, and the residue purified by flash column chromatogra-
phy eluting with 2:1 hexane:EtOAc to give 813 mg (74%) of
16: 1H NMR (CDCl
2H), 7.20-7.70 (m, 10H).
) δ 0.8-1.6 (m, 27H), 3.14 (s, 3H), 5.23 (s,
H
19
N
4
2
3
C, H, N.
2
-P h en yl-4-(4-p yr id yl)im id a zole (11). 4-(Bromoacetyl)-
2,5-Dip h en yl-1-(m eth oxym eth yl)-5-[4-(2-(m eth ylth io)-
p yr im id in yl)]im id a zole (17). A mixture of 16 (5.0 g, 9.04
mmol), 4-iodo-2-(methylthio)pyrimidine (3.417 g, 9.04 mmol),
and tetrakis(triphenylphosphine)palladium(0) (2.088 g, 1.807
mmol) in dry DMF (100 mL) was heated to 80 °C for 20 h.
The reaction mixture was cooled to room temperature, poured
pyridine hydrobromide (10 g, 3.55 mmol) and benzamidine (25
g, 20.8 mmol) were dissolved in DMF (150 mL) at room
temperature in DMF and heated to 40 °C for 45 min. The
cooled mixture was poured into saturated NaHCO
extracted with EtOAc (×3), the extracts were dried over
3
and

Imidazole Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2187
1
into water, and extracted with EtOAc (3 × 75 mL), the extracts
23 as a solid: mp 119-121 °C; H NMR (CD
3
OD) δ 6.6 (br s,
were dried over Na
2
SO
4
, the solvent was evaporated, and the
1H), 7.40-7.55 (m, 7H), 7.59 (dd, J ) 7.7,1.7 Hz, 2H), 8.00-
8.10 (m, 3H). Anal. (C19 ‚0.40Et O) C, H, N.
residue was purified by flash column chromatography eluting
H
15
N
5
2
with 2:1 hexane:EtOAc to give 2.549 g (73%) of 17: 1H NMR
N-Met h yl-N-m et h oxy[3-(t r iflu or om et h yl)p h en yl]ca r -
boxa m id e (24). To a suspension of N,O-dimethylhydroxy-
lamine hydrochloride (58.2 g, 0.60 mol) in DCM (1 L) at 0 °C
was added 3-(trifluoromethyl)benzoyl chloride (104.0 g, 0.50
mol), followed by a slow addition (e5 °C) of triethylamine
(152.3 mL, 1.09 mol). The reaction was stirred for 30 min at 5
°C and then allowed to warm to room temperature. The
reaction was then washed with 5% aqueous citric acid (500
(
5
(
CDCl
.1 Hz, 1H), 7.25-7.40 (m, 3H), 7.45-7.60 (m, 5H), 7.70-7.90
m, 2H), 8.36 (d, J ) 5.1 Hz, 1H).
,5-Dip h en yl-5-[4-(2-(m eth ylth io)p yr im id in yl)]im id a -
zole (18). A mixture of 17 (1.918 g, 4.937 mmol) and 3 M HCl
30 mL) was heated to reflux for 1.5 h, cooled, and concentrated
to a solid which was partitioned between EtOAc and saturated
aqueous NaHCO . The organic layer was dried over Na SO
3
) δ 2.59 (s, 3H), 3.20 (s, 3H), 5.65 (s, 2H), 6.93 (d, J )
2
(
3
2
4
mL) and 5% aqueous NaHCO , the aqueous extracts were
3
and the solvent evaporated to give 1.811 g of a solid. A small
portion of this was recrystallized from EtOAc/hexane to give
back-extracted with DCM (100 mL), the combined organic
extracts were dried over Na SO , and the solvent was evapo-
2
4
1
a pure sample of 18 as a yellow solid: mp 156-158 °C; H
rated. The oil was redissolved in toluene (2 × 100 mL) and
evaporated in vacuo to afford 24 (114.7 g, 98%) which was used
in the next step without further purification: 1H NMR (CDCl3)
δ 3.39 (s, 3H), 3.55 (s, 3H), 7.55 (t, J ) 7.8 Hz, 1H), 7.72 (d, J
) 7.8 Hz, 1H), 7.89 (d, J ) 7.8 Hz, 1H), 7.98 (s, 1H).
2-[2-(Meth ylth io)pyr im idin -4-yl]-1-[3-(tr iflu or om eth yl)-
p h en yl]eth a n on e (25). n-Butyllithium (209.7 mL, 2.5 M in
hexane, 0.524 mol) was added to a solution of diisopropylamine
(73.5 mL, 0.524 mol) in THF (980 mL) at -78 °C, followed
after 5 min by a solution of 2-(methylthio)-4-methylpyrimidine
(49 g, 0.35 mol) in THF (500 mL). After stirring for 15 min at
-78 °C, a solution of 24 (89.67 g, 0.385 mol) in THF (400 mL)
was added. After stirring for 5 min, the reaction was allowed
to warm to 0 °C and quenched by pouring into water (2000
mL) and EtOAc (2000 mL). The layers were separated, and
the aqueous layer was washed with EtOAc (1000 mL). The
NMR (CDCl ) δ 2.63 (s, 3H), 7.04 (d, J ) 5.4 Hz, 1H), 7.30-
3
7
.60 (m, 6H), 7.67 (dd, J ) 7.8,1.6 Hz, 2H), 7.98 (dd, J ) 7.8,1.6
Hz, 2H), 8.48 (d, J ) 5.1 Hz, 1H), 10.44 (br s, 1H). Anal.
S‚0.15H O) C, H, N.
,5-Dip h en yl-5-(4-p yr im id in yl)im id a zole (19). A mix-
ture of 18 (200 mg, 0.581 mmol), Raney nickel (0.46 g), EtOH
20 mL), and water (10 mL) was heated to reflux for 20 h. The
(C
20
H
16
N
4
2
2
(
reaction mixture was filtered, the solvent evaporated, and the
residue purified by flash column chromatography eluting with
1
:1 CHCl
solid: mp 232-234 °C; H NMR (DMSO-d
H), 8.03 (d, J ) 7.1 Hz, 2H), 8.32 (d, J ) 5.1 Hz, 1H), 8.44 (d,
J ) 7.6 Hz, 2H), 9.05 (d, J ) 5.4 Hz, 2H), 9.22 (s, 1H). Anal.
‚0.20H O) C, H, N.
,5-Dip h en yl-5-[4-(2-(m eth ylsu lfon yl)p yr im id in yl)]im i-
3
:EtOAc to provide 82 mg (47%) of 19 as a white
1
6
) δ 7.72-7.85 (m,
6
(C
19
H
14
N
4
2
2
d a zole (20). A cooled (0 °C) solution of 18 (1.36 g, 3.95 mmol)
2 4
combined organic extracts were dried over Na SO , and the
in MeOH (20 mL) was treated with a solution of Oxone (7.28
solvent evaporated to an oil/solid (133 g). Trituration with 10%
ether/hexane (1000 mL) gave 76 g (70%) of 25: H NMR
1
g, 11.84 mmol) in H
ture for 4 h. The reaction mixture was partitioned between
EtOAc and saturated aqueous NaHCO
(×3), the organic
phases were dried over Na SO and evaporated, and the
residue was recrystallized from EtOAc to give 1.134 g (77%)
2
O (20 mL) and stirred at room tempera-
(CDCl
3H), 6.0-8.5 (m, 6H, rotamers).
-[2-(Meth ylth io)pyr im idin -4-yl]-2-[3-(tr iflu or om eth yl)-
3
) (mixture of keto and enol tautomers) δ 2.4-2.7 (m,
3
2
4
1
p h en yl]eth a n e-1,2-d ion e 1-Oxim e (26). To 25 (67 g, 0.215
mol) in acetic acid (1000 mL) were added THF (800 mL) and
water (100 mL). The mixture was cooled to 5 °C and an
aqueous solution (130 mL) of sodium nitrite (20.0 g, 0.290 mol)
added dropwise while maintaining the temperature below 10
°C. Upon completion of addition, the reaction mixture was
allowed to warm to room temperature for 1 h, concentrated to
remove THF and acetic acid, and diluted with water (2000 mL)
and EtOAc (2000 mL). The pH was adjusted to 8 with 3 N
NaOH, the layers were separated, and the aqueous layer was
washed with EtOAc (500 mL). The combined organic layers
were dried over Na SO and evaporated to yield 73.2 g (100%)
1
of 20 as a yellow solid: mp 228-230 °C; H NMR (CD
3
OD) δ
.93 (br s, 3H), 7.40-7.60 (m, 7H), 7.66 (dd, J ) 7.6, 1.8 Hz,
H), 8.05 (d, J ) 6.63 Hz, 2H), 8.80 (br s, 1H). Anal.
S‚0.20H O) C, H, N.
,5-Dip h en yl-5-[4-(2-(m et h yla m in o)p yr im id in yl)]im i-
d a zole (21). A mixture of 20 (150 mg, 0.398 mmol) in EtOH
5 mL) and methylamine (5 mL) was heated to 140 °C in a
sealed tube for 3 h. The cooled reaction mixture was parti-
tioned between EtOAc and saturated NaHCO
(×3), the
combined extracts were washed with H O and brine and dried
over Na SO , and the solvent was evaporated. The residue was
purified by flash column chromatography eluting with 95:5:
.5 DCM:MeOH:NH OH and the product thus obtained tritu-
rated with ether overnight and filtered to give 73 mg (56%) of
2
2
(C
20
H
16
N
4
O
2
2
2
(
3
2
2
4
2
4
of 26 as an oil: 1H NMR (CDCl ) δ 1.60 (brs, 1H), 2.18 (s, 3H),
3
0
4
7.52 (d, J ) 5.4 Hz, 1H), 7.66 (t, J ) 8.6 Hz, 1H), 7.89 (d, J )
8.6 Hz, 1H), 8.06 (d, J ) 8.6 Hz, 1H), 8.18 (s, 1H), 8.58 (d, J
) 5.4 Hz, 1H).
1
2
3
1 as a solid: mp 182-184 °C; H NMR (CD
3
OD) δ 2.9 (br m,
H), 6.50 and 7.00 (br s, 1H total), 7.30-7.55 (m, 6H), 7.62 (d,
5
-[2-(Meth ylth io)pyr im idin -4-yl]-4-[3-(tr iflu or om eth yl)-
J ) 7.1 Hz, 2H), 8.02 (d, J ) 7.1 Hz, 2H), 8.10 (br s, 1H). Anal.
p h en yl]-2-[4-(N-(ben zyloxyca r bon yl)p ip er id in yl)]im id a -
zole (28). A mixture of ammonium acetate (333 g, 4.26 mol),
26 (72.5 g, 0.212 mol), and N-(benzyloxycarbonyl)piperidine-
4-carboxaldehyde (63.0 g, 0.255 mol) in acetic acid (1400 mL)
was heated to reflux for 2 h, cooled, and concentrated to remove
most of the acetic acid. The residue was quenched into
(C
20
H
17
N
5
‚0.10H
2
O) C, H, N.
2
,5-Dip h e n yl-5-[4-(4-(m e t h oxyb e n zyla m in o)p yr im i-
d in yl)]im id a zole (22). A mixture of 20 (100 mg, 0.266 mmol)
and 4-methoxybenzylamine (0.50 mL) was heated to 170 °C
for 1 h. The cooled reaction mixture was partitioned between
saturated aqueous NaHCO
organic layers were washed with H
Na SO , and the solvent was evaporated. The crude product
was purified by flash column chromatography eluting with 95:
3
and EtOAc (×3), the combined
concentrated NH OH (400 mL), ice (1 kg), and EtOAc (1500
4
2
O and brine and dried over
mL) and the pH adjusted to 9 with NH OH. The organic layer
4
2
4
was separated and the aqueous layer washed with EtOAc (500
mL). The combined organic phases were dried over Na SO
2
4
5
1
4
2
0
:0.5 DCM:MeOH:NH
4
OH to afford 22 as a white solid: mp
58-160 °C; H NMR (CD OD) δ 3.75 (s, 3H), 4.20 (br s, 1H),
.50 (br s, 1H), 6.5-7.55 (m, 11 H), 7.60 (dd, J ) 7.4, 1.8 Hz,
H), 8.00 (d, J ) 6.6 Hz, 2H), 8.10 (br s, 1H). Anal. (C27 O‚
.25H O) C, H, N.
,5-Diph en yl-5-[4-(2-am in opyr im idin yl)]im idazole (23).
and concentrated to 143.8 g of 27 as an oil. To a solution of
the crude 27 in methanol (1700 mL) at 20 °C was slowly added
titanium(III) chloride (223 mL, 0.258 mol, 15 wt % in 20-30%
HCl) dropwise over 20 min. The reaction mixture was stirred
for 3 h at 20 °C, concentrated to remove methanol, and
quenched into ice (1 kg) and EtOAc (3000 mL). The pH was
1
3
23 5
H N
2
2
A mixture of 22 (70 mg, 0.161 mmol) and 6 M HCl (8 mL) was
heated to 100 °C for 1 h. The cooled reaction mixture was
evaporated to dryness and azeotroped with EtOAc (×3) and
the residue purified by flash column chromatography eluting
4
adjusted to 8 with concentrated NH OH. After stirring for 30
min the organic layer was removed and aqueous extract
washed with EtOAc (2 × 1000 mL). The combined extracts
2 4
were dried over Na SO and concentrated to an oil (143 g).
with 95:5:0.5 DCM:MeOH:NH
4
OH to afford 27 mg (54%) of
The oil was dissolved in DCM (50 mL) and chromatographed

2
188 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Liverton et al.
on silica gel (4 kg) using 1:1 EtOAc/hexane to give 116.0 g (97%
from oxime) of 28 as a yellow foam: 1H NMR (CDCl
) δ 1.7-
.2 (m, 4H), 2.58 (s, 3H), 3.0 (br m, 3H), 4.30 (brs, 2H), 5.12
s, 2H), 6.95 (d, J ) 5.5 Hz, 1H), 7.2-7.8 (m, 9H), 8.28 (d, J )
.5 Hz, 1H), 10.18 (br s, 1H).
-[2-(Met h ylsu lfon yl)p yr im id in -4-yl]-4-[3-(t r iflu or o-
5.18 (s, 2H), 7.3-7.44 (m, 5H), 7.5-7.74 (m, 5H), 8.39 (d, J )
6.0 Hz, 1H).
5-[2-(Meth ylsu lfon yl)p yr im id in -4-yl]-1-m eth yl-4-[3-(tr i-
flu or om et h yl)p h en yl]-2-[4-(N-b en zyloxyca r b on yl)p ip e-
3
2
(
5
r id in yl)]im id a zole (34). This compound was prepared as
1
5
described in the synthesis of 29: H NMR (CDCl
3
) δ 2.0 (m,
m eth yl)ph en yl]-2-[4-(N-(ben zyloxycar bon yl)piper idin yl)]-
im id a zole (29). An aqueous solution (75 mL) of Oxone (8.32
g, 0.0135 mol) was slowly added to a stirred solution of 28 (2.5
g, 0.0045 mol) in methanol (75 mL) at 20 °C. After stirring for
4H), 3.01 (m, 3H), 3.40 (s, 3H), 3.95 (s, 3H), 4.34 (m, 2H), 5.14
(s, 2H), 7.24 (d, 1H), 7.3-7.4 (m, 5H), 7.48 (t, 1H), 7.60 (m,
2H), 7.76 (s, 1H), 8.58 (d, 1H).
(S)-5-[2-(1-P h en yleth yla m in o)p yr im id in -4-yl]-1-m eth -
yl-4-[3-(t r iflu or om et h yl)p h en yl]-2-[4-(N-(b en zyloxyca r -
bon yl)p ip er id in yl)]im id a zole (38). This compound was
4
h, the reaction mixture was concentrated in vacuo to remove
methanol, diluted with 10% aqueous NaHCO (100 mL), and
extracted with EtOAc (2 × 150 mL). The organic extracts were
combined, dried over Na SO , and concentrated to give 2.75 g
) δ 1.8-2.2 (m, 4H), 2.7-3.1 (m, 3H),
.35 (s, 3H), 4.30 (br s, 2H), 5.12 (s, 2H), 7.35 (m, 5H), 7.5-
.6 (m, 6H).
-[2-(4-(Met h oxyb en zyla m in o)p yr im id in yl)]-4-[3-(t r i-
3
1
prepared as described in the synthesis of 30: H NMR (CDCl3)
2
4
δ 1.59 (d, 3H, J ) 8.1 Hz), 1.8-2.0 (m, 4H), 2.8-3.1 (m, 3H),
3.32 (br s, 3H), 4.3 (br s, 2H), 5.1-5.2 (m, 3H), 5.55 (d, J ) 6.6
Hz, 1H), 6.35 (d, J ) 5.1 Hz, 1H), 7.2-7.4 (m, 11H), 7.48 (d, J
) 7.6 Hz, 1H), 7.58 (d, J ) 7.6 Hz, 1H), 7.79 (s, 1H), 8.12 (d,
J ) 5.1 Hz, 1H).
1
of 29: H NMR (CDCl
3
8
3
5
flu or om eth yl)p h en yl]-2-(4-p ip er id in yl)im id a zole (30). A
mixture of 29 (1.5 g, 2.56 mmol) and 4-methoxybenzylamine
(S)-5-[2-(1-P h en yleth yla m in o)p yr im id in -4-yl]-1-m eth -
yl-4-[3-(tr iflu or om eth yl)p h en yl]-2-(4-p ip er id in yl)im id a -
zole (39). A solution of 38 (10.9 g, 0.017 mol) in 2-propanol
(500 mL) containing 10% Pd/C (4.0 g) was hydrogenated at 1
atm of hydrogen for 24 h. The mixture was filtered, the catalyst
was washed with 2-propanol (200 mL), the combined filtrates
were concentrated, and the resulting foam (8.0 g, 0.0158 mol,
93% yield) was recrystallized from 400 mL of 50% methanol/
water to give upon filtration and drying at 40 °C in vacuo 7.04
(
1
3.51 g, 26 mmol) was heated in a pressure tube at 140 °C for
0 min. The mixture was cooled and residue purified by flash
column chromatography eluting with 5:95 MeOH/DCM to give
.52 g (92%) of the CBZ derivative of 30; 30% HBr/AcOH (16
mL) was slowly added to a stirred solution of the intermediate
1.0 g, 1.56 mmol) in DCM (16 mL) and the reaction mixture
stirred for 30 min and diluted with Et O (160 mL). The mixture
was stirred for 1 h and filtered and the solid washed with ether
1
(
2
1
g of 39 (88%): mp 97-99 °C; H NMR (CD OD) δ 1.58 (d, J )
3
(
(
10 mL) and partitioned between saturated aqueous NaHCO
40 mL) and DCM (50 mL). The aqueous layer was washed
3
7.6 Hz, 3H), 1.8-2.0 (m, 5H), 2.6-3.4 (m, 7H), 5.17 (m, 1H),
5.52 (brd, J ) 6.9 Hz, 1H), 6.35 (d, J ) 4.9 Hz, 1H), 7.2-7.3
(m, 6H), 7.48 (d, J ) 7.8 Hz, 1H), 7.60 (d, J ) 7.8 Hz, 1H),
7.81 (s, 1H). 8.11 (d, J ) 5.2 Hz, 1H). Anal. (C H N F ‚
with methylene chloride (25 mL); the combined organic
extracts were dried over Na SO and concentrated to a foam
0.80 g) which was purified by flash column chromatography
eluting with 95:10:2 DCM:MeOH:NH OH to give 180 mg (23%)
) δ 1.6-2.2 (m, 4H), 2.77 (m, 2H), 2.95
m, 1H), 3.22 (m, 2H), 3.80 (s, 3H), 4.60 (br s, 2H), 5.44 (br s,
2
4
2
8
29
6
3
(
2
0.75H O) C, H, N.
4
(
R)-5-[2-(1-P h en yleth yla m in o)p yr im id in -4-yl]-1-m eth -
1
of 30: H NMR (CDCl
3
yl-4-[3-(tr iflu or om eth yl)p h en yl]-2-(4-p ip er id in yl)im id a -
zole (41). This was prepared as described in the synthesis of
(
1
H), 6.78 (br s, 1H), 6.90 (d, J ) 8.1 Hz, 2H), 7.32 (d, J ) 8.1
3
9. The product was isolated as the hydrochloride salt by
Hz, 1H), 7.42 (t, J ) 8.1 Hz, 1H), 7.62 (d, J ) 8.1 Hz, 1H),
7
1
dissolving 0.20 g of the free base in 1.6 mL of 1 N HCl (4.0
.80 (d, J ) 8.1 Hz, 1H), 7.90 (s, 1H),), 8.12 (d, J ) 5.8 Hz,
H), 9.8 (br s, 1H). Anal. (C35 ‚0.20H O) C, H, N.
-(2-Am in op yr im id in yl)-4-[3-(tr iflu or om eth yl)p h en yl]-
equiv) and 5 mL of water. The resulting solution was filtered
H
33
N
6
O
2
F
3
2
1
and lyophilized to give 230 mg of solid: H NMR (CD
3
OD) δ
5
1.58 (br s, 3H), 2.3 (br m, 5H), 3.2-4.0 (m, 8H), 5.17 (m, 1H),
6.60 (s, 1H), 7.2-7.5 (m, 5H), 7.6-8.0 (m, 4H), 8.24 (s, 1H).
Anal. (C H N F ‚3.0HCl‚3.0H O) C, H, N.
2
-(4-p ip er id in yl)im id a zole (31). A mixture of 30 (0.125 mg,
0
.26 mmol) and 3 M HCl (35 mL) was heated to 100 °C for 12
2
8
29
6
3
2
h. The reaction was cooled, washed with diethyl ether (10 mL),
and concentrated in vacuo to a solid. Trituration from 80%
(
S)-4-[2-(1-P h en yleth yla m in o)p yr im id in -4-yl]-1-m eth -
yl-5-[3-(tr iflu or om eth yl)p h en yl]-2-(4-p ip er id in yl)im id a -
zole (42). This was prepared from 37 as described for the
synthesis of 39. Crystallization of the product as the sulfate
salt was accomplished by dissolving 4.0 g of the free base in
ethanol (40 mL) and adding 98% sulfuric acid (0.834 g, 1.05
equiv) dissolved in ethanol (10 mL). The resulting solid was
filtered cold at 0 °C and dried in vacuo at 60 °C to give 4.0 g
ether/ethanol (10 mL) gave the hydrochloride salt of 31 (0.102
1
g, 98%) as a yellow solid: mp 225-230 °C; H NMR (CD
3
OD)
δ 2.1-2.4 (m, 4H), 3.2-3.6 (m, 5H), 7.02 (d, J ) 6.6 Hz, 1H),
.77 (t, J ) 8.4 Hz, 1H), 7.89 (d, J ) 8.4 Hz, 1H), 7.96 (d, J )
.4 Hz, 1H), 8.0 (s, 1H), 8.19 (d, J ) 6.6 Hz, 1H).
7
8
(
S)-5-[2-(1-P h en yleth yla m in o)p yr im id in -4-yl]-4-[3-(tr i-
flu or om eth yl)ph en yl]-2-(4-piper idin yl)im idazole (32). This
compound was prepared as described in the synthesis of 30
of the sulfate salt: mp 193-196 °C; H NMR (CD OD) δ 1.25
1
3
(br d, J ) 7.6 Hz, 3H), 2.20 (m, 4H), 3.1-3.4 (m, 3H), 3.46 (s,
3H), 3.60 (m, 2H), 4.28 (m, 1H), 6.9-7.3 (m, 6H), 7.48 (d, J )
7.8 Hz 1H), 7.60 (d, J ) 7.8 Hz, 1H), 7.81 (s, 1H). 8.11 (d, J )
and 31: mp 157-161 °C; [R]
‚HCl‚1.50H O) C, H, N.
R)-5-[2-(1-P h en yleth yla m in o)p yr im id in -4-yl]-4-[3-(tr i-
D
) -165.1° (MeOH). Anal.
(C
27
H
26
N
6
F
3
2
(
5.2 Hz, 1H). Anal. (C28
H
29
N
6
F
3
‚H
2
SO
4
2
‚0.80H O) C, H, N.
flu or om eth yl)ph en yl]-2-(4-piper idin yl)im idazole (33). This
compound was prepared as described in the synthesis of 30
2-(2-F lu or op yr id in -4-yl)-1-[3-(tr iflu or om eth yl)p h en yl]-
eth a n on e (43). This was prepared as described in the
and 31: mp 155-160 °C; [R]
‚HCl‚1.50H O) C, H, N.
-[2-(Meth ylth io)p yr im id in -4-yl]-1-m eth yl-4-[3-(tr iflu o-
D
) +165.1° (MeOH). Anal.
synthesis of 25: H NMR (CDCl ) δ 4.37 (s, 2H), 6.86 (s, 1H),
1
3
(C
27
H
26
N
6
F
3
2
7.09 (d, J ) 5.1 Hz, 1H), 7.67 (t, J ) 7.8 Hz, 1H), 7.88 (d, J )
7.8 Hz, 1H), 8.18 (d, J ) 9.3 Hz, 1H), 8.20 (d, J ) 5.1 Hz, 1H),
8.25 (s, 1H).
5
r om et h yl)p h en yl]-2-[4-(N-(b en zyloxyca r b on yl)p ip er i-
d in yl)]im id a zole (36). To a stirred solution of 28 (24.0 g, 43.4
mmol) in toluene (300 mL) was added DMF dimethyl acetal
1-(2-F lu or op yr id in -4-yl)-2-[3-(tr iflu or om eth yl)p h en yl]-
eth a n e-1,2-d ion e 1-Oxim e (44). To a mixture of 43 (10.80
g, 0.038 mol) in ethanol (200 mL), at -10 °C, under argon,
were added tert-butyl nitrite (5.0 mL, 0.042 mol) and hydrogen
chloride (12.2 mL, 2.5 M in ethanol, 0.031 mol) dropwise while
maintaining the temperature below -5 °C. The reaction was
allowed to warm to room temperature, stirred for 2 h,
concentrated, diluted with water (100 mL) and saturated
(25 mL), and the mixture was heated to reflux for 48 h. The
reaction was cooled, concentrated in vacuo to a foam, and
chromatographed on silica gel (1 kg) using 1:33 acetone/DCM
1
to give 16 g (65%) of 36: mp 148-152 °C; H NMR (CDCl
3
) δ
2
2
7
.0 (m, 4H), 2.60 (s, 3H), 2.98 (m, 3H), 3.80 (s, 3H), 4.35 (br s,
H), 5.13 (s, 2H), 6.76 (d, J ) 6.0 Hz, 1H), 7.3-7.45 (m, 6H),
.5-7.6 (m, 2H), 7.78 (s, 1H), 8.33 (d, J ) 6.0 Hz, 1H).
aqueous NaHCO (200 mL), and extracted with EtOAc (3 ×
3
Additional elution of the above column chromatography with
400 mL). The organic layers were washed with water (300 mL)
1
1
1
:9 methanol/DCM gave 37: H NMR (CDCl
3
) δ 1.7 (s, 3H),
and brine (300 mL), dried over Na
afford 11.4 g of 44 (96%): H NMR (CD
2
SO
4
, and concentrated to
OD) δ 7.23 (s, 1H),
1
.9-2.1 (m, 4H), 2.7-3.1 (m, 3H), 3.38 (s, 3H), 4.35 (br s, 2H),
3

Imidazole Inhibitors of p38 MAP Kinase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2189
7
8
8
.40 (d, J ) 5.1 Hz, 1H), 7.71 (t, J ) 7.8 Hz, 1H), 7.92 (d, J )
.1 Hz, 1H), 8.24 (d, J ) 7.8 Hz, 1H), 8.29 (d, J ) 5.3 Hz, 1H),
.31 (s, 1H).
The soluble fraction was loaded onto a GammaBind Plus
column (Pharmacia) preloaded with 17 mg of anti-middle
T-antibody/mL of resin. The column was washed with three
column volumes of each of the following: buffer A; then 25
mM Tris (pH 8), 0.1 mM EGTA, 0.1 mM EDTA, protease
5
-(2-F lu or op yr id in -4-yl)-1-m eth yl-4-[3-(tr iflu or om eth -
yl)ph en yl]-2-[4-(N-(ben zyloxycar bon yl)piper idin yl)]im ida-
zole (45). This was prepared as described in the synthesis of
3 4
inhibitor cocktail, 1 mM DTT, 1 mM Na VO , 5 mM sodium
2
8 except that the first step was carried out at 80 °C instead
pyrophosphate, and 25 mM sodium glycerolphosphate (buffer
B) containing 0.01% Triton X-100; and buffer B without Triton.
Protein was eluted with 50 µg/mL Glu-Tyr-Met-Pro-Met-Glu
peptide in buffer B. Peak fractions were pooled and aliquots
stored at -70 °C in the presence of 0.1 mg/mL BSA and 20%
glycerol.
1
of reflux: H NMR (CD
Hz, 2H), 3.06 (m, 3H), 4.29 (d, J ) 13.4 Hz, 2H), 5.14 (s, 2H),
.09 (s, 1H), 7.26 (d, J ) 4.5 Hz, 1H), 7.38 (m, 5H), 7.63 (d, J
7.3 Hz, 1H), 7.68 (d, J ) 7.6 Hz, 1H), 7.70 (t, J ) 7.3 Hz,
H), 7.76 (s, 1H), 8.07 (d, J ) 5.1 Hz, 1H).
S)-5-[2-(1-P h en ylet h yla m in o)p yr id in -4-yl]-4-[3-(t r i-
3
OD) δ 1.87 (m, 2H), 2.04 (d, J ) 12.9
7
)
1
(
Wild-type Mek1 and a kinase-inactive form of Mek1 (Lys97
to Ala substitution) with amino-terminal middle T-epitopes
were cloned, expressed, and purified as described for c-Raf.
Activated Mek1 was obtained by coinfection with viruses
expressing c-Raf, Lck, and activated H-Ras at a multiplicity
of infection of 10:2:2:2. Wild-type and a kinase-inactive form
of Erk2 (Lys52 to Arg substitution) were expressed in Escheri-
chia coli as GST-fusion proteins in pGEX vectors (Promega)
and purified over glutathione resins. Wild-type GST-Erk2 was
activated in vitro with activated Mek1 using reaction condi-
tions as described above.
flu or om eth yl)ph en yl]-2-[4-(N-(ben zyloxycar bon yl)piper i-
d in yl)]im id a zole (46). A mixture of 45 (40.0 g, 0.0763 mol)
and (S)-(-)-(R)-methylbenzylamine (167 mL, 1.525 mol) was
heated at 150 °C for 100 h, cooled to room temperature, poured
in to 1.5 L of pH 4.5 solution of 10 N NaOH in 10% citric acid,
and extracted with EtOAc (3 × 1 L). The combined extracts
3
were washed with saturated aqueous NaHCO and brine (750
mL), dried over Na
purified by chromatography on silica gel (2 kg) eluting with
2 4
SO , and concentrated to an oil which was
1
7
5% EtOAc/hexane to give 46 (31.5 g, 66%): H NMR (CD
OD) δ 1.48 (d, J ) 6.8 Hz, 3H), 1.75-2.10 (m, 4H), 3.03 (m,
H), 4.29 (d, J ) 13.4 Hz, 2H), 4.63 (q, J ) 6.8 Hz, 1H), 5.14
s, 2H), 6.40 (s, 1H), 6.58 (d, J ) 5.4 Hz, 1H), 7.14-7.37 (m,
0H), 7.61 (d, J ) 5.9 Hz, 1H), 7.63 (d, J ) 7.3 Hz, 1H), 7.72
s, 1H), 7.86 (d, J ) 5.1 Hz, 1H).
S)-5-[2-(1-P h en yleth yla m in o)p yr id in -4-yl]-1-m eth yl-4-
3-(tr iflu or om eth yl)ph en yl]-2-[4-(N-(ben zyloxyca r bon yl)-
3
-
R a f, Mek , a n d E r k 2 Kin a se Assa ys. All three kinases
were assayed in the same basic assay buffer containing 25 mM
4
(
1
(
HEPES, pH 8.0, 5 mM MgCl
2 3
, 0.5 mM EDTA, 0.2 mM Na -
VO , 0.1 mg/mL bovine serum albumin, 2 mM â-mercaptoet-
4
33
hanol, and 10 µM [γ- P]ATP (20 µCi/mL). Raf, Mek, and Erk2
kinase assays used the following concentrations of sub-
strates: 25 µg/mL kinase-inactive Mek1, 30 µg/mL kinase-
inactive GST-Erk2, and 50 µg/mL myelin basic protein,
respectively. Inhibitor dilutions were made in DMSO. Final
DMSO concentrations were 2.5% for Mek assays and 5% for
all others. Reactions were run at room temperature and
terminated with an equal volume of 100 mM EDTA, 100 mM
sodium pyrophosphate. Phosphorylated product was captured
in 96-well Immobilon-P filter plates (Millipore). Filters were
(
[
p ip er id in yl)]im id a zole (47). Iodomethane (1.88 mL, 0.030
mol) was added dropwise to a mixture of 46 (18.32 g, 0.029
mol) and cesium carbonate (18.32 g, 0.056 mol) in DMF (150
mL) at -30 °C. This reaction mixture was slowly warmed to
room temperature over 3 h, diluted with EtOAc (1.5 L), washed
with water (5 × 100 mL) and brine (300 mL), dried over Na
SO , and concentrated to an oil. The oil was purified by column
chromatography on 1.5 kg of silica gel, eluting with 5% acetone
in DCM to give 11.50 g (61%) of 47: 1H NMR (CD
2
-
4
2
washed five times with H O and counted on a TopCount
microplate scintillation counter (Packard).
3
OD) δ 1.53
d, J ) 6.8 Hz, 3H), 1.95 (m, 4H), 3.05 (br s, 2H), 3.17 (m, 1H),
(
P KA a n d P KC Kin a se Assa ys. PKA and PKC were
3
1
7
1
1
.40 (s, 3H), 4.29 (d, J ) 13.4 Hz, 2H), 4.80 (q, J ) 6.8 Hz,
H), 5.14 (s, 2H), 6.32 (s, 1H), 6.44 (d, J ) 6.6 Hz, 1H), 7.22-
.38 (m, 101H), 7.44 (t, J ) 7.8 Hz, 1H), 7.48 (d, J ) 9.3 Hz,
H), 7.49 (d, J ) 7.8 Hz, 1H), 7.66 (s, 1H), 7.99 (d, J ) 5.9 Hz,
H).
3
3
assayed at room temperature in the presence of 10 µM [ P]-
ATP. PKA and PKC (mix of isozymes) were assayed as
recommended by the supplier (Upstate Biotechnology) using
1
0 µM kemptide (Sigma) and 70 µg/mL myelin basic protein
(Upstate Biotechnology) as substrates, respectively. Reactions
(
S)-5-[2-(1-P h en yleth yla m in o)p yr id in -4-yl]-1-m eth yl-4-
were terminated and quantitated as described for c-Raf, except
that PKA reactions were stopped with 75 mM phosphoric acid,
captured on 96-well P81 phosphocellulose filter plates (Milli-
pore), and washed with phosphoric acid. EGF receptor tyrosine
kinase (Promega) was assayed with poly(Glu,Ala,Tyr) (6:3:1)
[
(
3-(t r iflu or om et h yl)p h en yl]-2-(4-p ip er id in yl)im id a zole
48). This was prepared as described for 39 and characterized
D
as the sulfate salt: mp softens at 165 °C; [R] -45.60° (c )
0
3
1
.0109 g/mL, MeOH); H NMR (CD
3
OD) δ 1.53 (d, J ) 6.8 Hz,
H), 2.16 (m, 4H), 3.17 (m, 3H), 3.40 (s, 3H), 3.56 (d, J ) 12.7
(
Sigma) as substrate at a final concentration of 40 µM in a
buffer containing 25 mM HEPES, pH 7.4, 5 mM MgCl , 2 mM
MnCl . Phosphorylated peptide was captured and counted as
described for Raf.
Lck Assa ys. These were carried out in a buffer containing
50 mM MOPS (pH 7.0), 1 mM DTT, 10 mM MgCl , 1 mg/mL
Hz, 2H), 4.76 (q, J ) 6.8 Hz, 1H), 6.47 (s, 1H), 6.54 (d, J ) 5.9
Hz, 1H), 7.26 (m, 5H), 7.40 (t, J ) 7.7 Hz, 1H) 7.49 (d, J ) 8.1
Hz, 1H), 7.53 (d, J ) 7.6 Hz, 1H), 7.71 (s, 1H), 7.97 (d, J ) 5.9
2
2
Hz, 1H). Anal. (C29
H
30
N
5
F
3
‚H
2
SO
4
‚2.45H
2
O) C, H, N.
Biologica l Meth od s. p38 purification and assays were
2
8
2
carried out as described previously. For compounds with IC50
5 nM, the published conditions were altered by lowering
3
3
BSA, 0.2 µCi of [ P]ATP, 2 µM ATP, 2 µM Biotinyl-cdc2
peptide, and 1 nM Lck kinase domain. The reactions were
incubated at 23 °C for 40 min, stopped with 100 mM EDTA,
mixed with streptavidin-coated SPA beads, and counted on a
Packard Top Count.
<
the enzyme concentration and increasing the incubation period
by the same factor to give the same turnover.
P u r ifica tion of Ra f a n d Mek Kin a ses. Recombinant full-
length human c-Raf with a carboxyl-terminal middle T-epitope
tag, Glu-Tyr-Met-Pro-Met-Glu, was produced from baculovi-
rus-infected Sf9 cells. For that purpose c-Raf was cloned into
the pVL1393 vector and expressed using the BaculoGold
system (PharMingen). Activated Raf was produced by coin-
fection of Sf9 cells with virus expressing c-Raf, Lck, and
activated H-Ras (Val12) at a multiplicity of infection of 10:2:
LP S Ch a llen ge Assa y. Female CD1 mice (8/group, 8-10
weeks of age) were injected intraperitoneally with 10 µg of LPS
and 800 mg/kg D-galactosamine in saline (0.5 mL). Compound
was administered orally in 0.5 mL of 0.05 M aqueous citric
acid 6 h before injection of LPS or intravenously in 0.2 mL of
0
.5% methocel in saline immediately before LPS injection. Mice
were sacrificed by CO asphyxiation 90 min after LPS, and
2
2
1
. The cells were lysed by sonication in 20 mM Tris (pH 7.5),
50 mM NaCl, 1 mM Na VO , 50 mM NaF, 1 mM EDTA, 1
, 1% Triton X-100, protease inhibitor cocktail (10
heparinized blood was collected by cardiac puncture. Blood was
centrifuged at 1500g and 4 °C for 15 min, and plasma TNF-R
levels were determined by ELISA (Genzyme).
3
4
mM MgCl
2
µg/mL benzamidine, 5 µg/mL each of leupeptin, aprotinin,
pepstatin), 1 mM AEBSF, 1 mM DTT, 5 mM sodium pyro-
phosphate, and 25 mM sodium glycerolphosphate (buffer A).
Ra t a n d Rh esu s P h a r m a cok in etic Stu d ies. These were
carried out as described previously.30

2
190 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
In Vitr o p 450 In h ibition Stu d ies. These were performed
Liverton et al.
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3
1
as described previously.
Ack n ow led gm en t. We are grateful to the Depart-
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