Design, synthesis, and structure-activity relationships of aminopyridine N-oxides, a novel scaffold for the potent and selective inhibition of p38 mitogen activated protein kinase

Article abstract of DOI:10.1021/jm9008604

A novel series of aminopyridine N-oxides were designed, synthesized, and tested for their ability to inhibit p38α MAP kinase. Some of these compounds showed a significant reduction in the LPS-induced TNFR production in human whole blood. Structure-activit

Full text of DOI:10.1021/jm9008604

J. Med. Chem. 2009, 52, 5531–5545 5531
DOI: 10.1021/jm9008604
Design, Synthesis, and Structure-Activity Relationships of Aminopyridine N-Oxides, a Novel Scaffold
for the Potent and Selective Inhibition of p38 Mitogen Activated Protein Kinase†
‡
‡
‡
§
ꢀ §
Wenceslao Lumeras,*,‡ Francisco Caturla, Laura Vidal, Cristina Esteve, Cristina Balague, Adelina Orellana,
^
^
§
‡
ꢀ
ꢀ
Marıa Domınguez, Ramon Roca, Josep M. Huerta, Nuria Godessart, and Bernat Vidal
´ ´
‡Department of Medicinal Chemistry and §Department of Biology and Department of ADME and ^Department of Computational and Structural
Drug Discovery, Almirall Research Center, Almirall S.A., Ctra. Laureaꢁ Miroꢀ 408, E-08980 Sant Feliu de Llobregat, Barcelona, Spain
Received June 12, 2009
A novel series of aminopyridine N-oxides were designed, synthesized, and tested for their ability to
inhibit p38R MAP kinase. Some of these compounds showed a significant reduction in the LPS-induced
TNFR production in human whole blood. Structure-activity relationship studies revealed that N-oxide
oxygen was essential for activity and was probably a determinant factor for a marked selectivity against
other related kinases. Compound 45 was identified as a potent and selective p38R inhibitor with an
appropriate balance between potency and pharmacokinetics. In vivo efficacy of 45 was demonstrated in
reducing TNFR levels in an acute murine model of inflammation (ED50 = 1 mg/kg in LPS-induced
TNFR production when dosed orally 1.5 h prior to LPS administration). The oral efficacy of 45 was
further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when
administered orally (ED50 = 4.5 mg/kg).
Introduction
researchers into pursuing p38R inhibitors primarily as novel
anti-inflammatory drugs.8
The p38R mitogen activated protein (MAPa) kinase is an
intracellular serine/threonine (Ser/Thr) kinase that is acti-
vated by a range of environmental stimuli such as TNFR,
IL-1β, and stress.1,2 Activation of p38R occurs through
bisphosphorylation by the dual-specificity Ser/Thr MAP
kinases MKK3 and MKK6 on the Thr180-Gly181-Tyr182
motif located on the activation loop.2,3 In its activated state,
p38R phosphorylates a range of intracellular protein sub-
strates that post-transcriptionally regulate the biosynthesis
of TNFR and IL-1β. The pathophysiological consequence of
excessive production of TNFR and IL-1β is thought to be
significant mediation of the progression of many inflamma-
tory diseases such as rheumatoid arthritis, psoriasis, and
inflammatory bowel disease.4-7 The proven ability of p38R
MAP kinase to efficiently regulate both the release and the
activity of those pro-inflamatory cytokines has attracted
numerous pharmaceutical companies and independent
Since the first report in 1994 of the pyridylimidazole-based
p38R inhibitor SB203580(Figure1),9 several compounds have
advanced into clinical trials and some of them have dropped
out due to various reasons.10,11 Candidates from Vertex (VX-
745, Figure 1),12,13 Boehringer Ingelheim (BIRB-796),14,15
Pfizer (SD-006),16 Amgen (AMG-548),17,18 and Scios (SCIO-
469)19,20 have maintained, or in some cases improved, potency
and/or minimized liabilities that were identified in the original
pyridylimidazole series.
VX-745 was one of the first orally bioavailable discovered
compounds that progressed to phase IIb clinical trials. This
inhibitor was shown to have anti-inflammatory activity in
rodent models and established proof-of-principle in RA
patients21 before being discontinued due to issues associated
with adverse neurological effects in animal models.22
The prototypical p38R inhibitor SB203580 contained a
4-aryl 5-(4-pyridinyl) motif and was found to interact compe-
titively with the p38R ATP binding site. An example of the
binding orientation is shown in Figure 2, where key interac-
tions of this inhibitor with the active site of the enzyme are
depicted. Two key interactions with the hinge region connect-
ing the N- and C- terminal lobes of the kinase domain
are observed with this compound, as well as with most
known23 p38R inhibitors: a hydrogen bond through the
pyridyl nitrogen to the backbone amide NH of Met109 and
a significant lipophilic interaction of the inhibitor with the
deep hydrophobic pocket I. This pocket I (also called the
“selectivity pocket”) displayed in our proposed binding model
(Figure 2) is a region not occupied by the ATP when bound to
the kinase. It is known24 that the binding of an aryl group into
this pocket imparts selectivity for p38 against other related
kinases.
†Coordinates and structure factors have been deposited in the RCSB
Protein Data Bank (access code 3HRB for complex of p38R with 45).
*To whom correspondence should be addressed. Phone: 34 93 312 86
14. Fax: 34 93 312 86 35. E-mail: wenceslao.lumeras@almirall.com.
a Abbreviations: MAP, mitogen activated protein; TNFR, tumor
necrosis factor R; IL-1β, interleukin 1 β; MKK3, MAP kinase kinase-
3; MKK6, MAP kinase kinase-6; RA, rheumatoid arthritis; ERK1,
mitogen activated protein kinase 1; JNK1-3, c-Jun N-terminal kinase
1-3; MK2, MAP kinase activated protein kinase-2; COPD, chronic
obstructive pulmonary disease; TMEDA, tetramethylethylenediamine;
HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate; S-Phos, 2-dicyclohexylphosphino-20,60-dimethoxybi-
phenyl; LPS, lipopolysaccharide; THP-1, human acute monocytic
leukemia cell line; SAR, structure-activity relationships; HWB, human
whole blood; CHO, Chinese hamster ovarian; hERG, human ether-a-
go-go-related gene; HEK, human embryonic kidney; CI, confidence
interval; AIA, adjuvant-induced arthritis.
r
2009 American Chemical Society
Published on Web 08/14/2009
pubs.acs.org/jmc

5532 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Lumeras et al.
Many of the early compounds presented a diaryl hetero-
cyclic scaffold and have been shown to bind to the p38R active
site in an orientation similar to SB203580.25-28 Of the non-
vicinal diaryl scaffolds, Merck inhibitor 1 (Figure 3) has been
shown to be a potent and very selective inhibitor. This p38R
MAP kinase inhibitor displayed potent activity (p38R IC50
=
0.74 nM) and selectivity for p38R over a variety of other
closely related kinases, including ERK1 and JNK1-3
(>10000 fold).29 The exquisite selectivity of Merck’s com-
pound could be based on an optimum interaction with the
hydrophobic pocket I as well as on the ability of the carbonyl
oxygen of this ligand to establish two key hydrogen bond
contacts with both the backbone amide NH of Met109 and
the NH of Gly110, inducing a conformational change in the
hinge region of the enzyme, as depicted in Figure 4a. This
disposition can be clearly confirmed in the X-ray crystal-
lographic studies carried out with 1 in the active site of p38R.29
Although many kinases show a high degree of analogy at their
catalytic sites, this conformational flexibility is an almost
exclusive feature of p38R and p38β due to the presence of
the small Gly110. Other related kinases contain bulkier
residues on this position, which makes the conformational
change virtually impossible.29
Figure 1. Structure of some selected p38R MAP kinase inhibitors.
As part of our strategy to identify novel, potent, and
selective p38R MAP kinase inhibitors, we focused our interest
in the unprecedented level of selectivity exhibited in 1. This
compound is derived from a dihydroquinolinone core30,31 and
is characterized by the presence of two pendant aromatic rings
that are critical for the enzymatic potency. The combined fine-
tuned occupation of the hydrophobic pocket I with the
already mentioned conformational change (peptide flip of
the Gly110) approach provides a great opportunity to design
novel and selective p38R inhibitors, which should interact
within the catalytic site in a unique way.
Figure 2. Schematic representation of the interactions of prototy-
pical p38R inhibitor SB203580 in the active site of the enzyme, as
shown in X-ray complex 1A9U.23
With the aim of identifying a potent and selective p38R
inhibitor based on a simple core which displays a binding
mode similar to 1, we focused on published Bayer pyridinones
A (Figure 5). This original structure had been claimed by
Bayer scientists as p38 MAP kinase inhibitors for the treat-
ment of different inflammatory conditions (in particular
asthma and COPD), showing enzymatic inhibition values in
the submicromolar range.32,33 On the basis of this core, we
proposed an isosteric change, using a novel aminopyridine
N-oxide scaffold Bas a suitable alternative for the development
Figure 3. Merck’s p38R inhibitor 1.
Figure 4. (a) Schematic representation of key interactions of the dihydroquinolinone-based Merck’s inhibitor 1 in the active site of p38R,
derived from X-ray complex 1OVE.29 (b) Schematic drawing of predicted key interactions of the novel aminopyridine N-oxide inhibitor B in the
active site of p38R.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5533
Scheme 1a
Figure 5. Derivation of the novel aminopyridine N-oxide core B
from Bayer pyridinones A.32,33
of potential new p38R inhibitors (Figure 5). A plausible
intramolecular hydrogen bond between the carbonyl’s oxygen
and the amino group would arrange a pseudobicyclic scaffold
and place the rest of substituents in a suitable conformation
within the active site of the enzyme. As depicted in Figure 4b,
the N-oxide oxygen would be located in an appropriate
disposition to simultaneously interact with the backbone
amide NH of Met109 and the NH of Gly110 within the hinge
region, resulting in the described conformational change
(peptide flip of the Gly110) to reach good potency and an
improved selectivity profile, while the pendant aromatic rings
are assumed to make essential interactions with the enzyme by
occupying the two key hydrophobic regions.
a Reagents and conditions: (a) n-BuLi, TMEDA, Et2O, R1CHO, -78
to 0 ꢀC, 5 h, 35-65%. (b) MnO2, CHCl3, rt, 24-48 h, 85-100%. (c) HCl,
EtOH, 100 ꢀC, 6-24 h, 75-95%. (d) mCPBA, CH2Cl2, rt, 24-48 h,
70-90%. (e) POBr3, CH2Cl2, 60 ꢀC, 3 h, 40-60%. (f) Method A: R2-
B(OH)2 or 4,4,5,5-tetramethyl-2-R2-1,3,2-dioxaborolane, Pd2Cl2-
Chemistry
Aminopyridine N-oxides 34-61 were prepared following
the synthetic route depicted in Scheme 1. Starting from
N-(pyridin-3-yl)pivalamide 2,34 an ortho-directed lithiation
at the 4-position of the pyridine followed by subsequent
addition of the corresponding aldehydes gave carbinols
3a-e. Benzylic oxidation with MnO2 followed by the removal
of pivaloyl group in acidic media afforded ketones 4a-e,
which were converted into their corresponding N-oxides 5a-e
via a smooth oxidation with mCPBA. Treatment of the latter
with POBr3 furnished, in a regioselective way, key bromopyr-
idines 6a-e, which were reacted with the appropriate boronic
acids or esters (method A) under standard palladium-
mediated coupling conditions35,36 to afford compounds
7-33. For the preparation of 20 and 22, the arylation of
bromopyridine 6e with sterically hindered phenylboronic
acids was achieved in good to acceptable yields by using a
mixture of S-Phos and Pd2(dba)3 in toluene37 (method B). In
the case of 21 and 28, the introduction of the o,o-difluorophe-
nyl rings was successfully carried out by taking advantage of a
Negishi38,39 coupling reaction between 6e and the correspond-
ing in situ formed o,o-difluorophenyl zinc derivatives (method
C). Subsequent mCPBA oxidation at the pyridine nitrogen
cleanly provided N-oxides 34-61.
dppf DCM, 2M Cs2CO3, dioxane, 80-100 ꢀC, 18 h, 15-97%; method
3
B: R2-B(OH)2, Pd2(dba)3, S-Phos, K3PO4, toluene, 100 ꢀC, 18-72 h,
25-50%; method C: 1,3-difluorobenzene or 1,3-difluoro-5-methoxy-
benzene, n-BuLi, THF, -78 ꢀC, 30 min, then ZnCl2, THF, -50 ꢀC,
20 min, and then Pd(PPh3)4, THF, 40 ꢀC, 48-72 h, 21-93%; (g)
mCPBA, CH2Cl2, rt, 18 h, 40-100%.
Scheme 2a
a Reagents and conditions: (a) For 58: 4-(2-chloroethyl)morpholine
hydrochloride, 56, K2CO3, CH3CN, 80 ꢀC, 18 h, 54%. For 59: (2-
morpholin-4-ylethyl)amine, 57, HBTU, DIEA, DMF, rt, 18 h, <10%.
Alternatively, compounds 58 and 59 were synthesized in an
additional step by standard alkylation or amide formation
reactions, respectively, following the N-oxidation reaction
(Scheme 2) in order to avoid interferences with other basic
nitrogen atoms present in the molecule.
kinase. Some enzymatic inhibition was seen (p38R IC50
=
2.2 μM) and revealed 7 as an initial hit for further optimiza-
tion. To our delight, we could observe that the presence of an
oxygen attached to the nitrogen of the pyridine core
(aminopyridine N-oxide analogue 34, Table 1), which would
be responsible for the postulated interactions with the hinge
region, afforded a reasonable inhibitory potency in the enzy-
matic (p38R IC50 = 0.967 μM) and in the cellular (LPS-
induced TNFR release in THP-1 cells, IC50=1.63 μM) assays.
To our knowledge, this was the first example of a highly
Results and Discussion
On the basis of our initial design of a simple aminopyridine
template B (Figure 5), non-N-oxide compound 7 (precursor of
34) was tested for its ability to inhibit in vitro the p38R MAP

5534 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Lumeras et al.
Table 1. 3-Amino-2-aryl-4-benzoylpyridine 1-Oxides: R2 Group
Exploration
Table 2. 3-Amino-4-aroyl-2-(2-chlorophenyl)pyridine 1-Oxides: R1
Group Optimization
IC50 (nM)a obtained in the enzymatic and cellular
assays
IC50 (nM)a obtained in the enzymatic and cellular
assays
TNF-R
cell (THP-1)
TNF-R
HWB
TNF-R
cell (THP-1)
TNF-R
HWB
R1
p38R
compd
R2
p38R
compd
34
35
36
37
phenyl
967 (1.13)
174 (1.47)
411 (1.28)
2530 (1.60)
1633 (1.62) >10000
38
39
40
41
2-Cl-Ph
24 (1.26)
n.d.
152 (1.09)
>1000
nd
2-Cl-Ph
2-Me-Ph
4-Cl-Ph
168 (1.68)
705 (1.75)
1487 (2.44)
nd
2-MeO-Ph 149 (1.00)
2-CF3-Ph 1655 (1.77)
281 (1.51)
nd
75 (1.24)
nd
nd
2,4-diF-Ph
39 (1.33)
79 (1.09)
a Values are reported as the geometric mean of at least two indepen-
dent determinations along with the geometric SD (in brackets). nd = not
determined.
a Values are reported as the geometric mean of at least two indepen-
dent determinations along with the geometric SD (in brackets). nd = not
determined.
decorated 3-aminopyridine N-oxide scaffold to be identified
as an inhibitor of p38R MAP kinase.
inhibitor 1, exhibited good potency in both the enzymatic
(p38R IC50 = 39 nM) and cellular assays (TNFR in HWB
IC50=79 nM).
Preliminary SAR studies were developed in this series in
order to further explore its full potential. Our binding model
of B proposes a fit of the phenyl ring located into the
hydrophobic pocket II (Figure 4b), a relatively open solvent
exposed area. As demonstrated in Table 1, compounds 35 and
36, possessing ortho substituted phenyl rings in R2, were
markedly more potent than their unsubstituted analogue 34.
The role of this substituent seems to be the maintenance of the
ringin anappropriate(orthogonal) orientationwithrespect to
the aminopyridine core, increasing the lipophilic interactions
with the hydrophobic pocket II. The postulated binding
model is consistent with the observed SAR. Substitution at
the para position of the aryl ring by lipophilic groups with no
substitution at the ortho positions, as in 37, did not improve
the enzymatic inhibition (p38R IC50 = 2.53 μM).
The postulated binding model (Figure 4b) suggests that not
very large substituents would be tolerated in the para position
of the phenyl group in R1 due to its proximity toward the
hinge region.40 In contrast, relatively bulky substituents
would be allowed in the ortho position. To investigate the
effect of substitution on this group, a small number of
compounds (38-41) were synthesized, keeping the 2-Cl-
phenyl substitution pattern in R2, and their potency in the
enzymatic assay measured (Table 2). As postulated above, a
chlorine atom in the ortho position (38) was well tolerated and
significantly increased the p38 inhibitory potency (7-fold)
with respect to the R1 unsubstituted lead 35. This substituent
also afforded a substantial improvement (5-fold) with respect
to 35 in the LPS-induced TNFR production assay in human
wholeblood(HWB). Weputparticularemphasisonthisassay
as a key driver for compound selection41 because it provided
surrogateefficacy in a physiologically relevant environment in
the presence of serum albumin and other proteins.
After this preliminary exploration, the 2,4-difluorophenyl
ring was considered as an optimum R1 substitution pattern.
Taking this into consideration, a thorough analysis of the
SAR around the R2 moiety was thus carried out with the aim
of finding suitable substituents that would optimally fit the
hydrophobic pocket II, providing more potent analogues.
As we have previously mentioned, ortho substitution on the
R2 aryl groupispreferredover paraand meta, as canbeseenin
compounds 41-43 (Tables 2 and 3). The introduction of
bulkier groups in the ortho position resulted in a loss in
potency (analogue 46). Interestingly, the ortho substitution
by an electron-donating group, as in derivative 44, gave a very
potent compound in the enzymatic assay (IC50 = 23 nM) but
there was a clear shift in the cellular assay (TNFR in HWB
IC50 = 291 nM). However, 2-methyl analogue 45 displayed
good potency in both assays (p38R IC50 = 21 nM and TNFR
in HWB IC50 = 170 nM). We have already assessed that the
increased activity of these analogues is in agreement with the
fact that a substituent in ortho forces the orthogonality of the
rings, optimizing the lipophilic interactions with the hydro-
phobic pocket II.42-44 Following this premise, a series of 2,6-
disubstituted phenyl analogues in R2 were synthesized in
order to maximize the lipophilic interactions in this region.
Gratifyingly, we could observe that 2,6-dichlorophenyl ana-
logue 47 exhibited and excellent in vitro enzymatic potency
(p38R IC50 = 9 nM), 4-fold more potent than its monosub-
stituted analogue 41. Surprisingly, this compound suffered
from a considerable drop in potency in the cellular HWB
assay (77-foldloss). The presence ofthe smaller fluorineatoms
in 48 afforded the most potent compound of this series tested
in the cell-based assays (TNFR in HWB IC50 = 37 nM)
showing also a very good enzymatic activity (p38R IC50 = 17
nM). 2,6-Dimethylphenyl analogue 49 revealed a similar
trend to 48, with excellent values in all assays. In a comparable
manner to 44, electron-donating groups on the ring in 50 give
nice enzymatic values but fail again in the HWB assay (41-fold
drop). In general, the 2,6-disubstituted derivatives showed an
increase in the enzymatic potency with respect to their mono-
substituted analogues.
As the size of the ortho substituents on R1 notably in-
creased, there was a remarkable loss in potency (enzymatic
and cellular), as can be seen in compounds 39 and 40,
suggesting that this deep hydrophobic pocket is very sensitive
to accommodate larger substitutions on this ring.40 More
interestingly, compound 41, which combines two fluorine
substituents in the ortho and para positions, as in Merck

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5535
Table 3. 3-Amino-2-aryl-4-(2,4-difluorobenzoyl)pyridine-1-oxides: R2 Group Optimization
IC50 (nM)a obtained in the enzymatic and cellular assays
compd
R2
p38R
TNF-R cell (THP-1)
TNF-R HWB
42
43
44
45
46
47
48
49
50
51
52
53
54
55
3-Cl-Ph
4-Cl-Ph
2-MeO-Ph
228 (1.27)
802 (1.15)
23 (1.29)
21 (1.81)
208 (1.17)
9 (1.87)
1118 (1.08)
nd
nd
>1000
4460 (1.35)
291 (1.97)
170 (2.10)
>1000
2-Me-Ph
46 (1.43)
658 (1.52)
14 (2.37)
36 (1.16)
31 (1.83)
291 (1.58)
1483 (1.75)
215 (1.60)
nd
2-iPr-Ph
2,6-diCl-Ph
2,6-diF-Ph
692 (1.24)
37 (1.24)
50 (1.59)
616 (1.52)
>10000
17 (1.18)
17 (1.93)
15 (1.57)
37 (1.13)
24 (1.28)
126 (1.41)
38 (2.13)
5 (1.00)
2,6-diMe-Ph
2,6-diMeO-Ph
2,3-diMeO-Ph
1,3-benzodioxol-4-yl
2,4-diF-Ph
11076 (1.42)
283 (1.21)
470 (1.29)
1500 (1.07)
2-Me-4-Cl-Ph
2,6-diF-4-MeO-Ph
211 (1.08)
145 (1.05)
a Values are reported as the geometric mean of at least two independent determinations along with the geometric SD (in brackets). nd = not
determined.
Table 4. Solubility Values (μg/mL) of the Selected Compounds Mea-
sured at different pH
Table 5. 3-Amino-2-(2-methylphenyl)-4-(2,4-difluorobenzoyl)pyridine-1-
oxide: R3 Group Optimization
entry compda soln pH = 0.1 soln pH = 7.4 soln pH = 9.0
1
2
3
4
5
6
7
8
9
41
45
48
49
56
57
58
60
61
66 ( 6
110 ( 5
0 ( 0
40 ( 1
44 ( 6
59 ( 17
63 ( 1
4 ( 0
69 ( 6
37 ( 2
39 ( 4
74 ( 4
2 ( 0
70 ( 2
nd
41 ( 2
>1000 ( 17
24 ( 3
>1000 ( 10
51 ( 4
>1000 ( 3
nd
67 ( 1
46 ( 1
89 ( 3
49 ( 1
54 ( 2
a Results expressed as the mean of six independent determinations (
SD. nd = not determined.
Carrying out a variation at the phenyl substitution pattern,
it was seen that both the 2,3-disubstituted analogues 51 and 52
were potent against p38R activity but again showed a sig-
nificant shift in the cellular HWB assay. The 2,4-disubstitu-
tion pattern on the phenyl R2 ring offered a new approach to
interact with the hydrophobic pocket II. 2,4-Difluorophenyl
compound 53 displayed modest potency compared to its
2,6-difluorophenyl analogue 48 (7-fold drop in both assays).
A combination of methyl and chlorine in 54 gave a better p38
inhibitory potency (IC50 = 38 nM), but it was stillfar fromthe
2,6-disubstituted derivatives. Interestingly, the 2,4,6-trisubsti-
tuted compound 55 significantly increased the enzymatic
inhibitory activity, leading to the most potent molecule found
in the series (p38R IC50 = 5 nM); unfortunately, its lack of
potency in the HWB cellular assay (40-fold loss with respect to
48) meant that this compound was not characterized further.
Among all of the compounds synthesized up to this point,
analogues41, 45, 48, and49displayed the bestp38R inhibitory
and cellular (THP-1 and HWB) potency values. However,
these molecules exhibited rather limited solubility (Table 4,
entries 1-4), increasing the risk of a poor oral bioavailabil-
ity.45 For this reason, our next strategy focused on the
a Values are reported as the geometric mean of at least two indepen-
dent determinations along with the geometric SD (in brackets). nd = not
determined.
improvement of this physicochemical property, installing
either polar or ionizable functional groups directly or indir-
ectly attached (via an appropriate linker) to the para position
of the R2 aryl ring, pointing out toward the solvent exposed
area (Table 5). The presence of a hydroxyl group in 56 slightly
improved the enzymatic potency (2.6-fold compared to 45),
but there was a big drop in the cellular assays, particularly in

5536 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Lumeras et al.
Table 6. Pharmacokinetic Parameters of 41 and 45 in the Rat after Intravenous (iv) and Oral (po) Administrationa
dose po
(mg/kg)
dose iv
(mg/kg)
Cmax po
(μM)
tmax po
(h)
AUC0-6b po
Clc iv
(mL/min/kg)
Vssd iv
(L/kg)
compd
(μM h)
t
1/2 iv (h)
F0-6e (%)
41
45
10
10
1
0.5
11.9 ( 3.2
4.7 ( 0.3
3.5 ( 0.7
0.75 ( 0.3
54.4 ( 13
22.2 ( 4.7
14.8 ( 8
1.6 ( 0.02
1.7 ( 0
16.7 ( 3
1.8 ( 0.4
2.3 ( 0.1
23 ( 6
75 ( 1.3
a Results expressed as the mean ( SD of n = 2. b AUC = area under the curve. c Cl = clearance. d Vss = volume of distribution steady state. e F =
bioavailability.
Figure 6. X-ray overlay of compounds 45 (shown in light violet)46 and 1 (shown in yellow, taken from the published X-ray structure)29
complexed to p38R active site. The conformational change of Gly110 can be seen in both proteins, revealing significant hydrogen bond
interactions between Met109/Gly110 NHs and N-oxide oxygen (in 45) or amide carbonyl (in 1). Both compounds occupy in a similar manner
the deep hydrophobic pocket I (selectivity pocket) that is not accessed by ATP.
HWB (IC50 = 7 μΜ) and, to our surprise, displayed poorer
solubility values than its precursor 45 (Table 4, entry 5). When
a carboxylic acid was installed in this position (57), the
potency clearly decreased (24-fold loss in p38R with respect
to 45), although its solubility at neutral and basic pH drama-
tically increased (Table 4, entry 6). The insertion of a residue
containing a basic group (compounds 58 and 59), as expected,
led to a dramatic improvement in solubility at acidic pH
(Table 4, entry 7). Unfortunately, although 58 and 59 retained
or slightly improved the enzymatic activity, a tremendous
drop in the cellular assay potency was observed. On the basis
oftheseresults, theintroductionofnonbasic solubilizingpolar
substituents was considered. Unfortunately, solubility was
not increased (Table 4, entries 8 and 9) and the synthesized
derivatives 60 and 61 did not demonstrate noticeable advan-
tages with respect totheir basic counterparts 58 and 59. At this
point, we could speculate that the tremendous drop between
the enzymatic and HWB assays in 56-61 could be related to
the increased polarity of the compounds, although we were
not able to find a common trend to all the compounds of the
series.
Despite the efforts to increase the solubility, compounds 41
and 45 still displayed a useful compromise between desirable
physical properties (solubility) and potency in both enzymatic
and cellular (HWB) assays and were selected as suitable leads
for further characterization.
The pharmacokinetic parameters of compounds 41 and 45
in rat are summarized in Table 6. Compound 41 was slowly
absorbed (tmax = 3.5 h), showing very good oral exposure
(AUC0-6). The low values of clearance (Cl) and volume of
distribution in the steady state (Vss) translated into a very long
terminal half-life (t1/2 = 14.8 h). Moreover, this compound

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5537
Table 7. Summary of in Vivo Efficacy Assays
exhibited a modest bioavailability (F = 23%). On the
other hand, compound 45, with a more rapid absorption
(tmax =0.75 h) and still good oral exposure, displayed mod-
erate clearance and low volume of distribution in the steady
state, affording a shorter half-life (t1/2=1.6 h). Additionally,
analogue 45 exhibited a 3-fold increase in rat oral bioavail-
ability (F = 75%), which led to its selection as an advanced
lead for additional profiling.
rat LPS-induced TNF
ED50 (mg/kg)a
AIA ED50 (mg/kg) or %
inhibition
compd
34
45
8.4 (6-12)
1.03 (0.5-2)
7.9 (3-22)
nd
>10 (17% at 10 mg/kg)
4.5 (2-11)
8.6 (4-19)
>100
BIRB-796
VX-745
a Results represent the calculated ED50 along with the lower and
upper 95% confidence limits (in brackets). nd = not determined.
To confirm the proposed binding mode for our aminopyr-
idine N-oxide scaffold B (Figure 5), compound 45 was cocrys-
tallized with unphosphorylated p38R.46 Pleasantly, the X-ray
structure (Figure 6) revealed a novel binding mode according
to that previously proposed in Figure 4b and was consistent
with the structure activity relationships (SAR) generated. Of
particular interest were the two hydrogen bond interactions
generated between the N-oxide oxygen, the backbone amide
NH of Met109, and the NH of Gly110, resulting in the
described conformational change (“Gly flip”). Overlying
X-ray crystal structures of 4546 with that of Merck inhibitor
1 (Figure 6)28 clearly confirmed the similarities of the key
hydrogen bond interactions of both compounds with the
hinge region of the enzyme. More interestingly, the suggested
pseudobicyclic scaffold of B was nicely established in the
crystal structure of 45, with the formation of an intramolecular
hydrogen bond between the carbonyl oxygen and the amino
group, placing the pending aryl rings in a similar conformation
to those of 1 within the p38R active site.
The selectivity of 45 was assessed by the screening of this
compound against a panel of 21 tyrosine and serine/threonine
kinases.47 Impressively, 45 was found to be highly selective for
p38R/β over all tested kinases (e.g., c-Raf, JNK1-3, MK2,
IC50 > 10 μM).
Its ability to inhibit different cytochrome P450 isoforms
was also evaluated. Compound 45 did not inhibit any of the
most relevant P450 isoforms (1A2, 3A4, 2C9, 2C19, and 2D6)
at a concentration of 10 μM.
Compound 45 was tested for in vitro cytotoxicity against a
Chinese hamster ovarian (CHO) cell line, showing a clean
cytotoxic profile (IC50 > 200 μM).
Figure 7. Compound efficacy in the adjuvant-induced arthritis
model. Each point represents the mean ( SEM of individual values.
tested reference compounds like BIRB-796 and VX-745
when dosed in the same manner (Figure 7 and Table 7).
Conclusions
In relation to the cardiovascular safety package, analogue
45 did not have a significant effect on the inhibition of the
human ether-a-go-go-related gene (hERG) channel (4.7% at
3 μM) expressed in human embryonic kidney (HEK-293) cells.
In Vivo Pharmacological Evaluation. Key compounds were
tested in vivo in an acute model based on rat-LPS induced
TNFR (Table 7). In this model, compounds were dosed orally
one hour prior to LPS administration and the amount of
TNFR in plasma was measured 1.5 h later (coinciding with the
peak of TNFR production). Compound 34 dose-dependently
inhibited TNFR production with and ED50 similar to that
obtained for BIRB-796 reference compound. In contrast,
compound 45 was much more potent at inhibiting TNFR
production in this model with an ED50 of 1.03 mg/kg (95% CI,
0.5-2). These results are in accordance with those obtained in
enzymatic and cellular assays for both compounds.
The adjuvant-induced arthritis (AIA) model was selected
as a chronic disease model to evaluate compound efficacy
(Table 7). Upon arthritis induction, compounds were admi-
nistered orally once daily for 7 days. Compound 34 dosed at
10 mg/kg produced a slight inhibition (17%) of the contral-
ateral paw volumes (versus vehicle-treated animals). In con-
trast, 45 dose-dependently inhibited arthritis progression
with an ED50 of 4.5 mg/kg (95% CI, 2-11). These results
demonstrate a superior efficacy of 45 versus other clinically
The initial hypothesis of inducing a conformational change
in the hinge region of p38R to develop inhibitors with an
improved selectivity profile against other kinases has gener-
ated a variety of selective inhibitors based on a novel amino-
pyridine N-oxide scaffold. After an extensive SAR exercise,
compounds 41 and 45 were identified as potent p38R inhibi-
tors and were able to effectively inhibit the production of
TNFR in LPS-stimulated THP-1 cells as well as in human
whole blood. The proposed binding mode for this novel series
of inhibitors is consistent with the crystallography results
obtained from inhibitor 45 within the p38R active site. This
compound displayed an excellent selectivity for p38R/β versus
a panel of 21 related kinases as well as good pharmacokinetic
properties in Wistar rats.
In vivo pharmacological data for 45 confirms that this
compound is orally bioavailable and is active both in acute
and chronic inflammation models, clearly reflecting the con-
tribution of its potent cellular activity and the plasma levels
attained.
Experimental Section
Chemistry. Nonaqueous reactions were performed under
argon or nitrogen atmosphere at room temperature, unless
otherwise noted. All commercial reagents and anhydrous

5538 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Lumeras et al.
solvents were purchased from Aldrich and were used
without further purification or distillation unless otherwise
stated.
General Method for the Synthesis of N-{4-[(Aryl) hydroxy-
methyl]pyridin-3-yl}-2,2-dimethylpropanamides 6c and
6d. N-{4-[Hydroxy(2-methoxyphenyl)methyl]pyridin-3-yl}-2,2-
dimethylpropanamide (3c). n-BuLi (2.5 M in hexanes, 56 mL,
140.5 mmol) was dropwise added to a solution of 2,2-dimethyl-
N-pyridin-3-ylpropanamide (2) (10 g, 56.2 mmol) and N,N,
N0,N0-tetramethylethylenediamine (TMEDA) (20.93 mL,
140.5 mmol) in diethyl ether (338 mL) at -78 ꢀC under argon,
and the resulting mixture was stirred at that temperature for
15 min and at -10 ꢀC for 2 h. Then, the reaction mixture was
cooled down to -78 ꢀC, and 2-methoxybenzaldehyde (16.97
mL, 140.5 mmol) in 34 mL of dry tetrahydrofuran was carefully
added. After 15 min, the cooling bath was removed and the
mixture stirred at room temperature for 2 h. Subsequently,
water was added to the flask (400 mL) and it was extracted with
ethyl acetate (4 ꢀ 200 mL), the organic solution was washed with
brine, dried over sodium sulfate, and the solvent removed under
reduced pressure. The residue was treated with EtOAc (50 mL)
and stored at 4 ꢀC for 18 h. The final compound crystallized out
to yield the title compound (11.1 g, 63%) as a solid. LCMS (m/
z): 315 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 8.98 (s, 1H),
8.45 (brs, 1H), 8.30 (d, J = 5 Hz, 1H), 7.42-7.55 (m, 1H),
7.25-7.30 (m, 1H), 6.90-7.10 (m, 2H), 7.00 (d, J = 5 Hz, 1H),
6.05 (s, 1H), 3.75 (s, 3H), 1.12 (s, 9H).
N-{4-[Hydroxy(2-trifluoromethylphenyl)methyl]pyridin-3-yl}-
2,2-dimethyl-propanamide (3d). This compound was prepared
from 2,2-dimethyl-N-pyridin-3-ylpropanamide (2) and 2-tri-
fluoromethylbenzaldehyde as described in the synthesis of 3c.
Yield: 57%. LCMS (m/z): 353 (M þ 1)þ. 1H NMR (CDCl3, 300
MHz) δ 9.00 (s, 1H), 8.40 (brs, 1H), 8.35 (d, J = 5 Hz, 1H),
7.75-7.80 (m, 1H), 7.60-7.70 (m, 2H), 7.30-7.40 (m, 1H), 6.95
(d, J = 5 Hz, 1H), 6.05 (s, 1H), 1.10 (s, 9H).
General Method for the Synthesis of (3-Aminopyridin-4-yl)-
(aryl)methanones 4a-e. (3-Aminopyridin-4-yl)(phenyl)methanone
(4a). Compound 3a (2.16 g, 7.6 mmol) was dissolved in chloro-
form (65 mL) and activated manganese(IV) oxide (6.61 g,
76 mmol) was portionwise added during 1 h. The suspension
was stirred at room temperature for 16 h. The mixture was filtered
through celite, washed with more chloroform, and the solvent
evaporated to affordthe corresponding oxidized derivative, which
was directly dissolved in 23 mL of ethanol, treated with HCl 5N
(70 mL), and heated to 98 ꢀC for 6 h. The reaction mixture was
cooled down, poured into ice water, and the pH adjusted to 9-10
with concentrated aqueous ammonia. The solution was extracted
with ethyl acetate (2 ꢀ 250 mL), the organic layer was washed with
brine, dried over sodium sulfate, and the solvent removed under
reduced pressure. The residue was triturated with hexane/diethyl
ether (5:1) to yield the corresponding compounds 4a (0.9 g, 60%)
as a yellowish solid. LCMS (m/z): 199 (M þ 1)þ. 1H NMR
(CDCl3, 300 MHz) δ 8.35 (s, 1H), 7.95 (d, J = 5 Hz, 1H),
7.40-7.75 (m, 5H), 7.15-7.25 (m, 1H), 5.90 (brs, 2H).
Routine 1H nuclear magnetic resonance spectra were recorded
on the following instruments: Varian Gemini 300 MHz, in a
Varian Mercury plus NMR spectrometer operating at a frequency
of 200 MHz or in a Varian Mercury plus NMR spectrometer
at 400 MHz. Samples were dissolved in deuterated chloroform
(CDCl3) or deuterated methylsulfoxide (DMSO-d6) and tetra-
methylsilane (TMS) was used as reference.
Analytical thin-layer chromatography (TLC) was performed
on Merck silica gel 60 F254. Compounds were visualized by UV
light and/or stained with either potassium permanganate or
cerium molybdate solutions followed by heating. Flash column
chromatography was performed on SDS silica gel 60 (particle
size of 40-63 μm).
HPLC analysis was performed on a Waters Alliance 2795
chromatographer equipped with a Waters 2996 diode-array
detector and a Waters ZQ mass spectrometer detector. HPLC
analysis was conducted following the next method: chromato-
graphy performed on a Symmetry C18 column (100 mm ꢀ
2.16 mm, 3.5 μm). The mobile phase, at a flow of 0.4 mL/min,
was a 20 min binary gradient of water (containing 0.01 M
ammonium formate at pH 3.0) and a mixture acetonitri-
le-methanol 50:50 (containing 0.01 M ammonic formiate
0.01M) (0-95%). The total run time was 26 min. The retention
time (rt) is expressed in min and UV chromatograms were
processed at 210 nm with blank subtraction. All key compounds
were proven by this method to show g95% purity. Additionally,
elemental analyses were performed for key compounds. Ele-
mental analyses were performed in FISONS EA-1108 CHNS
analyzer using acetanilide as standard. A table summarizing
key target compounds can be found at the Supporting
Information.
General Method for the Synthesis of N-{4-[(Aryl) hydroxy-
methyl]pyridin-3-yl}-2,2-dimethylpropanamides 3a, 3b, and 3e.
N-{4-[Hydroxy(phenyl)methyl]pyridin-3-yl}-2,2-dimethylpro-
panamide (3a). n-BuLi (2.5 M in hexanes, 11.2 mL, 28 mmol) was
dropwise added to a solution of 2,2-dimethyl-N-pyridin-3-ylpro-
panamide (2) (2 g, 11.2 mmol) in dry tetrahydrofuran (28 mL) at
-78 ꢀC under argon and the resulting mixture was stirred at that
temperature for 15 min and at 0 ꢀC for 3 h. Then, the reaction
mixture was cooled down to -78 ꢀC and benzaldehyde (1.72 mL,
16.8 mmol) in 2.8 mL of tetrahydrofuran was carefully added.
After 15 min, the cooling bath was removed and the mixture
stirred overnight at room temperature. Subsequently, water was
added to the flask and it was extracted with ethyl acetate (3 ꢀ 50
mL), the organic solution was washed with brine, dried over
sodium sulfate, and the solvent removed under reduced pressure.
The residue was purified by column chromatography on silica
flash, using hexane/ethyl acetate (1:2 to ethyl acetate) as eluents, to
yield the title compound (2.16 g, 54%) as a solid. LCMS (m/z): 285
(M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 9.35 (brs, 1H), 8.82 (s,
1H), 8.30 (d, J = 5 Hz, 1H), 7.40 (d, J = 5 Hz, 1H), 7.18-7.38 (m,
5H), 6.78 (brs, 1H), 5.85 (s, 1H), 1.05 (s, 9H).
(3-Aminopyridin-4-yl)(2-chlorophenyl)methanone (4b). This
compound was prepared from 3b as described in the synthesis
of 4a. Yield: 92%. LCMS (m/z): 233, 235 (M þ 1)þ. 1H NMR
(CDCl3, 300 MHz) δ 8.31 (s, 1H), 7.86 (d, J = 6 Hz, 1H),
7.31-7.50 (m, 4H), 6.95 (d, J = 6 Hz, 1H), 6.31 (brs, 2H).
(3-Aminopyridin-4-yl)(2-methoxyphenyl)methanone (4c). This
compound was prepared from 3c as described in the synthesis of
4a. Yield: 84%. LCMS (m/z): 229 (M þ 1)þ. 1H NMR (CDCl3,
300 MHz) δ 8.25 (s, 1H), 7.85 (d, J = 5 Hz, 1H), 7.40-7.55 (m,
1H), 7.25-7.35 (m, 1H), 7.00-718 (m, 3H), 6.20 (brs, 2H), 3.78
(s, 3H).
(3-Aminopyridin-4-yl)[2-(trifluoromethyl)phenyl]methanone (4d).
This compound was prepared from 3d as described in the synthesis
of 4a. Yield: 78%. LCMS (m/z): 267 (M þ 1)þ. 1H NMR (CDCl3,
300 MHz) δ 8.28 (s, 1H), 7.77-7.83 (m, 2H), 7.59-7.69 (m, 2H),
7.34-7.38 (m, 1H), 6.83 (d, J = 5 Hz, 1H), 6.38 (brs, 2H).
(3-Aminopyridin-4-yl)(2,4-difluorophenyl)methanone (4e). This
compound was prepared from 3e as described in the synthesis of
4a. Yield: 74%. LCMS (m/z): 235 (M þ 1)þ. 1H NMR (CDCl3,
N-{4-[(2-Chlorophenyl) (hydroxy) methyl]pyridin-3-yl}-2,2-di-
methylpropanamide (3b). This compound was prepared from
2,2-dimethyl-N-pyridin-3-ylpropanamide (2) and 2-chloroben-
zaldehyde as described in the synthesis of 3a. Yield: 33%. LCMS
(m/z): 319, 321 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 9.10 (s,
1H), 8.21 (d, J = 6 Hz, 1H), 7.49-7.53 (m, 1H), 7.30-7.43 (m,
4H), 6.91 (d, J = 6 Hz, 1H), 6.15 (s, 1H), 1.31 (s, 9H).
N-(4-((2,4-Difluorophenyl)(hydroxy)methyl)pyridin-3-yl)-2,2-
dimethyl-propanamide (3e). This compound was prepared from
2,2-dimethyl-N-pyridin-3-ylpropanamide (2) and 2,4-difluoro-
benzaldehyde as described in the synthesis of 3a. Yield: 57%.
LCMS (m/z): 321 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 9.11
(s, 1H), 8.95 (brs, 1H), 8.18 (d, J = 5 Hz, 1H), 7.30-7.40 (m,
1H), 6.95 (d, J = 5 Hz, 1H), 6.80-7.00 (m, 2H), 6.08 (s, 1H),
1.27 (s, 9H).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5539
300 MHz) δ 8.35 (s, 1H), 7.90 (d, J = 6 Hz, 1H), 7.40-7.58 (m,
1H), 6.90-7.10 (m, 3H), 6.20 (brs, 2H).
General Method for the Synthesis of (3-Amino-1-oxidopyridin-
(CDCl3, 300 MHz) δ 7.78-7.83 (m, 1H), 7.61-7.68 (m, 3H),
7.33-7.37 (m, 1H), 6.91 (brs, 2H). 6.87 (d, J = 6 Hz, 1H).
(3-Amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone
(6e). This compound was prepared from 5e following the
experimental procedure described for the synthesis of 6a. Yield:
52%. LCMS (m/z): 313, 315 (M þ 1)þ. 1H NMR (CDCl3,
300 MHz) δ 7.70 (d, J = 6 Hz, 1H), 7.46-7.54 (m, 1H), 7.12
(dd, J = 2, 4 Hz, 1H), 6.88-7.09 (m, 2H), 6.75 (brs, 2H).
General Procedure for the Synthesis of (3-Amino-1-oxido-2-
arylpyridin-4-yl)(aryl)methanones 7-33. Method A. In a Schlenk
tube were charged the compounds 6a-e (0.50 mmol), the corre-
sponding boronic acids, or 4,4,5,5-tetramethyl-2-aryl-1,3,2-diox-
aborolanes (0.75 mmol), cesium carbonate (2 M aqueous solution,
1.50 mmol), and dioxane (1.4 mL). The mixture was submitted to
three vacuum-argon cycles, and then [1,10-bis(diphenyl-
phosphino)ferrocene]dichloropalladium(II) complex with dichlo-
romethane (1:1) (0.035 mmol) was added and the mixture purged
in the same way. The reaction was stirred at 80 ꢀC under argon for
17 h. Subsequently, water was added to the cold reaction mixture
and it was extracted with ethyl acetate (3 ꢀ 50 mL), the organic
solution was washed with brine, dried over sodium sulfate, and the
solvent removed under reduced pressure. The residue was purified
by column chromatography on silica flash, using a mixture of
hexanes and ethyl acetate as eluent, to yield the compounds 7-19,
24-27, 29, and 30.
Method B. In a Schlenk tube were charged the compounds
6a-e (0.50 mmol), the corresponding boronic acids (1.00 mmol),
potassium carbonate (1.50 mmol), and toluene (4 mL). The
mixture was submitted to three vacuum-argon cycles, and then
S-PHOS (0.030 mmol) and tris(dibenzylideneacetone)dipall-
adium(0) (0.015 mmol) were added and the mixture purged in
the same way. The reaction was stirred at 100 ꢀC under argon for 2
days. Subsequently, water was added to the cold reaction mixture
and it was extracted with ethyl acetate (3 ꢀ 50 mL), the organic
solution was washed with brine, dried over sodium sulfate, and the
solvent removed under reduced pressure. The residue was purified
by column chromatography on silica flash, using a mixture of
hexanes and ethyl acetate as eluent to afford compounds 20, 22,
and 23.
Method C. n-BuLi (2.5 M in hexanes, 1.20 mmol) was drop-
wise added to a solution of the corresponding benzene derivative
(1.30 mmol) in dry tetrahydrofuran (2 mL) at -78 ꢀC under
argon, and the resulting mixture was stirred at that temperature
for 30 min. Then, the reaction mixture was warmed up to -50 ꢀC
and ZnCl2 (0.5 M in THF, 1.30 mmol) carefully added. After
20 min, the corresponding compound 6a-e (0.65 mmol, in
1.5 mL of THF) and tetrakis(triphenylphosphine)palladium(0)
(0.061 mmol) were sequentially added. The mixture was then
submitted to three vacuum-argon cycles and warmed, first to
room temperature for 15 min and then to 40 ꢀC for 48 h. After
this time, the reaction was cooled down and the solvent evapo-
rated under reduced pressure. The resulting crude was purified
by column chromatography on silica flash using a mixture of
hexanes and ethyl acetate as eluent to yield the compounds 21
and 28.
(3-Amino-2-phenylpyridin-4-yl)(phenyl)methanone (7). This
compound was prepared starting from (3-amino-2-bromopyr-
idin-4-yl)(phenyl)methanone (6a) and phenylboronic acid fol-
lowing the general method A. Yield: 72%. LCMS (m/z): 275 (M
þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 8.07 (d, J = 5.3 Hz, 1H),
7.4-7.81 (m, 9H), 7.21 (d, J = 6.7 Hz, 2H), 6.1 (brs, 2H).
[3-Amino-2-(2-chlorophenyl)pyridin-4-yl](phenyl)methanone (8).
This compound was prepared starting from (3-amino-2-bromo-
pyridin-4-yl)(phenyl)methanone (6a) and (2-chlorophenyl)boro-
nic acid following the general method A. Yield: 67%. LCMS (m/z):
309-311 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 8.07 (d, J =
5.0 Hz, 1H), 7.53-7.59 (m, 1H), 7.38-7.49 (m, 7H), 7.26 (m, 1H),
7.05 (d, J = 5.0 Hz, 1H), 6.1 (brs, 2H).
4-yl)(aryl)methanones
5a-e. (3-Amino-1-oxidopyridin-4-yl)-
(phenyl)methanone (5a). To a solution of 4a (800 mg, 4 mmol)
in dichloromethane (20 mL) at 0 ꢀC was added portionwise
meta-chloroperbenzoic acid (6 mmol) and the reaction mixture
was stirred overnight at room temperature. Then, more dichloro-
methane was added (50 mL) and the solution was washed with
aqueous sodium bicarbonate 4% (3 ꢀ 30 mL) and brine. The
organic layer was dried over sodium sulfate and concentrated
under reduced pressure to give a residue that was triturated in a
mixture of hexane and ethyl acetate (9:1) and filtered to afford 5a
(778 mg, 90%) as a yellow solid. LCMS (m/z): 215 (M þ 1)þ. 1H
NMR (CDCl3, 300 MHz) δ 7.85 (s, 1H), 7.42-7.65 (m, 6H), 7.35
(d, J = 6 Hz, 1H), 6.35 (brs, 2H).
(3-Amino-1-oxidopyridin-4-yl)(2-chlorophenyl)methanone (5b).
This compound was prepared from 4b as described in the synthesis
1
of 5a. Yield: 88%. LCMS (m/z): 249, 251 (M þ 1)þ. H NMR
(DMSO-d6, 300 MHz) δ 7.90 (d, J = 4 Hz, 1H), 7.64 (brs, 2H),
7.45-7.57 (m, 4H), 7.31 (dd, J = 2 and 6 Hz, 1H), 6.86, (d, J = 6
Hz, 1H).
(3-Amino-1-oxidopyridin-4-yl)(2-methoxyphenyl)methanone (5c).
This compound was prepared from 4c as described in the synthesis
of 5a. Yield: 80%. LCMS (m/z): 245 (M þ 1)þ. 1H NMR (CDCl3,
300 MHz) δ 7.80 (s, 1H), 7.40-7.55 (m, 2H), 7.15-7.20 (m, 1H),
6.95-7.08 (m, 3H), 6.50 (brs, 2H), 3.78 (s, 3H).
(3-Amino-1-oxidopyridin-4-yl)[2-(trifluoromethyl)phenyl]-
methanone (5d). This compound was prepared from 4d as
described in the synthesis of 5a. Yield: 90%. LCMS (m/z): 283
(M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.82 (s, 1H), 7.77-7.83
(m,2H), 7.59-7.69(m,2H), 7.34-7.38(m,1H), 6.90(d, J = 5Hz,
1H), 6.58 (brs, 2H).
(3-Amino-1-oxidopyridin-4-yl)(2,4-difluorophenyl)methanone (5e).
This compound was prepared from 4e as described in the synthesis
of 5a. Yield: 80%. LCMS (m/z): 283 (M þ 1)þ. 1H NMR (CDCl3,
300 MHz) δ 7.82 (s, 1H), 7.77-7.83 (m, 2H), 7.59-7.69 (m, 2H),
7.34-7.38 (m, 1H), 6.90 (d, J = 5 Hz, 1H), 6.58 (brs, 2H).
General Method for the Synthesis of (3-Amino-2-bromopyr-
idin-4-yl)(aryl)methanones 6a-e. (3-Amino-2-bromopyridin-4-
yl)(phenyl)methanone (6a). (3-Amino-1-oxidopyridin-4-yl)-
(phenyl)methanone (5a) (520 mg, 2.43 mmol) was dissolved in
15 mL of dry dichloromethane and phosphorus oxybromide
(2.08 g, 7.28 mmol) added portionwise. The mixture was stirred
at 60 ꢀC for 3 h. The reaction was cooled down, poured into ice
water, and the pH adjusted to 10-11 with concentrated aqueous
ammonia. The solution was extracted with ethyl acetate (2 ꢀ 200
mL), the organic layer was washed with brine, dried over sodium
sulfate, and the solvent removed under reduced pressure. The
residue was purified by column chromatography on silica flash,
using hexane/ethyl acetate (3:1) as eluents, to yield 6a (390 mg,
58%) as a bright-yellow solid. LCMS (m/z): 277, 279 (M þ 1)þ.
1H NMR (CDCl3, 300 MHz) δ 7.40-7.78 (m, 6H), 7.22 (d, J = 6
Hz, 1H), 6.40 (brs, 2H).
(3-Amino-2-bromopyridin-4-yl)(2-chlorophenyl)methanone (6b).
This compound was prepared from 5b following the experimental
procedure described for the synthesis of 6a. Yield: 57%. LCMS
(m/z): 311, 313, 315 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.65
(d, J = 4 Hz, 1H), 7.29-7.49 (m, 4H), 6.98 (d, J = 4 Hz, 1H), 6.91
(brs, 2H).
(3-Amino-2-bromopyridin-4-yl)(2-methoxyphenyl)methanone (6c).
This compound was prepared from 5c following the experimental
procedure described for the synthesis of 6a. Yield: 61%. LCMS
(m/z): 307, 309 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.62 (d,
J = 6 Hz, 1H), 7.42-7.58 (m, 1H), 7.27-7.35 (m, 1H), 6.99-7.12
(m, 3H), 6.80 (bs, 2H).
(3-Amino-2-bromopyridin-4-yl)(2-trifluoromethylphenyl)-
methanone (6d). This compound was prepared from 5d following
the experimental procedure described for the synthesis of
6a. Yield: 49%. LCMS (m/z): 345, 347 (M þ 1)þ. 1H NMR
[3-Amino-2-(2-methylphenyl)pyridin-4-yl](phenyl)methanone (9).
This compound was prepared starting from (3-amino-2-bromo-

5540 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Lumeras et al.
pyridin-4-yl)(phenyl)methanone (6a) and (2-methylphenyl)boro-
nic acid following the general method A. Yield: 78%. LCMS (m/z):
7.31-7.37 (m, 4H), 7.13 (dd, J = 5.1 and 2.7 Hz, 1H), 6.91-7.09
(m, 2H), 6.17 (brs, 2H), 2.21 (s, 3H).
1
289 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ 8.03 (d, J =
[3-Amino-2-(2-isopropylphenyl)pyridin-4-yl](2,4-difluorophenyl)-
methanone (19). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone (6e)
and (2-isopropylphenyl)boronic acid following the general method
5.3 Hz, 1H), 7.47-7.80 (m, 5H), 7.33 (m, 4H), 5.83 (brs, 2H), 2.23
(s, 3H).
[3-Amino-2-(4-chlorophenyl)pyridin-4-yl](phenyl)methanone (10).
This compound was prepared starting from (3-amino-2-bromopyr-
idin-4-yl)(phenyl)methanone (6a) and (4-chlorophenyl)boronic
acid following the general method A. Yield: 71%. LCMS (m/z):
309-311 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 8.07 (d, J =
5.0 Hz, 1H), 7.46-7.80 (m, 9H), 7.26 (m, 1H), 6.07 (brs, 2H).
[3-Amino-2-(2-chlorophenyl)pyridin-4-yl](2-chlorophenyl)-
methanone (11). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2-chlorophenyl)methanone (6b)
and (2-chlorophenyl)boronic acid following the general method
A. Yield: 43%. LCMS (m/z): 343-345-347 (M þ 1)þ. 1H NMR
(CDCl3, 300 MHz) δ 7.98 (d, J = 6.0 Hz, 1H), 7.53-7.59 (m, 1H),
7.38-7.49 (m, 7H), 7.05 (d, J = 6.0 Hz, 1H), 6.30 (brs, 2H).
[3-Amino-2-(2-chlorophenyl)pyridin-4-yl](2-methoxyphenyl)-
methanone (12). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2-methoxyphenyl)methanone
(6c) and (2-chlorophenyl)boronic acid following the general
1
A. Yield: 80%. LCMS (m/z): 353 (M þ 1)þ. H NMR (CDCl3,
300 MHz) δ 8.00 (d, J = 5.5 Hz, 1H), 7.24-7.59 (m, 5H), 7.13 (dd,
J = 5.4 and 2.7 Hz, 1H), 6.91-7.09 (m, 2H), 6.16 (brs, 2H), 2.77
(hept, J=7.1 Hz, 1H), 1.21 (d, J=7.0 Hz, 3H), 1.16 (d, J=7.1 Hz,
3H).
[3-Amino-2-(2,6-dichlorophenyl)pyridin-4-yl](2,4-difluorophenyl)-
methanone (20). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone (6e)
and (2,6-dichlorophenyl)boronic acid following the general method
1
B. Yield: 50%. LCMS (m/z): 379, 381, 383 (M þ 1)þ. H NMR
(CDCl3, 300 MHz) δ 8.09 (d, J = 5.4 Hz, 1H), 7.33-7.61 (m, 4H),
7.23 (dd, J = 5.5 and 3.1 Hz, 1H), 6.91-7.10 (m, 2H), 6.06 (brs,
2H).
[3-Amino-2-(2,6-difluorophenyl)pyridin-4-yl](2,4-difluorophenyl)-
methanone (21). This compound was prepared starting from (3-
amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone (6e)
and 2,6-difluorobenzene following the general method C. Yield:
88%. LCMS (m/z): 347 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ
8.08 (d, J = 5.5 Hz, 1H), 7.39-7.59 (m, 2H), 7.22 (dd, J = 5.4 and
3.1 Hz, 1H) 6.93-7.14 (m, 4H), 6.20 (brs, 2H).
1
method A. Yield: 60%. LCMS (m/z): 339-341 (M þ 1)þ. H
NMR (CDCl3, 300 MHz) δ 7.98 (d, J = 6.0 Hz, 1H), 7.55-7.60
(m, 1H), 7.10-7.50 (m, 7H), 7.05 (d, J = 6.0 Hz, 1H), 6.30 (brs,
2H).
[3-Amino-2-(2-chlorophenyl)pyridine-4-yl](2-trifluoromethyl-
phenyl)methanone (13). This compound was prepared starting
from (3-amino-2-bromopyridin-4-yl)(2-trifluoromethylphenyl)-
methanone (6d) and (2-chlorophenyl)boronic acid following the
general method A. Yield: 72%. LCMS (m/z): 377-379 (M þ 1)þ.
1H NMR (CDCl3, 300 MHz) δ 7.98 (d, J = 6.0 Hz, 1H),
7.53-7.59 (m, 1H), 7.38-7.49 (m, 7H), 7.05 (d, J = 6.0 Hz,
1H), 6.30 (brs, 2H).
[3-Amino-2-(2,6-dimethylphenyl)pyridin-4-yl](2,4-difluorophenyl)-
methanone (22). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone (6e)
and (2,6-dimethylphenyl)boronic acid following the general method
1
B. Yield: 44%. LCMS (m/z): 339 (M þ 1)þ. H NMR (CDCl3,
300 MHz) δ 8.04 (d, J = 5.5 Hz, 1H), 7.55 (m, 1H), 6.91-7.30 (m,
6H) 6.08 (brs, 2H), 2.08 (s, 6H).
[3-Amino-2-(2,6-dimethoxyphenyl)pyridin-4-yl](2,4-difluorophe-
nyl)methanone (23). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone (6e)
and (2,6-dimethoxyphenyl)boronic acid following the general
[3-Amino-2-(2-chlorophenyl)pyridin-4-yl](2,4-difluorophenyl)-
methanone (14). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone
(6e) and (2-chlorophenyl)boronic acid following the general
1
method B. Yield: 44%. LCMS (m/z): 371 (M þ 1)þ. H NMR
1
method A. Yield: 49%. LCMS (m/z): 345-347 (M þ 1)þ. H
(CDCl3, 300 MHz) δ 8.03 (d, J = 4 Hz, 1H), 7.43-7.57 (m, 1H),
7.40 (t, J= 8 Hz, 1H), 7.10 (dd, J =4 and 2 Hz, 1H), 6.90-7.05 (m,
2H), 6.70 (d, J = 8 Hz, 2H), 6.20 (brs, 2H), 3.76 (s, 6H).
NMR (CDCl3, 300 MHz) δ 8.00 (d, J = 6.0 Hz, 1H), 7.88 (m,
1H), 7.47-7.75 (m, 4H), 7.18 (d, J = 5.5 and 2.8 Hz, 1H),
6.92-7.10 (m, 2H), 6.02 (brs, 2H).
[3-Amino-2-(2,3-dimethoxyphenyl)pyridin-4-yl](2,4-difluorophe-
nyl)methanone (24). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone (6e)
and (2,3-dimethoxyphenyl)boronic acid following the general
[3-Amino-2-(3-chlorophenyl)pyridine-4-yl](2,4-difluorophenyl)-
methanone (15). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone
(6e) and (3-chlorophenyl)boronic acid following the general
1
method A. Yield: 84%. LCMS (m/z): 371 (M þ 1)þ. H NMR
1
method A. Yield: 71%. LCMS (m/z): 345-347 (M þ 1)þ. H
(CDCl3, 300 MHz) δ8.00 (d, J= 5.1 Hz, 1H), 7.53 (m, 1H), 7.20 (d,
J = 8.2 Hz, 1H), 7.14 (dd, J = 5.5 and 3.2 Hz, 1H), 6.90-7.09 (m,
4H), 6.36 (brs, 2H), 3.94 (s, 3H), 3.71 (s, 3H).
NMR (CDCl3, 300 MHz) δ 8.01 (d, J = 5.5 Hz, 1H), 7.67 (m,
1H), 7.43-7.60 (m, 4H), 7.13 (dd, J = 5.1 and 2.8 Hz, 1H),
6.90-7.08 (m, 2H), 6.44 (brs, 2H).
[3-Amino-2-(1,3-benzodioxol-4-yl)pyridin-4-yl](2,4-difluorophe-
nyl)methanone (25). This compound was prepared starting from
from (3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)metha-
none (6e) and 1,3-benzodioxol-4-ylboronic acid following the
general method A. Yield: 56%. LCMS (m/z): 355 (M þ 1)þ. 1H
NMR (CDCl3, 300 MHz) δ 8.05 (d, J = 6 Hz, 1H) 7.46-7.57 (m,
1H), 7.16 (dd, J = 2 and 6 Hz, 1H), 6.90-7.11 (m, 5H), 6.8 (brs,
2H), 6.06 (s, 2H).
[3-Amino-2-(4-chlorophenyl)pyridine-4-yl](2,4-difluorophenyl)-
methanone (16). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone
(6e) and (4-chlorophenyl)boronic acid following the general
1
method A. Yield: 84%. LCMS (m/z): 345-347 (M þ 1)þ. H
NMR (CDCl3, 300 MHz) δ 8.00 (d, J = 6.7 Hz, 1H), 7.50-7.70
(m, 5H), 6.93-7.13 (m, 3H), 6.40 (brs, 2H).
[3-Amino-2-(2-methoxyphenyl)pyridin-4-yl](2,4-difluorophenyl)-
methanone (17). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone (6e)
and (2-methoxyphenyl)boronic acid following the general method
[3-Amino-2-(2,4-difluorophenyl-4-yl)pyridin-4-yl](2,4-difluoro-
phenyl)methanone (26). This compound was prepared starting
from from (3-amino-2-bromopyridin-4-yl)(2,4-difluorophe-
nyl)methanone(6e) and(2,4-difluorophenyl)boronic acid follow-
ing the general method A. Yield: 32%. LCMS (m/z): 347 (M þ
1
A. Yield: 81%. LCMS (m/z): 341 (M þ 1)þ. H NMR (CDCl3,
1
300 MHz) δ8.00 (d, J=5.5 Hz, 1H), 7.37-7.57 (m, 3H), 6.91-7.14
(m, 5H), 6.30 (brs, 2H), 3.85 (s, 3H).
1)þ. H NMR (CDCl3, 300 MHz) δ 8.03 (d, J = 6 Hz, 1H)
7.46-7.59 (m, 2H), 7.18 (dd, J = 2 and 4 Hz, 1H), 6.90-7.11 (m,
4H), 6.25 (brs, 2H).
[3-Amino-2-(2-methylphenyl)pyridin-4-yl](2,4-difluorophenyl)-
methanone (18). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone
(6e) and (2-methylphenyl)boronic acid following the general
method A. Yield: 85%. LCMS (m/z): 325 (M þ 1)þ. 1H NMR
(CDCl3, 300 MHz) δ 8.00 (d, J = 5.1 Hz), 7.48-7.59 (m, 1H),
[3-Amino-2-(2-methyl-4-chlorophenyl-4-yl)pyridin-4-yl](2,4-di-
fluorophenyl)methanone (27). Thiscompound waspreparedstart-
ing from from (3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)-
methanone (6e) and (2-methyl-4-chlorophenyl)boronic acid follow-
ing the general method A. Yield: 83%. LCMS (m/z): 359 (M þ 1)þ.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5541
1H NMR (CDCl3, 300 MHz) δ 8.00 (d, J = 6 Hz, 1H) 7.49-7.60
(m, 1H), 7.25-7.36 (m, 3H), 7.15 (dd, J = 2 and 4 Hz, 1H),
6.90-7.25 (m, 2H), 6.12 (brs, 2H), 2.19 (s, 3H).
J = 2 and 4 Hz, 1H), 6.87-7.04 (m, 4H), 6.20 (brs, 2H), 4.18 (t,
J = 6 Hz, 2H), 3.79 (t, J = 6 Hz, 2H), 3.48 (s, 3H), 2.18 (s, 3H).
(2-[4-(2-Methoxyethoxy)-2-methylphenyl]-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane was prepared starting from 1-bromo-4-(2-
methoxyethoxy)-2-methylbenzene following the general method
used for (3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzoic acid (see 30). Yield: 64%).
[3-Amino-2-(2,6-difluoro-4-methoxyphenyl-4-yl)pyridin-4-yl]-
(2,4-difluorophenyl)methanone (28). This compound was pre-
pared starting from from (3-amino-2-bromopyridin-4-yl)(2,4-
difluorophenyl)methanone (6e) and 2-bromo-1,3-difluoro-5-
methoxybenzene following the general method C. Yield: 21%.
LCMS (m/z): 377 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 8.05
(d, J = 6 Hz, 1H) 7.47-7.58 (m, 1H), 7.19 (dd, J = 2 and 4 Hz,
1H), 6.90-7.00 (m, 2H), 6.62 (m, 2H), 6.23 (brs, 2H), 3.86 (s,
3H).
[3-Amino-2-(2-methyl-4-hydroxyphenyl-4-yl)pyridin-4-yl](2,4-
difluorophenyl)methanone (29). This compound was prepared
starting from from (3-amino-2-bromopyridin-4-yl)(2,4-difluor-
ophenyl)methanone (6e) and (2-methyl-4-hydroxyphenyl)boro-
nic acid following the general method A. Yield: 58%. LCMS
(m/z): 341 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.97 (d, J =
6 Hz, 1H) 7.48-7.59 (m, 1H), 7.18 (dd, J = 2 and 4 Hz, 1H),
6.90-7.10 (m, 3H), 6.58-6.62 (m, 2H), 6.25 (brs, 2H), 2.08 (s,
3H).
4-[3-Amino-4-(2,4-difluorobenzoyl)pyridin-2-yl]-N-(2-meth-
oxyethyl)-3-methyl-benzamide (33). To a mixture of 4-[3-amino-
4-(2,4-difluorobenzoyl)pyridin-2-yl]-3-methylbenzoic acid (30)
(100 mg, 0.27 mmol), 2-methoxyethanamine (24 μL, 0.27 mmol),
and HATU (104 mg, 0.27 mmol) in dimethylformamide (2 mL)
was added DIEA (107 μL, 0.61 mmol), and the resulting suspen-
sionwas stirredat room temperature for 2 h. Ethyl acetate (40mL)
and water (20 mL) were added, the aqueous phase was separated,
and the organic phase was washed with water (20 mL) and brine
(20 mL), dried over anhydrous sodium sulfate, and the solvent
removed under reduced pressure. The residue was purified by
column chromatography on silica flash, using hexane/ethyl acet-
ate (7:3), to yield the title compound as a yellow solid (85 mg,
73%). LCMS (m/z):426 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ
8.02 (d, J=6 Hz, 1H) 7.70-7.80 (m, 2H), 7.50-7.60 (m, 1H), 7.42
(d, J = 6 Hz, 1H), 7.16 (dd, J = 4 and 6 Hz, 1H), 6.90-7.00 (m,
2H), 6.58 (brt, J = 6 Hz, 1H), 6.10 (brs, 2H), 3.68 (m, 2H), 3.62
(dd, J = 4 and 8 Hz, 2H), 3.42 (s, 3H), 2.26 (s, 3H).
(3-Amino-1-oxido-2-phenylpyridin-4-yl)(phenyl)methanone (34).
To a solution of (3-amino-2-phenylpyridin-4-yl)(phenyl)meth-
anone (7) (137 mg, 0.5 mmol) in dichloromethane (3 mL) at
0 ꢀC was portionwise added meta-chloroperbenzoic acid (130
mg, 0.75 mmol), and the reaction mixture was stirred overnight
at room temperature. Then, more dichloromethane was added (30
mL) and the solution was washed with aqueous sodium bicarbo-
nate 4% (3 ꢀ 30 mL) and brine. The organic layer was dried over
sodium sulfate and concentrated under reduced pressure to give a
residue that was purified by crystallization from a mixture of
hexane, diethyl ether, and ethyl acetate to yield the title compound
(113 mg, 75%) as a yellow solid. LCMS (m/z): 291 (M þ 1)þ. 1H
NMR (CDCl3, 300 MHz) δ 7.45-7.68 (m, 12H), 7.36 (d, J = 6.0
Hz, 1H), 6.32 (brs, 2H).
4-[3-Amino-4-(2,4-difluorobenzoyl)pyridin-2-yl]-3-methylben-
zoic Acid (30). This compound was prepared starting from
(3-amino-2-bromopyridin-4-yl)(2,4-difluorophenyl)methanone
(6e) and 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzoic acid following the general method A. Yield: 60%.
1
LCMS (m/z): 369 (M þ 1)þ. H NMR (CD3OD, 300 MHz) δ
8.00-8.09 (m, 2H) 7.88 (d, J = 6 Hz, 1H), 7.58-7.70 (m, 1H),
7.42 (d, J = 8 Hz, 1H), 7.26 (dd, J = 2 and 4 Hz, 1H), 7.10-7.20
(m, 2H), 2.23 (s, 3H).
(3-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic
acid was prepared as follows: In a Biotage microwave vial (10-
20 mL) was placed a mixture of 4-bromo-3-methylbenzoic acid
(1 g, 4.65 mmol), 4,4,40,40,5,5,50,50-octamethyl-2,20-bi(1,3,2-diox-
aborolane) (1.77 g, 6.97 mmol), potassium acetate (2.3 g, 23.2
mmol), and [1,10-bis(diphenylphosphino)ferrocene]dichloropall-
adium(II) complex with dichloromethane (1:1) (379 mg,
0.46 mmol) in dry DMF (20 mL). The mixture was submmited
tomicrowave irradiation at 120 ꢀC for 15 min on an Initiator Sixty
system from Biotage. The solvent was evaporated and the residue
suspended on a 1:1 mixture 2N HCl/EtOAc. The aqueous phase
was extracted with EtOAc. The combined organic layers were
dried and the solvents removed to afford a brown oil, which was
submitted to column chromatography on silica flash using a
mixture of hexanes and ethyl acetate (8:2) as eluent to yield the
title compound (1.1 g, 90%) as a white solid).
{3-Amino-2-[2-methyl-4-(2-morpholin-4-ylethoxy)phenyl]-
pyridin-4-yl}(2,4-difluorophenyl)methanone (31). This compound
was prepared starting from from (3-amino-2-bromopyridin-4-yl)-
(2,4-difluorophenyl)methanone (6e) and 4-{2-[3-methyl-4-(4,4,
5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]ethyl}morpho-
line following the general method A. Yield: 61%. LCMS (m/z):
454 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.98 (d, J = 4 Hz,
1H) 7.47-7.58 (m, 1H), 7.24 (d, J = 4 Hz, 1H), 7.10 (dd, J =
2 and 4 Hz, 1H), 6.84-7.04 (m, 4H), 6.19 (brs, 2H), 4.18 (t, J =
6 Hz, 2H), 3.77 (t, J = 6 Hz, 4H), 2.85 (t, J = 6 Hz, 2H), 2.62 (t,
J = 6 Hz, 4H), 2.18 (s, 3H).
(4-{2-[3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenoxy]ethyl}-morpholine was prepared starting from 4-[2-(4-
bromo-3-methylphenoxy)ethyl]-morpholine following the general
method used for (3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)benzoic acid (see 30). Yield: 71%).
{3-Amino-2-[4-(2-methoxyethoxy)-2-methylphenyl]pyridin-4-
yl}(2,4-difluoro-phenyl)methanone (32). This compound was
prepared starting from from (3-amino-2-bromopyridin-4-yl)-
(2,4-difluorophenyl)methanone (6e) and 2-[4-(2-meth-
oxyethoxy)-2-methylphenyl]-4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane following the general method A. Yield: 42%. LCMS
(m/z): 399 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.98 (d, J =
6 Hz, 1H) 7.47-7.58 (m, 1H), 7.25 (d, J = 6 Hz, 1H), 7.10 (dd,
[3-Amino-2-(2-chlorophenyl)-1-oxidopyridin-4-yl](phenyl)-
methanone (35). This compound was prepared starting from
[3-amino-2-(2-chlorophenyl)pyridin-4-yl](phenyl)methanone (8)
following the experimental procedure described for the synthesis
1
of 34. Yield: 98% LCMS (m/z): 325, 327 (M þ 1)þ. H NMR
(CDCl3, 300 MHz) δ 7.40-7.69 (m, 11H), 6.3 (brs, 2H).
[3-Amino-2-(2-methylphenyl)-1-oxidopyridin-4-yl](phenyl)-
methanone (36). This compound was prepared starting from
[3-amino-2-(2-methylphenyl)pyridin-4-yl](phenyl)methanone (9)
following the experimental procedure described for the synthesis
of 34. Yield: 96%. LCMS (m/z): 305 (M þ 1)þ. 1H NMR (CDCl3,
300 MHz) δ 7.23-7.73 (m, 11H), 6.27 (brs, 2H), 2.23 (s, 3H).
Anal. (C19H16N2O2) C, H, N.
[3-Amino-2-(4-chlorophenyl)-1-oxidopyridin-4-yl](phenyl)-
methanone (37). This compound was prepared starting from
[3-amino-2-(4-chlorophenyl)pyridin-4-yl](phenyl)methanone (10)
following the experimental procedure described for the synthesis of
1
34. Yield: 100%. LCMS (m/z): 325, 327 (M þ 1)þ. H NMR
(CDCl3, 300 MHz) δ 7.33-7.73 (m, 11H), 6.3 (brs, 2H).
[3-Amino-2-(2-chlorophenyl)-1-oxidopyridin-4-yl](2-chlorophenyl)-
methanone (38). This compound was prepared starting from
[3-amino-2-(2-chlorophenyl)pyridin-4-yl](2-chlorophenyl)meth-
anone (11) following the experimental procedure described for
the synthesis of 34. Yield: 48%. LCMS (m/z): 359-361-363 (M
þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.60 (d, J = 8 Hz, 1H),
7.37-7.67 (m, 8H), 7.08 (d, J = 6 Hz, 1H), 6.47 (brs, 2H).
[3-Amino-2-(2-chlorophenyl)-1-oxidopyridin-4-yl](2-methoxy-
phenyl)methanone (39). This compound was prepared starting
from [3-amino-2-(2-chlorophenyl)pyridin-4-yl](2-methoxyphe-
nyl)methanone (12) following the experimental procedure de-
scribed for the synthesis of 34. Yield: 83%. LCMS (m/z):

5542 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Lumeras et al.
355-357 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.68-7.61
(m, 1H), 7.60 (d, J = 6 Hz, 1H), 7.54-7.31 (m, 5H), 7.19 (d, J =
6 Hz, 1H), 7.12-7.01 (m, 2H), 3.82 (s, 3H), 6.39 (brs, 2H).
[3-Amino-2-(2-chlorophenyl)-1-oxidopyridin-4-yl](2-trifluoro-
methylphenyl)methanone (40). This compound was prepared
starting from [3-amino-2-(2-chlorophenyl)pyridin-4-yl](2-tri-
fluoromethylphenyl)methanone (13) following the experimen-
tal procedure described for the synthesis of 34. Yield: 54%.
LCMS (m/z): 393-395 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz)
δ 7.80-7.84 (m, 1H), 7.61-7.71 (m, 3H), 7.58 (d, J = 6 Hz, 1H),
7.53-7.40 (m, 4H), 6.98 (d, J = 6 Hz, 1H), 6.42 (brs, 2H).
[3-Amino-2-(2-chlorophenyl)-1-oxidopyridin-4-yl](2,4-difluoro-
phenyl)methanone (41). This compound was prepared starting
from [3-amino-2-(2-chlorophenyl)pyridin-4-yl](2,4-difluorophe-
nyl)methanone (14) following the experimental procedure de-
scribed for the synthesis of 34. Yield: 72%. LCMS (m/z):
[3-Amino-2-(2,6-difluorophenyl)-1-oxidopyridin-4-yl](2,4-di-
fluorophenyl)methanone (48). This compound was pre-
pared starting from [3-amino-2-(2,6-dichlorophenyl)pyridin-4-
yl](2,4-difluorophenyl)methanone (21) following the experimen-
tal procedure described for the synthesis of 34. Yield: 73%.
1
LCMS (m/z): 363 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ
7.67 (d, J = 7.1 Hz, 1H), 7.46-7.60 (m, 2H), 7.27 (m, 1H),
6.92-7.17 (m, 4H), 6.49 (brs, 2H). Anal. (C18H10F4N2O2)
C, H, N.
[3-Amino-2-(2,6-dimethylphenyl)-1-oxidopyridin-4-yl](2,4-
difluorophenyl)methanone (49). This compound was prepared
starting from [3-amino-2-(2,6-dimethylphenyl)pyridin-4-yl](2,4-
difluorophenyl)methanone (22) following the experimental
procedure described for the synthesis of 34. Yield: 73%. LCMS
(m/z): 355 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz): δ 7.67 (d, J =
7.1 Hz, 1H), 7.53 (m, 1H), 7.19-7.38 (m, 4H), 6.92-7.10 (m, 2H),
6.35 (brs, 2H), 2.14 (s, 6H).
1
361-363 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ 7.62-7.67
(m, 2H), 7.38-7.58 (m, 4H), 7.24 (dd, J = 7.0 and 2.8 Hz, 1H),
6.92-7.10 (m, 2H), 6.38 (brs, 2H). Anal. (C18H11ClF2N2O2) C,
H, N.
[3-Amino-2-(2,6-dimethoxyphenyl)-1-oxidopyridin-4-yl](2,4-
difluorophenyl)methanone (50). This compound was prepared
starting from [3-amino-2-(2,6-dimethoxyphenyl)pyridin-4-yl]-
(2,4-difluorophenyl)methanone (23) following the experimen-
tal procedure described for the synthesis of 34. Yield: 83%.
[3-Amino-2-(3-chlorophenyl)-1-oxidopyridin-4-yl](2,4-difluoro-
phenyl)methanone (42). This compound was prepared starting
from [3-amino-2-(3-chlorophenyl)pyridin-4-yl](2,4-difluorophe-
nyl)methanone (15) following the experimental procedure de-
scribed for the synthesis of 34. Yield: 75%. LCMS (m/z):
1
LCMS (m/z): 387 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ
7.60 (d, J = 8 Hz, 1H), 7.55-7.43 (m, 2H), 7.14 (dd, J = 2,4
Hz, 1H), 6.90-7.08 (m, 2H), 6.72 (d, J = 8 Hz, 2H), 6.46 (brs,
2H), 3.80 (s, 6H). Anal. (C20H16F2N2O4) C, H, N: calcd, 62.18;
found, 61.68.
1
361-363 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ 7.63 (d, J
= 7.0Hz, 1H), 7.45-7.56(m, 4H), 7.37 (m, 1H), 7.20 (dd, J = 7.0
and 3.1 Hz, 7H), 6.91-7.10 (m, 2H), 6.47 (brs, 2H).
[3-Amino-2-(2,3-dimethoxyphenyl)-1-oxidopyridin-4-yl](2,4-
difluorophenyl)methanone (51). This compound was prepared
from [3-amino-2-(2,3-dimethoxyphenyl)pyridin-4-yl](2,4-di-
fluorophenyl)methanone (24) following the experimental pro-
cedure described for the synthesis of 34. Yield: 67%. LCMS
[3-Amino-2-(4-chlorophenyl)-1-oxidopyridin-4-yl](2,4-difluor-
ophenyl)methanone (43). This compound was prepared starting
from [3-amino-2-(4-chlorophenyl)pyridin-4-yl](2,4-difluoro-
phenyl)methanone (16) following the experimental procedure
described for the synthesis of 34. Yield: 79%. LCMS (m/z):
361-363 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.43-7.70
(m, 6H), 7.20 (dd, J = 7.0 and 3.1 Hz, 1H), 6.90-7.10 (m, 2H),
6.5 (brs, 2H).
1
(m/z): 387 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ 7.64 (d,
J = 7.0 Hz, 1H), 7.51 (m, 1H), 7.24-7.32 (m, 1H), 7.19 (dd,
J = 7.0 and 2.7 Hz, 1H), 6.85-7.13 (m, 4H), 6.44 (brs, 2H),
3.94 (s, 3H), 3.84 (s, 3H).
[3-Amino-2-(2-methoxyphenyl)-1-oxidopyridin-4-yl](2,4-di-
fluorophenyl)methanone (44). This compound was prepared
starting from [3-amino-2-(2-methoxyphenyl)pyridin-4-yl](2,4-
difluorophenyl)methanone (17) following the experimental pro-
cedure described for the synthesis of 34. Yield: 79%. LCMS
(m/z): 357 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.62 (d, J =
7.0 Hz, 1H), 7.44-7.57 (m, 2H), 7.31 (m, 1H), 7.31 (m, 1H), 6.45
(brs, 2H), 3.85 (s, 3H).
[3-Amino-2-(2-methylphenyl)-1-oxidopyridin-4-yl](2,4-di-
fluorophenyl)methanone (45). This compound was prepared
starting from [3-amino-2-(2-methylphenyl)pyridin-4-yl](2,4-di-
fluorophenyl)methanone (18) following the experimental proce-
dure described for the synthesis of 34. Yield: 95%. LCMS (m/z):
341 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.64 (d, J = 7.0 Hz,
1H), 7.35-7.57 (m, 4H), 7.29 (m, 1H), 7.20 (dd, J = 7.0 and 3.1
Hz, 1H), 6.91-7.10 (m, 2H), 6.40 (brs, 2H), 2.21 (s, 3H). Anal.
(C19H14F2N2O2) C, H, N.
[3-Amino-2-(1,3-benzodioxol-4-yl)-1-oxidopyridin-4-yl](2,4-
difluorophenyl)methanone (52). This compound was prepared
from [3-amino-2-(1,3-benzodioxol-4-yl)pyridin-4-yl](2,4-di-
fluorophenyl)methanone (25) following the experimental pro-
cedure described for the synthesis of 34. Yield: 43%. LCMS
1
(m/z): 371 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ 7.64 (d,
J = 8.0 Hz, 1H), 7.43-7.55 (m, 1H), 7.19 (dd, J = 4.0 and 8.0
Hz, 1H), 6.88-7.09 (m, 5H), 6.62 (brs, 2H), 6.06 (dd, J = 2 and
12 Hz, 2H).
[3-Amino-2-(2,4-difluorophenyl)-1-oxidopyridin-4-yl](2,4-di-
fluorophenyl)methanone (53). This compound was prepared
from
[3-amino-2-(2,4-difluorophenyl)pyridin-4-yl](2,4-di-
fluorophenyl)methanone (26) following the experimental pro-
cedure described for the synthesis of 34. Yield: 90%. LCMS
1
(m/z): 363 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ 7.65 (d,
J = 8.0 Hz, 1H), 7.38-7.57 (m, 2H), 7.24 (dd, J = 2.0 and 4.0
Hz, 1H), 6.91-7.15 (m, 4H), 6.47 (brs, 2H).
[3-Amino-2-(2-isopropylphenyl)-1-oxidopyridin-4-yl](2,4-di-
fluorophenyl)methanone (46). This compound was prepared
starting from [3-amino-2-(2-isopropylphenyl)pyridin-4-yl]-
(2,4-difluorophenyl)methanone (19) following the experimen-
tal procedure described for the synthesis of 34. Yield: 93%.
[3-Amino-2-(4-chloro-2-methylphenyl)-1-oxidopyridin-4-yl]-
(2,4-difluorophenyl)methanone (54). This compound was pre-
pared from [3-amino-2-(4-chloro-2-methylphenyl)pyridin-4-yl]-
(2,4-difluorophenyl)methanone (27) following the experimental
procedure described for the synthesis of 34. Yield: 84%. LCMS
(m/z): 375 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz) δ 7.64 (d, J =
8.0 Hz, 1H), 7.36-7.57 (m, 3H), 7.19-7.25 (m, 2H), 6.90-7.10
(m, 2H), 6.37 (brs, 2H), 2.20 (s, 3H).
[3-Amino-2-(2,6-difluoro-4-methoxyphenyl)-1-oxidopyridin-
4-yl](2,4-difluorophenyl)methanone (55). This compound was
prepared from [3-amino-2-(2,6-difluoro-4-methoxyphenyl)-
pyridin-4-yl](2,4-difluorophenyl)methanone (28) following
the experimental procedure described for the synthesis of 34.
Yield: 72%. LCMS (m/z): 393 (M þ 1)þ. 1H NMR (CDCl3, 300
MHz) δ 7.65 (d, J = 8.0 Hz, 1H), 7.45-7.56 (m, 1H), 7.23 (dd,
J = 8.0 and 4.0 Hz, 1H), 6.92-7.10 (m, 2H), 6.63-6.72 (m,
2H), 6.54 (brs, 2H), 3.88 (s, 3H).
1
LCMS (m/z): 369 (M þ 1)þ. H NMR (CDCl3, 300 MHz) δ
7.66 (d, J = 7.4 Hz, 1H), 7.36-7.58 (m, 4H), 7.18-7.23 (m,
2H), 6.92-7.10 (m, 2H), 6.38 (brs, 2H), 2.59 (hep, J = 7.0 Hz,
1H), 1.27 (d, J = 7.0 Hz, 3H), 1.19 (d, J = 6.6 Hz, 3H).
[3-Amino-2-(2,6-dichlorophenyl)-1-oxidopyridin-4-yl](2,4-di-
fluorophenyl)methanone (47). This compound was prepared
starting from [3-amino-2-(2,6-dichlorophenyl)pyridin-4-yl]-
(2,4-difluorophenyl)methanone (20) following the experimen-
tal procedure described for the synthesis of 34. Yield: 87%.
1
LCMS (m/z): 395, 397, 399 (M þ 1)þ. H NMR (CDCl3, 300
MHz) δ 7.67 (d, J = 7.0 Hz, 1H), 7.40-7.59 (m, 4H), 7.28 (dd,
J = 7.0 and 2.7 Hz, 1H), 6.92-7.11 (m, 2H), 6.35 (brs, 2H).

Article
[3-Amino-2-(4-hydroxy-2-methylphenyl)-1-oxidopyridin-4-yl]-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5543
1
Yield: 60%. LCMS (m/z): 442 (M þ 1)þ. H NMR (CDCl3,
(2,4-difluorophenyl)methanone (56). This compound was pre-
pared from [3-amino-2-(4-hydroxy-2-methylphenyl)pyridin-4-
yl](2,4-difluorophenyl)methanone (29) following the experi-
mental procedure described for the synthesis of 34. Yield:
99%. LCMS (m/z): 357 (M þ 1)þ. 1H NMR (CDCl3, 300 MHz)
δ 7.60-7.70 (m, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.40-7.50 (m,
1H), 7.20-7.30 (m, 1H), 7.14 (dd, J = 8.0 and 2.0 Hz, 1H), 6.97
(d, J = 8.0 Hz, 1H), 6.88 (brs, 2H), 6.70-6.80 (m, 2H), 1.94
(s, 3H).
4-[3-Amino-4-(2,4-difluorobenzoyl)-1-oxidopyridin-2-yl]-
3-methylbenzoic Acid (57). This compound was prepared from
4-[3-amino-4-(2,4-difluorobenzoyl)pyridin-2-yl]-3-methylbenzoic
acid (30) following the experimental procedure described for the
synthesis of 34. Yield: 57%. LCMS (m/z): 385 (M þ 1)þ. 1H NMR
(CDCl3, 300 MHz) δ 7.01 (brs, 2H), 7.24-7.70 (m, 6H),
7.89-7.98 (m, 2H).
300 MHz) δ 7.75-7.84 (m, 2H), 7.65 (t, J = 4.0 Hz, 2H),
7.49-7.60 (m, 1H), 7.37 (t, J = 8.0 Hz, 2H), 7.23 (t, J = 4.0
and 8.0 Hz, 2H), 6.92-7.10 (m, 2H), 6.57 (brt, J = 6.0 Hz, 1H),
6.34 (brs, 2H), 3.67 (m, 2H), 3.60 (dd, J = 8.0 and 4.0 Hz), 3.41
(s, 3H), 2.26 (s, 3H).
Biological Methods. p38r Kinase Inhibition Assay. Enzy-
matic activity assay was performed in 96-well microtiter plates
(Corning, catalogue no. 3686) using a total volume of 50 μL of
an assay buffer composed of 50 mM HEPES pH 7.5, 10 mM
MgCl2, 1.75 mM Na3VO4.
Various concentrations of the test compound or vehicle
controls were preincubated for one hour with 0.055 μg/mL of
the human p38R (SAPKa) enzyme (obtained from University of
Dundee). The reaction started by addition of biotinylated ATF2
substrate and ATP in concentrations around their Km values
(final concentration 0.62 and 60 μM, respectively) and took
place for one hour at 25 ꢀC. Addition of the detection rea-
gents, streptavidin-XL665 and antiphosphoresidue antibody
coupled to Europium cryptate, caused the juxtaposition of the
cryptate and the XL665 fluorophore, resulting in fluorescence
energy transfer (FRET). The FRET intensity depends on the
amount of bounded cryptate antibody, which is proportional to
the extent of substrate phosphorylation. FRET intensity was
measured using Victor 2 V spectrofluorometer.
{3-Amino-2-[2-methyl-4-(2-morpholin-4-ylethoxy)phenyl]-1-
oxidopyridin-4-yl}(2,4-difluorophenyl)methanone (58). To a so-
lution of [3-amino-2-(4-hydroxy-2-methylphenyl)-1-oxido-
pyridin-4-yl](2,4-difluorophenyl)methanone (56) (200 mg,
0.56 mmol) in 6 mL of acetonitrile were added 4-(2-chloro-
ethyl)morpholine hydrochloride (156 mg, 0.84 mmol) and
potassium carbonate (301 mg, 2.18 mmol), and the mixture
was heated to 80 ꢀC for 18 h. The reaction was cooled down
and filtered through a pad of celite, washing with acetonitrile
(10 mL). The solvent was removed under reduced pressure to
give a crude oil, which was purified by column chromatogra-
phy on silica flash using dichloromethane/ methanol (95:5) as
eluents. The resulting solid was further purified by crystal-
lization from a mixture of diisopropylether and ethyl acetate
(2/1) to yield the title compound (142 mg, 54%) as a bright-
Data were analyzed by nonlinear regression (Hill equation) to
generate a dose-response curve. The calculated IC50 value is the
concentration of the test compound, which caused a 50%
decrease in the maximal FRET intensity.
Inhibition of TNFr Production Induced by LPS in the Human
Monocytic Cell Line THP-1 Assay. For this purpose, 2 ꢀ 105
cells/well were plated in tissue-culture treated round-bottom
96-well plates in RPMI (containing 10% FCS, L-Gln 2 mM,
Hepes buffer 10 mM, sodium pyruvate 1 mM, glucose 4.5 g/L,
NaHCO3 1.5 g/L and β-mercaptoethanol 50 μM), together with
compounds at the desired test concentration and LPS (Sigma,
L2630) at a final 10 μg/mL concentration. Compounds were
resuspended in 100% DMSO at a concentration of 1 mM and
titrated thereof in 10ꢀ dilutions in medium. Controls included
unstimulated and stimulated cells treated with the highest
concentration of compound vehicle (1% DMSO). Cells were
incubated for 5 h at 37 ꢀC in a 5% CO2 atmosphere. Cell
supernatant was recovered by centrifugation and diluted 5-fold
prior to testing in a standard human TNFR ELISA (RnD
systems).
Inhibition of TNFr Production Induced by LPS in Human
Whole Blood Assay. Healthy volunteer donor blood was col-
lected by venipuncture in heparinized tubes. Two μL of 10-fold
compound concentrations in 100% DMSO were mixed with 200
μL of LPS-stimulated blood in microtiter plates. Controls
included unstimulated and stimulated blood treated with the
highest concentration of compound vehicle (1% DMSO). Plates
were incubated for 24 h at 37 ꢀC with shaking. Supernatant was
recovered by centrifugation and diluted one-sixth prior to
testing in a standard TNFR ELISA (RnD systems).
1
yellow solid. LCMS (m/z): 470 (M þ 1)þ. H NMR (CDCl3,
300 MHz) δ 7.62 (d, J = 6.0 Hz, 1H), 7.45-7.56 (m, 1H),
7.14-7.21 (m, 2H), 6.90-7.10 (m, 4H), 6.43 (brs, 2H), 4.17 (t,
J = 4.0 Hz, 2H), 3.76 (t, J = 4.0 Hz, 2H), 2.84 (t, J = 4.0 Hz,
2H), 2.61 (t, J = 6.0 Hz, 2H), 2.17 (s, 3H).
4-[3-Amino-4-(2,4-difluorobenzoyl)-1-oxidopyridin-2-yl]-3-
methyl-N-(2-morpholin-4-ylethyl)benzamide (59). To a solu-
tion of 4-[3-amino-4-(2,4-difluorobenzoyl)-1-oxidopyridin-2-yl]-
3-methylbenzoic acid (57) (50 mg, 0.14 mmol) in 2 mL of N,
N-dimethylformamide were added (2-morpholin-4-ylethyl)amine
(25 mg, 0.19 mmol), O-benzotriazol-1-yl-N,N,N0,N0-tetramethyl-
uronium hexafluorophosphate (HBTU) (65 mg, 0.17 mmol), and
diisopropyl ethyl amine (301 mg, 2.18 mmol) and the mixture was
stirred overnight under argon. The reaction was diluted with ethyl
acetate, washed with 5% citric acid, water, brine, and dried over
sodium sulfate. Removal of the solvent under reduced pressure
afforded a residue which was purified by column chromatography
on silica flash, using dichloromethane/ethanol (95:5) as eluent, to
yield 4-[3-amino-4-(2,4-difluorobenzoyl)-1-oxidopyridin-2-yl]-3-
methyl-N-(2-morpholin-4-ylethyl)benzamide (59) (1%) as a
bright-yellow solid. LCMS (m/z): 497 (M þ 1)þ. 1H NMR
(CD3OD, 300 MHz) δ 7.83-7.90 (m, 2H), 7.56-7.68 (m, 2H),
7.36-7.43 (m, 2H), 7.12-7.21 (m, 2H), 3.72-3.77 (m, 4H), 3.61
(t, J = 8.0 Hz, 2H), 2.64-2.75 (m, 6H), 2.23 (s, 3H).
{3-Amino-2-[4-(2-methoxyethoxy)-2-methylphenyl]-1-oxido-
pyridin-4-yl}(2,4-difluorophenyl)methanone (60). This com-
pound was prepared from {3-amino-2-[4-(2-methoxyethoxy)-2-
methylphenyl]pyridin-4-yl}(2,4-difluorophenyl)methanone (32)
following the experimental procedure described for the synthesis
of 34. Yield: 72%. LCMS (m/z): 415 (M þ 1)þ. 1H NMR (CDCl3,
300 MHz) δ 7.63 (d, J = 8.0 Hz, 1H), 7.45-7.56 (m, 1H),
7.14-7.21 (m, 2H), 6.91-7.01 (m, 4H), 6.43 (brs, 2H), 4.18 (t,
J = 4.0 Hz, 2H), 3.78 (t, J =4.0Hz, 2H), 3.47 (s, 3H), 2.17 (s, 3H).
4-[3-Amino-4-(2,4-difluorobenzoyl)-1-oxidopyridin-2-yl]-N-(2-
methoxyethyl)-3-methylbenzamide (61). This compound was pre-
pared from 4-[3-amino-4-(2,4-difluorobenzoyl)pyridin-2-yl]-
N-(2-methoxyethyl)-3-methylbenzamide (33) following the ex-
perimental procedure described for the synthesis of 34.
Data were analyzed by nonlinear regression (Hill equation) to
generate a dose-response curve. The calculated IC50 value
corresponds to the concentration of the test compound, causing
a 50% decrease in the maximal TNFR production (absolute
IC50).
In Vivo Assays. LPS-Induced TNFr in the Rat. One hour
prior to LPS administration, rats were dosed orally with the
compounds suspended in 0.5% methylcellulose/0.1% Tween-
80. LPS (5 mg/kg) was administered intraperitoneally, and 1.5 h
later, rats were anesthesized and retroorbital blood collected in
heparin tubes. Plasma was separated by centrifugation and
diluted one-fifth prior to assaying in a standard rat TNF-alpha
ELISA (RnD Systems).
Adjuvant-Induced Arthritis (AIA) Model. Arthritis was in-
duced by intraplantar administration of 100 μL Mycobacter-

5544 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Lumeras et al.
ium tuberculosis suspension (5 mg/mL in paraffin oil) in the left
paw of male Wistar rats (Day 0). On day 11 postinoculation,
animals were weighed and paw volume measured by plethis-
mometry. Animals with left paw volumes ranging between 3.5
and 5 mL and right paw volumes between 2 and 3 mL were
randomized in the required treatment groups (7 animals/
group). Treatment was started and continued for 7 consecu-
tive days. On each day, animals were weighed and appropri-
ately dosed orally with the indicated doses of compounds
suspended in 0.5% methylcellulose/0.1% Tween 80. Paw
volumes were monitored every other day. A healthy control
group was inoculated with paraffin oil on day 0 and monitored
thereafter for paw volumes and weight. At the end of the
study, animals were euthanized with CO2. Inhibition percen-
tage was calculated using last day contralateral (right) paw
volumes.
(12) Ferraciola, G. F. VX-745 Vertex Pharmaceutical. Curr. Opin. Anti-
Inflammatory Immunomodul. Invest. Drugs 2000, 2, 74–77.
(13) Haddad, J. J. VX-745 (Vertex Pharmaceuticals). Curr. Opin.
Investig. Drugs 2001, 2, 1070–1076.
(14) Zimmiti, C. S.; Schwartz, R.; Torcellini, C. A.; Pargellis, C. A.;
Madwed, J. B.; Weldon, S. M. Suppression of p38 activity in vitro
and THF alpha production in vivo with BIRB-796 BS, a novel p38
kinase inhibitor. Arthritis Rheum. 2001, 44, S114.
(15) Regan, J.; Capolino, A.; Cirillo, P. F.; Gilmore, T.; Graham, A. G.;
Hickey, E.; Kroe, R. R.; Madwed, J.; Moriak, M.; Nelson, R.;
Pargellis, C. A.; Swinamer, A.; Torcellini, C.; Tsang, M.; Moss, N.
Structure-Activity Relationships of the p38R MAP Kinase In-
hibitor 1-(5-tert-Butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpho-
lin-4-yl-ethoxy)napthalen-1-yl]urea (BIRB-796). J. Med. Chem.
2003, 46, 4676–4686.
(16) Devraj, R. V. Discovery and Development of Orally Active p38
Kinase Inhibitors as Anti-TNF Agents. Abstract of Papers, 229th
National Meeting of the American Chemical Society, San Diego, CA,
March 13-17, 2005; American Chemical Society: Washington, DC,
2005; MEDI-296.
Acknowledgment. We thank Victor Segarra from the
Department of Computational and Structural Drug Discov-
ery for his kind and valuable support. We gratefully acknowl-
edge Phil Leonard, Marieke Lamers and Martin Fisher from
BioFocus DPI (formerly at Sareum) for their kind assistance
in the crystallographic studies of compound 45.
(17) Zack, D. J.; Campagnuolo, G.; Middleton, S.; Koch, A.; Zhu, L.;
Bolon, B.; Feige, U. Efficacy of AMG 548, a novel p38 kinase
inhibitor, in rats with adjuvant-induced or collagen-induced ar-
thritis. 67th Annual Scientific Meeting of the American College of
Rheumatology, Orlando, FL, October 23-28, 2003, Abstract 834.
(18) Domınguez, C; Liu, L.; Zhang, D.; Tamayo, N.; Powers, D.; Min,
´
W.; Feige, F.; Harris, R.; Wild, S.; Neervannan, S.; Rashid, S.;
Harvey, T. p38 MAP kinase: an exciting target for the treatment of
inflammatory diseases. Abstract of Papers, 228th National Meeting
of the American Chemical Society, Philadelphia, PA; American Che-
mical Society: Washington, DC, 2004; MEDI-189.
Supporting Information Available: Detailed elemental analy-
sis and HPLC purity data for final compounds and kinase
selectivity panel for 45. This material is available free of charge
via the Internet at http://pubs.acs.org.
(19) Nikas, S. N.; Drossos, A. A. SCIOS-469 (Scios Inc). Curr. Opin.
Investig. Drugs 2004, 5, 1205–1212.
(20) Perumattan, J. 2-[6-Chloro-5-[4-(4-fluoro-benzyl)-2R,5S-dimeth-
yl-piperazine-1-carbonyl]-1H-indol-3-yl]-2-oxo-acetamides as po-
tent inhibitors of p38 alpha MAP kinase. Drugs Future 2006, 31
(Suppl. A), Abstract O19.
(21) Weisman, M.; Furst, D.; Schiff, M.; Kauffman, R.; Merica, E.;
Martin-Munley, S. FRI0018 A double-blind, placebo controlled
trial of VX-745, an oral p38 MAP kinase inhibitor, in patients with
rheumatoid arthritis (RA). Ann. Rheum. Dis. 2002, 61, Suppl. 1.
(22) Vertex Pharmaceuticals, Inc. Press Release (September 24, 2001).
(23) Wang, Z.; Canagarajah, B. J.; Boehm, J. C.; Kassisa, S.; Cobb, M.
H.; Young, P. R.; Abdel-Meguid, S.; Adams, J. L.; Goldsmith, E. J.
Structural basis of inhibitor selectivity in MAP kinases. Structure
1998, 6, 1117–1128.
(24) Gallagher, T. F.; Seibel, G. L.; Kassis, S.; Laydon, J. T.;
Blumenthal, M. J.; Lee, J. C.; Lee, D.; Boehm, J. C.;
Fier-Thompson, S. M.; Abt, J. W.; Sorenson, M. E.; Smietana,
J. M.; Hall, R. F.; Garigipati, R. S.; Bender, P. E.; Erhard, K. F.;
Krog, A. J.; Hoffmann, G. A.; Sheldrake, P. L.; McDonnell, P. C.;
Kumar, S.; Young, P. R.; Adams, J. L. Regulation of stress-
induced cytokine production by pyridinylimidazoles; inhibition
of CSBP kinase. Bioorg. Med. Chem. 1997, 5, 49–64.
(25) Liverton, N. J.; Butcher, J. W.; Claiborne, C. F.; Claremon, D. A.;
Libby, B. E.; Nguyen, K. T.; Pitzenberger, S. M.; Selnick, H. G.;
Smith, G. R.; Tebben, A.; Vacca, J. P.; Varga, S. L.; Agarwal, L.;
Dancheck, K.; Forsyth, A. J.; Fletcher, D. S.; Frantz, B.; Hanlon,
W. A.; Harper, C. F.; Hofsess, S. J.; Kostura, M.; Lin, J.; Luell, S.;
O’Neill, E. A.; Orevillo, C. J.; Pang, M.; Parsons, J.; Rolando, A.;
Sahly, Y.; Visco, D. M.; O’Keefe, S. J. Design and Synthesis of
Potent, Selective, and Orally Bioavailable Tetrasubstituted Imida-
zole Inhibitors of p38 Mitogen-Activated Protein Kinase. J. Med.
Chem. 1999, 42, 2180–2190.
References
(1) Han, J.; Lee, J. D.; Bibbs, L.; Ulevitch, R. J. A MAP kinase
targeted by endotoxin and hyperosmolarity in mammalian cells.
Science 1994, 265, 808–811.
(2) Raingeaud, J.; Gupta, S.; Rogers, J. S.; Dickens, M.; Han, J.;
Ulevitch, R. J.; Davis, R. J. Characterization of the structure and
function of a novel MAP kinase kinase (MKK6). J. Biol. Chem.
1996, 271, 2886–2891.
(3) Raingeaud, J.; Whitmarsh, A. J.; Barrett, T.; Derijard, B.; Davis,
R. J. MKK3- and MKK6-regulated gene expression is mediated by
the p38 mitogen-activated protein kinase signal transduction path-
way. Mol. Cell. Biol. 1996, 16, 1247.
(4) Badger, A. M.; Bradbeer, J. N.; Votta, B.; Lee, J. C.; Adams, J. L.;
Griswold, D. E. Pharmacological profile of SB 203580, a selective
inhibitor of cytokine suppressive binding protein/p38 kinase, in
animal models of arthritis, bone resorption, endotoxin shock and
immune function. J. Pharmacol. Exp. Ther. 1996, 279, 1453–1461.
(5) Feldmann, M.; Brennan, F. M.; Maini, R. N. Role of cytokines in
rheumatoid arthritis. Annu. Rev. Immunol. 1996, 14, 397–440.
(6) 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.; et al. Efficacy and safety of retreatment with
anti-tumour necrosis factor antibody (Infliximab) to maintain
remission in Chron’s disease. Gastroenterology 1999, 117, 761–769.
(7) Foster, M. L.; Halley, F.; Souness, J. E. Potential of p38 inhibitors
in the treatment of rheumatoid arthritis. Drug News Perspect. 2000,
13, 488–497.
(8) Kumar, S.; Boehm, J.; Lee, J. C. p38 MAP kinases: key signaling
molecules as therapeutic targets for inflammatory diseases. Nature
Rev. Drug Discovery 2003, 2, 717–726.
(26) Jackson, P. F.; Bullington, J. L. Pyridinylimidazole based p38
MAP kinase inhibitors. Curr. Top. Med. Chem. 2002, 2, 1011–1020.
(27) Collis, A. J.; Foster, M. L.; Halley, F.; Maslen, C.; McLay, I. M.;
Page, K. M.; Redford, E. J.; Souness, J. E.; Wilsher, N. E.
(9) Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T. F.;
Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J.; Heyes, 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. A protein kinase involved in the regulation of
inflammatory cytokine biosynthesis. Nature 1994, 372, 739–746.
(10) Vertex USA: Vertex has suspended development of VX-745, a p38
MAP kinase inhibitor, which was in phase II trials in the USA and
Europe as potential treatmentfor rheumatoid arthritis. Vertex Inc.,
press release (September, 2001).
RPR203494
a Pyrimidine Analogue of the p38 Inhibitor
RPR200765A with an Improved in Vitro Potency. Bioorg. Med.
Chem. Lett. 2001, 11, 693–696.
(28) Revesz, L.; Dipadova, F. E.; Buhl, T.; et al. SAR of 4-Hydro-
xypiperidine and Hydroxyalkyl Substituted Heterocycles as Novel
p38 MAP Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2000, 10,
1261–1264.
(11) SmithKlineBeecham(nowGlaxoSmithKline) hasdiscontinueddevel-
opment of SB 242235,
(29) Fitzgerald, C. E.; Patel, S. B.; Becker, J. W.; Cameron, P. M.;
Zaller, D.; Pikounis, V. B.; O’Keefe, S. J.; Scapin, G. Structural
Basis for p38R MAP Kinase Quinazolinone and Pyrido-Pyrimidine
Inhibitor Specificity. Nat. Struct. Biol. 2003, 10, 764–769.
(30) Hunt, J. A.; Kallashi, F.; Ruzek, R. D.; Sinclair, P. J.; Ita, I.;
McCormick, S. X.; Pivnichny, J. V.; Hop, C. E. C.; Kumar, S.; m
a cytokine suppressor (SmithKline
Beecham, April 1999). This product was in phase I clinical studies in
the UK (SmithKline Beecham, March 1999) and was being investi-
gated as a potential treatment for rheumatoid arthritis (SmithKline
Beecham, April 1999). R&D Focus Report, March 27, 2001.

Article
Wang, Z.; O’Keefe, S. J.; O’Neill, E. A.; Porter, G.; Thompson,
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5545
(39) Negishi, E.-I.; Luo, F.-T.; Frisbee, R.; Matsushita, H. A Regios-
pecific Synthesis of Carbosubstituted Heteroaromatic Derivatives
via Pd-Catalyzed Cross Coupling. Heterocycles 1982, 18, 117–122.
(40) Wang, Z.; Canagarajah, B. J.; Boehm, J. C.; Kassisa, S.; Cobb, M.
H.; Young, P. R.; Abdel-Meguid, S.; Adams, J. L.; Goldsmith, E. J.
Structural Basis of Inhibitor Selectivity in MAP Kinases. Structure
(London) 1998, 6, 1117–1128.
(41) McClure, K. F.; Letavic, M. A.; Kalgutkara, A. S.; Gabel, C. A.;
Audoly, L.; Barberiaa, J. T.; Braganzaa, J. F.; Cartera, D.; Cartya,
T. J.; Cortinaa, S. R.; Dombroskia, M. A.; Donahuea, K. M.;
Elliotta, N. C.; Gibbonsa, C. P.; Jordana, C. K.; Kupermana, A.
V.; Labasia, J. F.; LaLibertea, R. E.; McCoya, J. M.; Naimana, B.
M.; Nelsona, K. L.; Nguyena, H. T.; Peesea, K. M.; Sweeneya, F.
J.; Taylora, T. J.; Trebinoa, C. E.; Abramova, Y. A.; Lairda, E. R.;
Volberga, W. A.; Zhoua, J.; Bacha, J.; Lombardo, F. Structure-
-activity relationships of triazolopyridine oxazole p38 inhibitors:
Identification of candidates for clinical development. Bioorg. Med.
Chem. Lett. 2006, 16, 4339–4344.
(42) Hauser, D. R.; Scior, T.; Domeyer, D. M.; Kammerer, B.; Laufer,
S. A. Synthesis, Biological Testing, and Binding Mode Prediction
of 6,9-Diarylpurin-8-ones as p38 MAP Kinase Inhibitors. J. Med.
Chem. 2007, 9, 2060–2066.
(43) Wan, Z.; Boehm, J. C.; Bower, M. J.; Kassis, S.; Lee, J. C.; Zhao,
B.; Adams, J. L. N-Phenyl-N-purin-6-yl ureas: The design and
synthesis of p38R MAP kinase inhibitors. Bioorg. Med. Chem. Lett.
2003, 13, 1191–1194.
J. E.; Woods, A.; Zaller, D. M.; Doherty, J. B. p38 Inhibitors:
piperidine- and 4-aminopiperidine-substituted naphthyridinones,
quinolinones, and dihydroquinazolinones. Bioorg. Med. Chem.
Lett. 2003, 13, 467–470.
(31) Bao, J.; Hunt, J. A.; Miao, S.; Rupprecht, K. M.; Stelmach, J. E.;
Liu, L.; Ruzek, R. D.; Sinclair, P. J.; Pivnichny, J. V.; Hop, C. E. C.
A.; Kumar, S.; Zaller, D. M.; Shoop, W. L.; O’Neill, E. A.;
O’Keefe, S. J.; Thompson, C. M.; Cubbon, R. M.; Wang, R.;
Zhang, W. X.; Thompson, J. E.; Doherty, J. B. p38 MAP kinase
inhibitors: Metabolically stabilized piperidine-substituted quinoli-
nones and naphthyridinones. Bioorg. Med. Chem. Lett. 2006, 16,
64–68.
(32) Alonso-Alija, C.; Michels, M.; Schirok, H.; Schlemmer, K.-H.;
Bell, J.; Fitzgerald, M. F.; Dodd, S.; Gill, A. Monocyclic aroylpyr-
idinones as antiinflammatory agents. Patent WO03076405, Sep-
tember, 2003.
(33) Schirok, H.; Alonso-Alija, C.; Benet-Buchholz, J.; Goller, A. H.;
Grosser, R.; Michels, M.; Paulsen, H. Efficient Regioselective
Synthesis of 6-Amino-5-benzoyl-1-Substituted 2(1H)-Pyridinones.
J. Org. Chem. 2005, 70, 9463–9469.
(34) Guengoer, T.; Marsais, F.; Queguiner, G. ortho-Functionalization
of Aminopyridines. Regioselective Lithiation of 3-Pivaloylamino-
pyridines. Synthesis 1982, 6, 499–500.
(35) (a) Miyaura, N.; Suzuki, A. Palladium-Catalyzed Cross-Coupling
Reactions of Organoboron Compounds. Chem. Rev. 1995, 95,
2457-2483 (Review). (b) Stanforth, S. P. Catalytic cross-coupling
reactions in biaryl synthesis. Tetrahedron 1998, 54, 263-303 (review).
(36) Jin, R.; Bian, Z.; Kang, C.; Guo, H.; Gao, L. Synthesis of 3,30-Di(2-
pyridyl)-1,10-bi-2-naphthol Derivatives. Synth. Commun. 2005, 35,
1897–1902.
(37) Walker, S. D.; Barder, T. E.; Martinelli, J. R.; Buchwald, S. L. A
Rationally Designed Universal Catalyst for Suzuki-Miyaura
Coupling Processes. Angew. Chem., Int. Ed. 2004, 43, 1871–1876.
(38) Negishi, E.-I.; King, A. O.; Okukado, N. Selective carbon-carbon
bond formation via transition metal catalysis. 3. A highly selective
synthesis of unsymmetrical biaryls and diarylmethanes by the
nickel- or palladium-catalyzed reaction of aryl- and benzylzinc
derivatives with aryl halides. J. Org. Chem. 1977, 42, 1821–1823.
(44) Natarajan, S. W.; Wisnoski, D. D.; Singh, S. B.; Stelmach, J. E.;
O’Neill, E. A.; Schwartz, C. D.; Thompson, C. M.; Fitzgerald, C.
E.; O’Keefe, S. J.; Cornelis, S. K.; Hop, E. C.; Zaller, D. M.;
Schmatz, D. M.; Doherty, J. B. p38 MAP kinase inhibitors. Part 1:
Design and development of a new class of potent and highly
selective inhibitors based on 3,4-dihydropyrido[3,2-d]pyrimidone
scaffold. Bioorg. Med. Chem. Lett. 2003, 13, 273–276.
(45) Clark, R. D.; Wolohan, P. R. Molecular Design and Bioavailabil-
ity. Curr. Top. Med. Chem. 2003, 3, 1269–1288.
(46) Crystallographic coordinates for the structure of unphosphory-
lated p38R complexed with 45 have been deposited at the Protein
Data Bank (www.rscb.org) with entry accession code 3HRB.
(47) See Supporting Information (p S32).