Discovery of N-substituted pyridinones as potent and selective inhibitors of p38 kinase

Article abstract of DOI:10.1016/j.bmcl.2009.08.082

The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhibitors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR explored. Their design was guided by ligand bound co-

Full text of DOI:10.1016/j.bmcl.2009.08.082

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5851–5856
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-substituted pyridinones as potent and selective inhibitors
of p38 kinase
a
b
a
a
Shaun R. Selness a, , Rajesh V. Devraj , Joseph B. Monahan , Terri L. Boehm , John K. Walker ,
Balekudru Devadas a, Richard C. Durley a, Ravi Kurumbail c, Huey Shieh c, Li Xing c, Michael Hepperle a,
Paul V. Rucker a, Kevin D. Jerome a, Alan G. Benson a, Laura D. Marrufo a, Heather M. Madsen a,
Jeff Hitchcock a, Tom J. Owen a, Lance Christie a, Michele A. Promo a, Brian S. Hickory a, Edgardo Alvira a,
Win Naing a, Radhika Blevis-Bal a
*
a Department of Medicinal Chemistry, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
b Inflammation Biology, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
c Structural and Computational Chemistry, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhib-
Received 8 June 2009
Revised 21 August 2009
Accepted 24 August 2009
Available online 27 August 2009
itors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR
explored. Their design was guided by ligand bound co-crystals of p38a. These efforts resulted in the iden-
tification of 12r and 19 as orally active inhibitors of p38 with significant efficacy in both acute and
chronic models of inflammation.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
p38 kinase
Pyridinones
The overexpression of the pro-inflammatory cytokines TNF-
a
1 against p38a was modest (1 lM). However, evaluation of 1
and IL-1b has been linked to the development and progression of
several inflammatory diseases such as rheumatoid arthritis (RA)
and Chrohn’s disease.1 The efficacy demonstrated by several mar-
keted protein therapeutics such as etanercept, adalimumab and
inflixamab in RA suggests that small molecule modulators of path-
ways leading to the production of these cytokines could have po-
tential therapeutic benefit.2 p38 has been identified as a central
kinase in the stress induced MAPKAP pathways leading to the pro-
duction of these key mediators.3,4 Inhibition of p38 has been
against a panel of 48 kinases revealed that the compound was
selective for p38 (less than 30% inhibition at 10
tire panel).
lM against the en-
The co-crystal structure of 1 bound to p38
a highlighted the key
structural features associated with its binding mode (Fig. 2).9
A
dual hydrogen bond to Met 109 and Gly 110 was observed for 1
which requires a backbone flip of Gly 110. This binding mode is
consistent with the induced flip observed with 2 (Fig. 3).10 The
benzyloxyl group occupied the lipophilic pocket and the N-benzyl
group was directed toward the solvent front. This binding mode
suggested several structure-based design strategies. These strate-
gies included targeting residues Asp112 and Asn115 with appro-
priate functionality via substitution on the N-benzyl group of 1.
The role of the C3 bromine was probed with simple halogen
isosteres.
shown to reduce the production of cytokines such as TNF-a, IL-6
and IL-8 in monocytic cell systems.5 In vivo anti-inflammatory
activity has been demonstrated with numerous p38 inhibitors in
several rodent models of inflammation. This data has driven much
research in this area and diverse classes of small molecule inhibi-
tors of p38 have been identified.6
We herein describe the identification and generation of highly
selective p38 inhibitors from the N-benzyl pyridinone high-
throughput screening hit, SC-25028, 1. Recently described inhibi-
tors of p38 kinase such as VX-745, 2, have demonstrated superior
kinase selectivity as compared to historical diaryl heterocyclic
inhibitors such as SB-203580, 3 (Fig. 1).7,8 The enzyme activity of
Metabolic stability of 1 was recognized as a primary liability as
revealed by its incubation with human liver microsomes (52% par-
ent remaining after 45 min of incubation). Efforts to improve on
the metabolic stability of 1 focused on halogen substitution on
the benzyloxy ring and placement of electron-withdrawing groups
on the N-benzyl ring to block oxidative cleavage of the C–O and C–
N bonds.
The general preparation of these N-substituted pyridinones is
shown in Schemes 1 and 2. Displacement of 4-chloropyridine-1-
* Corresponding author. Tel.: +1 636 247 6856.
E-mail address: shaun.r.selness@pfizer.com (S.R. Selness).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.082

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S. R. Selness et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5851–5856
Figure 1. SC-25028, VX-745 and SB-203580.
Figure 2. Crystal structure of compound 1 bound in the active site of p38
of p38 were obtained by soaking experiment. The structure has been refined to a
Rfree of 25.1% at 2.15 Å resolution (Rcrystal: 32.1%). Some of the key side chains of
p38 are displayed (C: green, N: dark blue and O: red). The inhibitor is represented
with carbon, nitrogen, oxygen and bromine atoms displayed in gold, blue, red, and
pink, respectively. The hydrogen bonds formed by the inhibitor are shown in dotted
white lines.
a. Crystals
Figure 3. Crystal structure of compound 2 bound in the active site of p38
of p38 were obtained by soaking experiment. The structure has been refined to a
Rfree of 22.3% at 2.3 Å resolution (Rcrystal: 30.4%). Some of the key side chains of p38
are displayed (C: green, N: dark blue and O: red). The inhibitor is represented with
carbon, nitrogen, oxygen, sulfur, fluorine, chlorine and bromine atoms displayed in
gold, blue, red, rose, green, purple and pink, respectively. The hydrogen bonds
formed by the inhibitor are shown in dotted white lines.
a. Crystals
a
a
a
a
Scheme 1. Synthesis of N-benzyl pyridinones from 4-chloropyridine-1-oxide. Reagents and conditions: (a) benzyl alcohol, NaH/DMF, 100 °C; (b) Ac2O, reflux then MeOH/
H2O; (c) Br2, AcOH, 0 °C or NBX, AcCN; (d) arylCH2X, K2CO3/DMF 110 °C; (e) vinylSnBu3, PdCl2(PPh3)2, DMF, 80 °C; (f) H2, Pd/C, EtOH, 1 atm; (g) Me4Sn, PdCl2(PPh3)2, DMF; (h)
CuI, NaOC(O)CF3; (i) Zn(CN)2, Pd(OTf)2, Zn, racemic-2-di-t-butylphosphino-1.10binaphthyl, DMAC, 95 °C, 3 h.

S. R. Selness et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5851–5856
5853
Scheme 2. Synthesis of N-benzyl pyridinones from 4-hydroxy-6-methylpyran-2-one. Reagents and conditions: (a) NH4OH (aq); (b) ArylCH2X, K2CO3/DMF; (c) Br2, AcOH, 0 °C
or NBX, AcCN; (d) ArylCH2X or heteroarylmethylene halide, NaH/THF, reflux; (e) ArylCH2NH2 or heteroarylCH2NH2, H2O, reflux.
oxide with the appropriate benzyl alcohol affords the correspond-
ing aryloxypyridine-N-oxide, 4. Acetic anhydride mediated rear-
rangement of 4 produces the pyridinone, 5. Halogenation of 5
can be accomplished with molecular bromine or NBX (X = Br, I or
Cl) to yield the corresponding 3-halopyridinone, 6. Alkylation of
6 with various benzyl halides gives the 3-halo-N-substituted
pyridinone, 7. The iodo precursor, 7b, is converted to the C3 vinyl
pyridinone via a Stille coupling reaction to afford pyridinone 8a.
Catalytic hydrogenation of 8a afforded the C3 ethyl analog, 8b.
The C3 methyl, trifluoromethyl and cyano analogs (8c–e) were pre-
pared via metal mediated coupling reactions (Scheme 1).
Two alternative syntheses were pursued to generate a series of
6-methyl-N-substituted pyridinone derivatives as outlined in
Scheme 2. Condensation of 4-hydroxy-6-methylpyran-1-one with
aqueous NH4OH gives the hydroxy pyridinone, 9. Alkylation of 9
with a benzyl halide affords the benzyloxy pyridinone, 10. Haloge-
nation of 10 under the conditions in Scheme 1 generates the 3-
halopyridinone, 11. Initial attempts at alkylation of 11 with a ben-
zyl or heteroarylmethylene halide under several conditions re-
sulted in mixtures of O- and N-alkylated products. We found that
the use of NaH/THF favored the formation of the desired N-substi-
tuted pyridinone, 12. Alternatively, condensation of 4-hydroxy-6-
methylpyran-1-one with a substituted benzyl or heteroarylmeth-
ylene amine yields the N-substituted pyridinone, 13 in moderate
to good yields. This condensation was reported to proceed in n-bu-
tyl alcohol.11 We observed bis-addition of the amine under these
conditions which was avoided by using water as the solvent. This
minimized the bis-adduct formation and facilitated isolation of
13. Installation of the halide is accomplished via treatment with
bromine or NCX to afford the 3-halo-N-substituted pyridinone,
14. Benzylation of 14 with a substituted benzyl halide provided
the desired pyridinone, 12a–f.
substituted benzyloxy groups were investigated.7,8 We concluded
that the 2,4-difluorobenzyloxy group was optimal for potency
and focused our attention on the N-benzyl substituent.
As shown in Table 2, ortho-substituted N-benzyl groups were
not tolerated compared to the meta- or para-substituted N-benzyl
groups (12a–f). Carboxamide substitution in the para-position
afforded several potent compounds (12f, 12h–k) with activity be-
low 100 nM. Compounds 12h, 12i and 12k were shown to be active
in a rat lipopolysaccharide (rLPS) model of inflammation.12,13
A
series of heterocyclic replacements were investigated in an effort
to reduce the log D of the series and to improve the metabolic sta-
bility of the series. The pyridines 12o and 12p showed modest
activity against p38 but exhibited good activity in rLPS. This was
attributed to improved metabolic stability and better bioavailabil-
ity relative to 1.
In an effort to engage Asp 112 the amines 16, 17, 18 and 19
were prepared (via BH3/dimethylsulfide reductions of 12b, 12c,
12g and 12h, respectively). Although their enzyme activities
against p38a did not improve relative to the neutral carboxamides,
significant in vivo activity in rLPS was achieved for 16 and 19.
Investigation of neutral analogs of 16 and 17 identified 20, 21
and 22 as potent inhibitors of p38a. These were prepared via acyl-
ation of 16, 17 and the chloro analog of 17 with glycolic acid.
Table 1
Potency and selectivity (as measured by inhibition of JNK2) of 4-benzyloxy-3-halo-N-
substituted pyridinones and 4-benzyloxy-3-alkyl-N-substituted pyridinones
Compd
R
Ar
Ph
Ar0
Ph
p38
a
IC50
(
l
M) JNK2 IC50 (lM)
1
2
3
Br
0.68
0.07
0.07
0.90
3.10
43
>200
>200
0.32
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
Cl
I
H
Br
Br
Br
Ph
Ph
Ph
4-Cl–Ph
4-F–Ph
Ph
Ph
Ph
NA
Initially, we investigated the role of the C3 position and its con-
tribution to the activity of the series. Chloro and bromo groups
were identified as preferred as shown by the activity of 1 and 7a
(Table 1). The halogen group at C3 occupies a shallow lipophilic
pocket adjacent to the deep selectivity pocket as defined by
Thr106. Additionally, we used activity against JNK2 to evaluate
the potential for kinase selectivity. As shown in Table 1 this series
was highly selective versus JNK2. Based on historical data from
both the diaryl five-membered heterocycles such as 3 and the
new class of fused ring systems such as 2, a limited number of halo
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
4-F–Ph 1.90
3-F–Ph 0.62
2,4-DiF–Ph 3-F–Ph 0.13
CHCH2 Ph
Et
Me
CF3
CN
Ph
Ph
Ph
72
6.1
5.3
Ph
Ph
Ph
3-F–Ph 50
2,4-DiF–Ph 3-F–Ph 9.9
NA—not assessed.

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S. R. Selness et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5851–5856
Table 2
Potency and in vivo activity (as measured by inhibition of TNF
a in rLPS) of 4-(halo-benzyloxy)-3-halo-N-substituted benzyl pyridinones and substituted 4-(halo-benzyloxy)-3-
halo-N-substituted heteroaryl-methylene pyridinones13
Compd
X
Ar
Ar0
p38
a
IC50
(
lM)
JNK2 IC50
(
lM)
hPBMC IC50
(
lM)
% rLPS TNFa Inhibition @–4 h @ (mpk)
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
15
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
4-F–Ph
2-CN–Ph
3-CN–Ph
4-CN–Ph
1.20
0.11
0.19
2.67
0.27
0.022
0.25
0.066
0.079
0.20
0.025
1.00
0.38
0.39
0.15
0.21
0.45
0.45
0.18
1.26
0.065
0.12
0.063
0.059
>200
>200
>200
>200
167
NA
NA
0.118
NA
NA
0.046
NA
0.044
NA
0.115
0.13
NA
NA
NA
NA
NA
0.576
0.566
NA
NA
NA
NA
NA
NA
NA
NA
40 (5)
0 (5)
69 (5)
79 (5)
0 (5)
84 (5)
0 (20)
30 (20)
0 (20)
81 (20)
80 (20)
80 (20)
89 (30)
63 (5)
NA
2-NH2C(@O)–Ph
3-NH2C(@O)–Ph
4-NH2C(@O)–Ph
3-NMe2C(@O)–Ph
4-NMe2C(@O)–Ph
4-NMe2C(@O)–Ph
3-NMeHC(@O)–Ph
4-NMeHC(@O)–Ph
2-Pyridine
3-Pyridine
4-Pyridine
3-Pyridine
4-Pyridine
2-(5-Me-pyrazine)
2-(5-HOCH2–pyrazine)
5-(2-Me–pyrimidine)
2-H2NCH2-Ph
3-H2NCH2–Ph
4-H2NCH2–Ph
3-Me2NCH2–Ph
4-Me2NCH2–Ph
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>171
4-F–Ph
4-F–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
16
17
18
19
90 (20)
57 (5)
61 (5)
87 (5)
0.039
0.101
0.044
20
21
22
Br
Cl
Br
2,4-DiF–Ph
2,4-DiF–Ph
2,4-DiF–Ph
0.046
0.055
0.049
>200
>200
>200
0.023
0.028
0.102
93 (5)
69 (5)
32 (15)
NA—not assessed.
Compound 20 possessed significant in vivo activity in rLPS. Assess-
ment of the metabolic stability (as measured by incubation with
human and rat microsomes) of a series of pyridinones indicated
that significant improvements were achieved for several analogs
when compared to 1 (Table 3). The relatively low stabilities ob-
served for compounds 12h and 18 are attributed to demethylation
of each to their more stable metabolites, 12f and 16, respectively.
Both 12r and 19 were profiled against a panel of over 200 kinases
to assess their selectivities. The compounds showed no significant
cross-reactivity with any of the kinases tested with the exception
of p38b (5–10 more selective for p38 vs p38 b; Table 4).
Several pyridinones were selected for assessment of their phar-
macokinetic profiles in male Sprague–Dawley rats (Table 5).13
range of clearances were observed for the compounds from low
to high. Characterization of plasma samples after oral dosing of
12q indicated that the major metabolite is the longer-lived 12r.
Similarly, analysis of plasma samples after oral dosing of 19 re-
vealed that the mono-methylamine, 23, and primary amine, 17
were generated (Fig. 4). Both 17 and 23 demonstrated activity
a
A
against p38a (IC50s of 120 nM and 52 nM, respectively), activity
in hPBMCs (IC50s of 59 nM and 49 nM) and were active in rLPS
Table 3
at ꢀ4 h at 5 mpk (95% and 91%, respectively). Evaluation of 23
Metabolic stability (percent remaining after incubation with human or rat liver
microsomes for 45 min) of selected pyridinones14
against JNK2 indicated that it was not very potent (IC50 = 191 lM).
Compounds 12r and 19 were evaluated in a rat Strep Cell Wall
(rSCW) model of chronic inflammation (Table 6). Both compounds
demonstrated significant efficacy in this model. The higher dose re-
Compd
cLog D
HLM (% remaining)
RLM (% remaining)
1
7e
7f
12f
12h
12k
12q
12r
16
3.81
3.92
4.01
3.07
2.76
3.19
2.51
0.86
1.49
2.97
2.91
2.88
52
76
48
89
59
75
98
96
89
50
57
73
3
26
16
94
51
63
95
100
99
39
53
82
quired for 12r is attributed to its inferior potency against p38
a
whereas the activity of 19, in spite of its high clearance, is attrib-
uted to the generation of the active metabolites, 23 and 17.
In summary, a series of potent, selective and stable inhibitors of
p38
was metabolically unstable and moderately active against p38
This new class of inhibitors exhibit a unique binding mode which
imparts a high degree of selectivity for p38 over other kinases.
These compounds demonstrated significant activity in both acute
a were generated from a high-throughput screening hit that
a.
18
19
20
a

S. R. Selness et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5851–5856
5855
Table 4
Kinase selectivity panel for 12r and 19 (percent inhibition <50% at 10
l
M for all kinases tested, except for p38a and p38b)
CAMK1
CDK5/p35
CHK1
CHK2
CLK1
CLK2
CLK3
CSF1R
CSK
FGFR3 K650E
FGFR4
FGR
FLT1
FLT3
FLT3 D835Y
FLT4
mTOR
FRK
FYN
HGK
PDGFR alpha
PDGFRA D842V
PDGFRA T674I
PDGFRA V561D
PDGFR beta
PDK1
PDK1 Direct
PHKG1
PHKG2
MSK2
MSK1
RSK4
p70S6K
SGK1
SGK2
SGK3
SNF1LK2
SRC
CDK7/cyclin H/MNAT1
CDK9/cyclin T1
IKK alpha
DAPK1
GSG2
IRAK1
LRRK2
LRRK2 G2019S
NUAK1
KHS1
ERK2
JNK3
p38 beta 82; 99
p38 gamma
p38 delta
p38 alpha 100; 100
p38 alpha Direct 100; 100
ERK1
CSNK1A1
CSNK1D
CSNK1E
PIM1
PIM2
SRC N1
Srm
ABL1
GRK4
JNK1
ABL1 E255K
ABL1 G250E
ABL1 T315I
ABL1 Y253F
ABL2
ALK4
GRK2
GRK3
AKT1
AKT2
AKT3
ALK
AMPK A1/B1/G1
AMPK A2/B1/G1
AURKA
AURKB
AURKC
CSNK1G1
CSNK1G2
CK1 gamma 3
CK2 alpha 1
CK2 alpha 2
ZIPK
GRK5
GRK6
GRK7
JNK2
PRK1
PLK1
PLK2
PLK3
SRPK1
SRPK2
TSSK2
TSSK1
MSSK1)
MST3
YSK1
MST2)
MST1)
SYK
TAO1
TBK1
Tie2
TYK2
RSE
YES1
MAPKAPK2
MAPKAPK3
PRAK
MARK1
MARK2
MARK3
MARK4
MATK
MELK
MERTK
cMet
MET M1250T
MINK1
RON
GSK3A
GSK3B
HCK
HIPK1
HIPK2
HIPK4
IGF1R
IKK beta
IKK epsilon
INSR
PKA
PKC alpha
PKC beta I
PKC beta II
PKC delta
PKC epsilon
PKC gamma
PKC eta
PKC iota
PKD3
PKC theta
PKC zeta
PKC mu
PKD2
DCK2
DYRK1A
DYRK1B
DYRK3
DYRK4
EEF2K
EGFR (ErbB1)
EGFR (ErbB1) L858R
EGFR (ErbB1) L861Q
EGFR (ErbB1) T790M
EGFR (ErbB1) T790M
EPHA1
INSRR
IRAK4
ITK
JAK1
MST4
MUSK
MYLK2
ZAP70
AXL
JAK2
BLK
BMX
BRAF
EPHA2
EPHA3
EPHA4
JAK2 JH1 JH2
JAK2 JH1 JH2 V617F
JAK3
NEK1
NEK2
NEK4
PRKG1
PKG2
PRKX
BRAF V599E
BRSK1
EPHA5
EPHA8
KDR (VEGFR2)
KIT
NEK6
NEK7
FAK
FAK2
BTK
EPHB1
EPHB2
EPHB3
EPHB4
ERBB2 (HER2)
ERBB4 (HER4)
FER
KIT T670I
LCK
LTK (TYK1)
LYN A
LYN B
NEK9
TRKA
TRKB
TRKC
PAK1
PAK2
PAK3
Brk
cRAF
RET
RET V804L
RET Y791F
ROCK1
CAMK1D
CAMK2A
CAMK2B
CAMK2D
CAMK4
MEK1
MEK2
MRCKA
ROCK2
MRCKB
FES (FPS)
FGFR1
FGFR2
MKK6
COT
MLK1
GCK
PAK4
PAK6
PAK7 (KIAA1264)
PASK
ROS1
RSK1
RSK3
RSK2
CDK1/cyclin B
CDK2/cyclin A
CDK5/p25
FGFR3
Table 6
Efficacy in rat Strep Cell Wall (rSCW, female Lewis) of selected pyridinones11
Table 5
Rat pharmacokinetics (male Sprague–Dawley) of selected pyridinones13
Compd
Dose (mpk)
Inhibition of paw swelling (%)
Compd
CL (ml/min/kg)
t
(h)
F (%)
½
12r
19
120
60
87
88
12q
12r
19
21.50
6.25
70
0.39
4.71
4.97
4.1
30
44
91
33
20
26.2
Acknowledgements
We acknowledge the reviewers for providing feedback on the
incorporation of key data. We acknowledge Sheri Bonar for gener-
ating the hPBMC data. We acknowledge Elizabeth Webb and Gary
Anderson for conducting the rLPS and rSCW experiments.
(rLPS) and chronic (rSCW) models of inflammation. Forthcoming
publications will describe additional improvements in potency
and pharmacokinetic profiles of this pyridinone class of p38
inhibitors.
a
Figure 4. Observed metabolites of 19.

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S. R. Selness et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5851–5856
8. Adams, J. L.; Boehm, J. C.; Kassis, S.; Gorycki, P. D.; Webb, E. F.; Hall, R.;
References and notes
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9. The atomic coordinates for the X-ray structure of p38a in complex with 1 have
been deposited in the RCSB: rcsb id code—a RCSB053406; PDB code—
3HP2.
10. The atomic coordinates for the X-ray structure of p38a in complex with 2 have
been deposited in the RCSB: rcsb id code—RCSB053409; PDB code—
3HP5.
11. Castillo, S.; Ouadahi, H.; Herault, V. Bull. Soc. Chim. Fr. 1982, 7–8, 257.
12. Compounds were pre-dosed in male Lewis rats at various time points prior to
an iv challenge with lipopolysaccharide. Blood was drawn 1.5 h post-challenge
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and TNF-a levels were evaluated by ELISA.
13. The Pfizer Institutional Animal Care and Use Committee reviewed and
approved the animal use in these studies. The animal care and use program
is fully accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care, International.
14. Metabolic stability was assessed in vitro by incubating 2 lM test compound
with human or rat liver microsomes, NADPH and buffer at 37 °C for 45 min and
measuring percent compound remaining by a precipitation procedure followed
by LC/MS analysis.