Design, synthesis and activity of a potent, selective series of N-aryl pyridinone inhibitors of p38 kinase

Article abstract of DOI:10.1016/j.bmcl.2011.04.120

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.

Full text of DOI:10.1016/j.bmcl.2011.04.120

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4059–4065
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and activity of a potent, selective series of N-aryl
pyridinone inhibitors of p38 kinase
Shaun R. Selness a, , Terri L. Boehm a, John K. Walker a, Balekudru Devadas a, Richard C. Durley a,
⇑
Ravi Kurumbail b, Huey Shieh b, Li Xing b, 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, Rajesh V. Devraj a,
Dean Messing c, John F. Schindler d, Jeffrey Hirsch d, Matthew Saabye d, Sheri Bonar d, Elizabeth Webb d,
Gary Anderson d, Joseph B. Monahan b,d
a Department of Medicinal Chemistry, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
b Structural and Computational Chemistry, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 3017, USA
c Department of Pharmcokinetics and Drug Metabolism, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
d Inflammation Biology, 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:
A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and
prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of
the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connec-
tivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization
produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both
acute and chronic models of inflammation.
Received 2 March 2011
Revised 23 April 2011
Accepted 26 April 2011
Available online 13 May 2011
Keywords:
p38 kinase
Ó 2011 Elsevier Ltd. All rights reserved.
Inflammation
Rheumatoid arthritis
TNFa
Pyridinones
The correlation of elevated levels of the cytokine tumor necrosis
factor- (TNF ) with the pathophysiology of a number of inflamma-
tory diseases has been studied for over two decades.1 The efficacy
observed for marketed anti-TNF therapies, such as etanercept,
adalimumab and inflixamab, in rheumatoid arthritis, psoriasis and
Crohn’s disease has established the important role of TNF in these
mode of 1 with p38a has been disclosed in a previous communica-
a
a
tion.4 This structural data was used to optimize the potency of the
a
a
diseases and validates approaches to limit its production as poten-
tial therapeutic mechanisms.2 The role of a number of stress-acti-
vated pathways in the production of TNF
investigated and has yielded several potential therapeutic targets
such as p38a 3
In this Letter, we discuss our progression from the
a has been extensively
.
screening hit, SC-25028 (1) to the N-phenyl pyridinones such as 2
and 3 (Fig. 1).
The initial lead, 1, was identified from a high-throughput full
file screen and demonstrated low micro molar activity against
p38a. Additional profiling of 1 revealed that it possessed a high de-
gree of selectivity against a panel of over 200 kinases. The binding
⇑
Corresponding author.
E-mail address: shaun.selness@gmail.com (S.R. Selness).
Figure 1. HTS hit SC-25028 (1) and N-aryl pyridinone leads.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.120

4060
S. R. Selness et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4059–4065
Scheme 1. Reagents and conditions: (a) 2,6-difluoroaniline, o-dichlorobenzene,
165 °C, 20 min, 35%; (b) 2,6-dimethyl or 2,6-dichloroaniline, o-dichlorobenzene,
p-toluenesulfonic acid, 165 °C, 3 h, 28–30%; (c) NBS, CH2Cl2, 1 h, 25 °C, 77–79%; (d)
benzyl, 4-fluorobenzyl, or 2,4-difluorobenzylbromide, DMF, K2CO3, 25 °C, 45–65%;
(e) NCS, CH2Cl2, isopropanol, reflux, 3 h, 62%; (f) 2,4-difluorobenzylbromide, DMF,
K2CO3, 25 °C, 65%.
Figure 2. Historical p38 inhibitors.
series and was used to identify key binding contacts that provide
the rational for the high degree of selectivity and specificity exhib-
ited by this series. Improving the metabolic stability of 1 was a pri-
mary objective and drove the decision to replace the N-benzyl
group with an N-phenyl group. Previous work on this series had
identified the 2,4-difluoro substitution on the O-benzyl group as
optimal for both potency and metabolic stability. These investiga-
tions led to the generation of 4, a potent and selective inhibitor of
stitution was tolerated and afforded improved potency compared
to that observed for 4 (p38a IC50 = 46 nM) as illustrated by 13.
Scheme 1 outlines the synthetic sequences used to prepare the
initial N-aryl analogs 8–13 (Table 1). Condensation of the corre-
sponding 2,6-disubstituted aniline with 4-hydroxy-6-methyl-2H-
pyran-2-one followed by halogenation with either NCS or NBS
yielded the N-phenyl-3-halo-4-hydroxy-6-methyl pyridinones
15–18. O-Alkylation of the 4-hydroxy group with the suitable ben-
zyl halide afforded the N-phenyl-3-chloro-4-benzyloxy or N-phe-
nyl-3-bromo-4-benzyloxy pyridinones 8–13.
p38a
(Fig. 2).4 High clearance (in rats) was identified as one of the
liabilities of this N-benzyl pyridinone class. We hypothesized that
N-debenzylation was a potential contributor to the observed high
clearances in rat pharmacokinetic studies. In order to test this
hypothesis, a series of N-phenyl pyridinone derivatives were pre-
pared and evaluated for their activity, selectivity, in vitro stability
and pharmacokinetic profiles.
Based on docking of 9 with p38a, targeting Asp112 and Asn115
(to improve potency) appeared to be feasible via elaboration of the
4-position of the N-phenyl group (Fig. 3). As shown in Table 2
these analogs possessed significant activity against p38. The piper-
azine derivatives, 19 and 20, did not provide any advantage over
the smaller N,N-dimethyl derivative, 21, the phenol 22 or the phe-
nyl ethers, 23 and 24. However, based on the activity (and lower
metabolic stability) of these analogs 2,4,6-trisubstitution did not
appear to offer any significant advantages over the 2,6-disubsti-
tuted analogs.
The N-phenyl trisubstituted analogs 19–24 were prepared as
shown in Scheme 2. Under similar condensation conditions 4-hy-
droxy-6-methyl-2H-pyran-2-one was allowed to react with
2,4,6-trifluoroaniline followed by alkylation with 2,4-difluoroben-
Initially, 2,6-disubstituted N-phenyl analogs were prepared
based on comparisons to the fused biaryl class of p38 inhibitors
represented by 5, 6 and 7.5 The data for these analogs is shown
in Table 1. The potency exhibited by these compounds required
the development of an assay with improved sensitivity relative
to previously reported assays.6 A cascade assay with p38
a
and
MK-2 was developed to accomplish this objective (with a readout
of phosphor-HSP27 peptide).7 The final concentration of p38
in
the assay was 100 pM, thus allowing for the detection of low nM
inhibitors of p38 . Compounds 8–13 demonstrated that 2,6-disub-
a
a
Table 1
2,6-Bis ortho substituted 4-benzyloxy-N-aryl pyridinones
Compound
R1
R2
R3
X1, X2
p38a (lM)
cascadea IC50
8
9
10
11
12
13
2,4-Di-F–PhCH2
2,4-Di-F–PhCH2
PhCH2
4-F–PhCH2
2,4-Di-F–PhCH2
2,4-Di-F–PhCH2
Br
Cl
Br
Br
Br
Br
CH3
CH3
CH3
CH3
CH3
CH3
F, F
F, F
Cl, Cl
Cl, Cl
Cl, Cl
CH3, CH3
0.003
0.004
0.030
0.006
0.001
0.002
a
For assay conditions please see Ref. 7.

S. R. Selness et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4059–4065
4061
Figure 3. Docked model of 9 with p38a showing residues Asp112 and Asn 115.
Table 2
4-Substituted 2,6-difluoro N-aryl pyridinones
Scheme 2. Reagents and conditions: (a) 2,4,6-trifluoroaniline, o-dichlorobenzene,
165 °C, 1 h, 33%; (b) 2,4-difluorobenzyl bromide, K2CO3, DMF, 25 °C, 77%; (c) excess
4-methylpiperazine or 2.0 N dimethylamine in THF, K2CO3, 25 °C, 34–52%; (d) NBS,
CH2Cl2, 25 °C, 50–65%; (e) NCS, dichloroacetic acid, CH2Cl2, 25 °C, 16 h, 57–62%; (f)
KOSiMe3, THF, 45 °C, 1 h, 37%; (g) BrCH2CH2OH (5 equiv), K2CO3 (6 equiv), DMF,
25 °C, 48 h, 27%; (h) BrCH2C(O)NH2 (1.3 equiv), K2CO3, DMF, 25 °C, 16 h, 51%.
Compound
R1
R2
p38
IC50
a
(
cascadea
lM)
19
20
21
22
23
24
4-N-Me–piperazine
4-N-Me–piperazine
–N(CH3)2
–OH
–OCH2CH2OH
–OCH2C(O)NH2
Br
Cl
Br
Br
Br
Br
0.014
0.023
0.008
0.015
0.007
0.003
Table 3
6-Substituted pyridinones
a
For assay conditions please see Ref. 7.
zyl bromide to afford 25. Displacement of the 4-fluoro group with
either N-methylpiperazine or dimethylamine followed by subse-
quent halogenation with either NCS or NBS gave the N-phenyl 4-
substituted pyridinones 19–21. Alternatively, bromination with
NBS and displacement with potassium trimethylsilanoate pro-
vided the phenol, 22. Alkylation of 22 with either 2-bromoethanol
or
2-bromoacetamide generated the alkoxyl analogs 23 and 24.
The C-6 position of the pyridinone was identified as a potential
site for cytochrome P450-mediated metabolism. Chemistry was
developed to prepare the C-6 oxidized analogs, 26 and 2, as well
as the methylamine derivatives, 27–29, (Table 3). The potential
metabolites showed retention of activity consistent with a model
that orients the C-6 substituents towards the solvent front
( Fig. 3). The alkyl amino derivatives also possessed significant
Compound
R
X
p38a (lM)
cascadea IC50
26
2
27
28
–CH2OH
–CH2OH
–CH2N(CH3)2
–CH2N(CH3)2
Br
Cl
Br
Cl
0.005
0.008
0.006
0.008
29
Br
0.009
a
For assay conditions please see Ref. 7.
activity against p38a.
to be optimal for occupying the lipophilic pocket beyond the gate-
keeper residue, Thr103. Both the benzylamine and ethylene linkers
were investigated and found to be significantly less active than the
corresponding benzyloxy linker.
We then turned our attention to the necessity of the 2,6-disub-
stitution on the N-phenyl group (Table 4). A set of compounds lack-
ing ortho-substitution on the N-phenyl ring were prepared. These
compounds were designed with the potential to engage Asp112
and/or Asn113 from either the 3- or the 4-position of the N-phenyl
ring. These analogs were accessed via the readily available amino
benzoates. The activity profiles showed a consistent dependence
on the substitution pattern and the trajectory of the functional
group. The parent aminomethyl compounds, 32 and 33, as well
Alkylation of 14 with 2,4-difluorobenzyl bromide generated the
pyridinone intermediate 30 (Scheme 3). Oxidation of the C-6
methyl of 21 with SeO2 followed by reduction with NaBH4 cleanly
gave the alcohol 31 (initial oxidation with SeO2 generated a mix-
ture of the alcohol and the aldehyde). Halogenation of 31 with
either NBS or NCS afforded the pyridinones 26 and 2, respectively.
Oxidation of the primary alcohol in 26 and 2 followed by reductive
amination with dimethylamine or morpholine yielded the C-6 ami-
nomethyl derivatives, 27–29.
We next focused on the importance of the benzyloxy linker.
Based on modeling of the amine and ethylene linkers versus the
benzyloxy linker, the trajectory of the benzyloxy group appeared

4062
S. R. Selness et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4059–4065
Table 5
Cell activity (hPBMCs) and metabolic stability (as measured by incubation with
human or rat liver microsomes) of selected N-aryl pyridiones
Compound
TNF release from
HLM
RLM
hPBMCs IC50
(l
M)a
(% remaining)b
(% remaining)b
13
8
9
26
2
23
27
34
36
3
0.025
0.022
0.017
0.025
0.045
0.061
0.050
0.091
0.118
0.064
0.045
3
60
74
39
70
87
0
88
97
100
95
1
80
87
65
69
96
0
83
91
97
92
42
a
For assay conditions please see Ref. 8.
For assay conditions please see Ref. 9.
b
with 2-acetoxy acetylchloride gave the amide 47. Hydrolysis of
47 provided the hydroxy acetamide derivative 34. Coupling of 32
with N-(tert-butoxycarbonyl)glycine gave the amide 48 which
was deprotected to afford 36.
Scheme 3. Reagents and conditions: (a) SeO2, dioxane, sealed tube, blast shield,
170 °C, 1.5 h; (b) NaBH4, MeOH, 0 °C ? rt, 4 h, 44% for two steps; (c) NBS, CH3CN,
0 °C, 2 h, 91%; (d) NCS, CH3CN, 25 °C, 4 h, 59%; (e) Dess–Martin periodinane, CH2Cl2,
0 °C, 4 h, 70–90%; (f) morpholine or 2 N dimethylamine in THF, CH2Cl2, NaBH(OAc)3,
HOAc, 25 °C, 12–16 h, 30–50%.
An analogous series of N-(3-substituted)phenyl pyridinones
was prepared as outlined in Schemes 5 and 6. Condensation of
ethyl-3-aminobenzoate with 4-hydroxy-6-methyl-2H-pyran-2-
one followed by alkylation with 2,4-difluorobenzyl bromide gave
the pyridinone intermediate 49. Halogenation and hydrolysis of
49 afforded the carboxylic acid 50. Coupling of 50 with ammonia,
methylamine or dimethylamine provided the amide analogs, 3,
42 and 44, respectively. Condensation of 3-hydroxymethylaniline
with 4-hydroxy-6-methyl-2H-pyran-2-one followed by subse-
quent benzylation with 2,4-difluorobenzyl bromide generated the
alcohol 51. The primary amine, 52, was generated from 51 via
conversion of the alcohol to the corresponding chloride with
2,4,6-trichlorotriazine followed by treatment with ammonia. Halo-
genation of 52 with NBS generated the bromide 33. The amine 33
was acylated with acetoxyacetyl chloride to generate the amide 53.
Subsequent hydrolysis of 53 provided the hydroxyacetamide 35.
Polymer bound carbodiimide catalyzed coupling of 33 with
N-(tert-butoxycarbonyl)glycine yielded the amide 54 which, upon
treatment with 4 N HCl in dioxane, generated the glycinamide
as their corresponding acyl and urea derivatives 34–39, exhibited
greater activity for the 4-substituted compounds. In contrast to
this trend, the 3-carboxamide derivatives were more active than
the corresponding 4-carboxamides as demonstrated by the analogs
3 and 40–44.
Scheme 4 shows the preparation of the N-(4-substituted) phe-
nyl pyridinones. Condensation of methyl-4-aminobenzoate with
4-hydroxy-6-methyl-2H-pyran-2-one followed by alkylation with
2,4-difluorobenzyl bromide generated the pyridinone intermedi-
ate, 45. Bromination of 45 with NBS followed by hydrolysis of
the ester provided the carboxylic acid, 46. The acid intermediate
46 was coupled with ammonia, methylamine or dimethylamine
to afford the amides 40, 41 and 43. Reduction of 40 with borane
yielded the amino methyl derivative 32. Treatment of 32 with
2,2,2-trichloroacetyl isocyanate followed by addition of ammo-
nium hydroxide generated the primary urea, 38. Acylation of 32
Table 4
3- And 4-substituted N-phenyl pyridinones
Compound
R1
R2
p38a (lM)
cascadea IC50
32
33
34
35
36
37
38
39
40
3
–CH2NH2
H
–CH2NHC(O)CH2OH
H
–CH2NHC(O)CH2NH2
H
–CH2NHC(O)NH2
H
–C(O)NH2
H
–C(O)NHCH3
H
–C(O)N(CH3)2
H
H
0.051
0.681
0.020
0.175
0.015
0.261
0.142
0.251
0.122
0.012
0.126
0.017
0.746
0.102
–CH2NH2
H
–CH2NH(O)CCH2OH
H
–CH2NH(O)CCH2NH2
H
–CH2NH(O)CNH2
H
–C(O)NH2
H
–C(O)NHCH3
H
–C(O)N(CH3)2
41
42
43
44
a
For assay conditions please see Ref. 7.

S. R. Selness et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4059–4065
4063
Scheme 5. Reagents and conditions: (a) ethyl-3-aminobenzoate, 1,2-dichloroben-
zene, 165 °C, 15 min, 37%; (b) 2,4-difluorobenzylbromide, DMF, K2CO3, 25 °C, 50%;
(c) NBS, CH3CN, 1 h, 25 °C, 90%; (d) KOSiMe3, THF, 75%; (e) 2-chloro-4,6-dimeth-
oxytriazine, 4-methylmorpholine, THF, 1.5 h, then NH4OH, 30 min, 93%; (f) methyl
or dimethylamine, PS-carbodiimide resin, HOBt, DMF rt, 15 min; then add, THF,
PS-polyamine resin, PS-polyisocyante resin, ꢀ60% two steps.
Scheme 4. Reagents and conditions: (a) methyl-4-aminobenzoate, 1,2-dichloro-
benzene, 165 °C, 15 min, 35%; (b) 2,4-difluorobenzylbromide, DMF, K2CO3, 25 °C,
50%; (c) NBS, CH3CN, 1 h, 25 °C, 90%; (d) KOSiMe3, THF, 70%; (e) methyl or
dimethylamine, PS-carbodiimide resin, HOBt, DMF rt; then filter, THF PS-polyamine
resin, PS-polyisocyante resin, 20-25% two steps; (f) 2-chloro-4,6-dimethoxy-1,3,5-
triazine, 4-methylmorpholine, THF, 1.5 h, then NH4OH, 30 min, 95%; (g) BH3ÁTHF,
THF, 0 °C ? reflux, 56%; (h) acetoxyacetyl chloride or N-(tert-butoxycarbonyl)-
glycine, PS-carbodiimide resin, HOBt, iPr2Net, DMF rt, 16 h; (i) K2CO3, MeOH, 84%;
(j) 4 N HCl–Dioxane, 24%; (k) 2,2,2-trichloroacetyl isocyanate, CH2Cl2, rt, 30 min,
then add NH4OH, rt, 3 h, 14%.
derivative, 37. Treatment of 33 with trimethylsilyl isocyanate in
the presence of N-methylmorpholine afforded the primary urea 39.
Potent compounds in the enzyme assay were evaluated for cel-
lular activity and metabolic stability (Table 5).8,9 In general, most
compounds potently (<100 nM) inhibited the release of TNFa from
Scheme 6. Reagents and conditions:(a) 3-hydroxymethyl aniline, H2O, 100 °C, 48 h,
21%; (b) 2,4-difluorobenzylbromide, acetone, Cs2CO3, 25 °C, 38%; (c) 2,4,6-trichloro-
1,3,5-triazine, 4-methylmorpholine, DMF, CH2Cl2, 2 h, 85%; (d) NH3, MeOH, rt, 24 h,
99%; (e) NBS, CH3CN, 25 °C, 0 °C ? rt, 1.5 h, 43%; (f) acetoxyacetyl chloride or
N-(tert-butoxycarbonyl)-glycine, PS-carbodiimide resin, HOBt, N-methylmorpho-
line, DMAC–CH2Cl2, rt, 4 h; (g) K2CO3, MeOH, 84%; (h) 4 N HCl–Dioxane, 24%; (i)
trimethylsilyl isocyante, N-methylmorpholine, THF, rt, 2 h, 93%.
human peripheral blood monocytes (hPBMC). Relative to 13, the
compounds in Table 6 exhibited improved stability upon incuba-
tion with either human or rat liver microsomes. The sole exception
being 27 which, presumably, suffered from oxidative demethyla-
tion of the N,N-dimethylamino group. Compounds 3, 36 and 42
exhibited remarkable metabolic stability while retaining cellular
activity.
and/or shorter pre-dose times. Compounds such as 8 and 12 (data
not shown for 12) exhibited modest inhibition at 5 mpk. In con-
trast to the Vertex series, the 2,6-disubstituted N-aryl pyridinone
analogs possessed excellent in vitro activity but suffered from a
lack of translation in vivo (with compound 9 being an exception).
The more stable compounds, such as 9 and 3, showed robust inhi-
bition at longer pre-dose times and lower doses (À4 h and 5 mpk).
Several exceptions to this trend included compounds 23 and 40.
This same set of compounds from above was evaluated in a rat
lipopolysaccharide (rLPS) model of inflammation (Table 6).10 The
rLPS model served as a convenient pharmacodynamic screen for
our team and allowed for the evaluation of compounds for their
duration of effect at various pre-dose time points prior to challenge
with LPS. A wide range of activity was observed and was consistent
with the trends established for metabolic stability. Not surpris-
ingly, the less stable compounds, 13 and 33, required higher doses

4064
S. R. Selness et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4059–4065
Table 6
Pharmacokinetics (male Sprague–Dawley rats) and pharmacodynamics (acute rat lipopolysaccharide model of inflammation) of selected N-aryl
pyridiones11
Compound
Rat pharmacokinetics (i.v. and p. o.)
Rat LPS model (p. o.)b
CL (mL/min/kg)
t
(h)
Suspension dose F (%)
Dose (mpk)
Predose time (@ Àh)
Inhibition of TNFa release (%)
½
13
8
9
26
2
23
27
34
36
3
—
—
1.9
1.8
8.9
1.5
—
20
5
5
5
5
5
15
5
5
5
2
4
6
4
4
4
4
4
4
4
4
66
38
83
93
81
44
69
43
64
89
70
28.7
12.8
4.0
5.9
—
—
—
—
9.34
8.1
19
21
19
32
—
—
—
—
—
—
—
—
3.4
3.1
7, 93a
3
42
5
a
Dosed in solution (i.v. 5 mpk and p.o. 5 mpk).
For assay conditions please see Ref. 10.
b
2008.; (b) Bemis, G. W.; Salituro, F. G.; Duffy, J. P.; Harrington, E. M. U.S. Patent
6,147,080, 2000.; (c) Colletti, S. L.; Frie, J. L.; Dixon, E. C.; Singh, S. B.; Choi, B. K.;
Scapin, G.; Fitzgerald, C. E.; Kumar, S.; Nichols, E. A.; O’Keefe, S. J.; O’Neill, E. A.;
Porter, G.; Samuel, K.; Schmatz, D. M.; Schwartz, C. D.; Shoop, W. L.; Thompson,
C. M.; Thompson, J. E.; Wang, R.; Woods, A.; Zaller, D. M.; Doherty, J. B. J. Med.
Chem. 2003, 46, 349; (d) Natarajan, S. R.; Heller, S. T.; Nam, K.; Singh, S. B.;
Scapin, G.; Patel, S.; Thompson, J. E.; Fitzgerald, C. E.; O’Keefe, S. J. Bioorg. Med.
Chem. Lett. 2006, 16, 5809.
The corresponding primary alcohols of 23 and 40 are potential sites
for phase II metabolism which is not detected typically in liver
microsomal assays.
The pharmcokinetic profiles of a select number of compounds
were determined in male Sprague–Dawley rat (i.v. and p.o. routes
of administration, Table 6). The data are consistent with the
in vitro RLM stability data as exemplified by 8 and 26. The more
stable 26 had a very low clearance (4.0 mL/min/kg) compared to
8 (28.7 mL/min/kg). The low to moderate bioavailabilities exhib-
ited by this class of compounds is primarily attributed to the
relatively low solubilities associated with these compounds
6. Full disclosure of earlier assay methods is made in the following patent
application: Devadas, Balekudru; Walker, John; Selness, Shaun, R.; Boehm, Terri
L.; Durley, Richard C.; Devraj, Rajesh; Hickory, Brian S.; Rucker, Paul V.; Jerome,
Kevin D.; Madsen, Heather M.; Alvira, Edgardo; Promo, Michele A.; Blevis-Bal,
Radhika M.; Marrufo, Laura D.; Hitchcock, Jeff; Owen, Thomas; Naing, Win;
Xing, Li; Shieh, Huey S.; Sambandam, Aruna; Liu, Shuang; Scott, Ian L.; Mcgee,
Kevin F. PCT Int. Appl. 2005, 968, pp. WO 2005018557.
(<10 lg/mL). This is supported by the oral suspension dosing com-
7. p38
was evaluated using a p38
p38 was determined by its ability to phosphorylate/activate unactivated MK2.
a
/MK2 cascade assay: The ability of compounds to inhibit activated p38
a
pared to oral solution dosing for 3. Crystalline 3, when dosed via
oral suspension, had very low bioavailability; however, oral solu-
tion dosing of this compound demonstrated high bioavailability.
This data suggests oral absorption is limited mainly by solubility,
not permeability or metabolism. Both 2 and 3 demonstrated
efficacy in a rat strep cell wall model of arthritis (33% and 40%
incidence, respectively, at 60 mpk/day).12 The overall kinase selec-
tivities of 2 and 3 were comparable to that previously reported for
the N-benzyl analog, 4.4,13
a/MK2 cascade assay format. The kinase activity of
a
Activation of MK2 by p38a was measured by following the phosphorylation of
a fluorescently-labelled, MK2 specific peptide substrate, Hsp27 peptide (FITC-
KKKALSRQLSVAA). The phosphorylation of the Hsp27 peptide was quantified
using the Caliper LabChip 3000. Kinase reactions were carried out in a 384-well
plate (Matrical, MP101-1-PP) in kinase buffer (20 mM HEPES pH 7.5, 10 mM
MgCl2, 0.0005% Tween-20, 0.01% BSA, 1 mM DTT, and 2% DMSO). The inhibitors
were varied between 0.2 and 10,000 nM, while the Hsp27 peptide substrate,
MgATP, and unactivated MK2 were held constant at 1, 10, and 1 nM,
respectively. Reactions were initiated by the addition of activated p38
a to a
final concentration of 6 pM. Kinase reactions were incubated at room
temperature and quenched after 60 minutes by the addition of stop buffer
(180 mM HEPES, 30 mM EDTA, and 0.2% Coating Reagent-3).
In summary, a series of potent, selective and stable N-aryl
pyridinone inhibitors of p38a were generated to address liabilities
8. In vitro cell activity: Human whole blood (HWB) was collected from two healthy
donors in sodium heparinized tubes (BD Biosciences, Franklin Lakes, NJ), and
PBMCs were isolated by Ficoll separation. Cells were washed in DPBS,
resuspended in DMEM containing 5% endotoxin-free fetal bovine serum and
associated with our previously described N-benzyl pyridinone ser-
ies. This series was metabolically stable in vitro and in vivo. These
compounds demonstrated significant activity in both acute (rLPS)
and chronic (rSCW) models of inflammation. Work in this that ser-
ies ultimately led to the identification of a clinical candidate which
will be disclosed in subsequent publications.
10
l
L penicillin-streptomycin, and plated at 2.5 Â 105 cells/well in 96-well
tissue culture plates. Cells were pretreated with increasing concentrations of
compound (0.0001–25
lipopolysaccharide (LPS, Sigma Aldrich, St. Louis, MO). Final Me2SO
concentration in cell assay was 0.25%. Secreted TNF- was measured by MSD
lM) for 1 h before the 18 h stimulation with 22 ng/ml
a
technology (MSD, Gaithersburg, MD). IC50s were determined using an internal
data analysis program (Pfizer, St. Louis).
References and notes
9. Metabolic stability was assessed in vitro by incubating 2 lM test compound
1. (a) Arend, W. P.; Dayer, J. M. Arthritis Rheum. 1990, 33, 305; (b) Firestein, G. S.;
Alvaro-Gracia, J. M.; Maki, R. J. Immunol. 1990, 144, 3347; (c) Rutgeerts, P.;
D’Haens, G.; Targan, S.; Vasiliauskas, E.; Hanauer, S. B.; Present, D. H.; Mayer, L.;
Van Hogezand, R. A.; Braakman, T.; DeWoody, K. L.; Shaible, T. F.; Van Deventer,
S. J. H. Gastroenterology 1999, 117, 761.
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.
10. Adult male Lewis rats (Harlan Sprague Dawley, Indianapolis, IN) (225–250 g)
were used in these studies. Rats were fasted 18 h prior to oral dosing, and
allowed free access to water throughout the experiment. Each treatment group
2. Ulfgren, A. K.; Andersson, U.; Engstrom, M.; Klareskog, L.; Maini, R. N.; Taylor, P.
C. Arthritis Rheum. 2000, 43, 2391.
consisted of five animals. 10 was prepared as
a suspension in a vehicle
3. (a) Foster, M. L.; Halley, F.; Souness, J. E. Drug News Perspect. 2000, 13, 488; (b)
Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T. F.; Kumar, S.; Green, D.;
MeNulty, D.; Blumenthal, M. J.; Heys, J. R.; Landvatter, S. W.; Strickler, J. E.;
McLaughlin, M. M.; Siemens, I. R.; Fisher, S. M.; Livi, G. P.; White, J. R.; Adams, J.
L.; Young, P. R. Nature 1994, 372, 739.
4. Selness, S. R.; Devraj, R. V.; Monahan, J. B.; Boehm, T. L.; Walker, J. K.; Devadas,
B.; Durley, R. C.; Kurumbail, R.; Shieh, H.; Xing, L.; Hepperle, M.; Jerome, K. D.;
Benson, A. G.; Marrufo, L. D.; Madsen, H. M.; Hitchcock, J.; Owen, T. J.; Christie,
L.; Promo, M. A.; Hickory, B. S.; Alvira, E.; Naing, W.; Blevis-Bal, R. Bioorg. Med.
Chem. Lett. 2009, 19, 5851.
consisting of 0.5% methylcellulose, (Sigma, St. Louis, MO), 0.025% Tween 20
(Sigma). The compound or vehicle was administered by oral gavage in a
volume of 1 mL. Two vehicle groups were used per experiment to control for
intra-experiment variability, and three experiments were performed. LPS
(E. coli serotype 0111:B4, Sigma) was administered two, four or six hours later
by intravenous injection at a dose of 1 mg/kg in 0.5 mL sterile saline (Baxter
Healthcare, Deerfield, IL). Blood was collected in serum separator tubes via
cardiac puncture ninety minutes after LPS injection,
corresponding to maximal TNF production (data not shown). After clotting,
serum was withdrawn and stored at À20 °C until it was assayed for TNF
TNF levels in serum were quantified from a recombinant rat TNF (Biosource
International) standard curve using a four parameter fit generated by an Excel
a time point
a
a.
5. (a) Bemis, G. W.; Salituro, F. G.; Duffy, J. P.; Cochran, J. E.; Harrington, E. M.;
Murcko,. M. A.;Wilson, K. P.; Su, M.; Galullo, V. P. U.S. Patent 7,365,072 B2,
a
a

S. R. Selness et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4059–4065
4065
(Microsoft, Redmond, WA) macro. The limit of detection for the ELISA was
approximately 41 pg TNF /mL.
11. 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.
plethysmometer. Each compound was prepared as an aqueous suspension in
0.5% methylcellulose and 0.025% Tween 20(sigma–aldrich). The compound
was administered by oral gavage in a volume of 0.5 mL beginning on day 10
post-SCW injection and continuing daily until day 21. Methylcellulose/Tween
20 vehicle was used for comparison. Group size was four to eight animals per
group. Two paw volumes were taken for each animal. Paw volume was
measured on day 21 by using a water displacement plethysmometer. Three to
four paws from each treatment group were scanned for bone density
evaluation. Plasma samples were collected on day 21 for determination of
compound levels.
a
12. Compounds 2 and 3 assayed in Streptococal Cell Wall (SCW) induced arthritis in
rats as follows: Arthritis was induced in female Lewis rats by
intraperitoneal administration of peptidoglycan-polysaccaride complexes
isolated from group a SCW (15 g/g bodyweight). The SCW preparation was
a single
l
purchase from Lee Labs (Grayson, GA). The disease course is biphasic in which
an acute inflammatory arthritis develops within days 1–3 (nonT-cell-
dependent phase) followed by a chronic erosive arthritis (T-cell- dependent
phase) developing on days 14–28. Only animals developing the acute phase
were treated with compound from days 10 to 21 after SCW injection. Paw
13. Compounds 2 and 3 exhibited less than 50% at 10 lM against over 200 targets
including, but not limited to, the following kinases: BTK, CDK2, AURKA, EGFR,
FLT1, ERK1, mTOR, IKKb, IRAK4, JAK1, JAK2, JAK3, TYK2, VEGFR2, LCK, MEK1,
p38d, p38
c
, ERK1, JNK, MK-2, TRKA, PDGFR, cRAF, ROCK1, ROCK2 and ZAP70.
and p38b.
The only significant inhibition observed was for p38
a
volume was measured on day 21 by using
a water displacement