The design and synthesis of novel α-ketoamide-based p38 MAP kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.02.033

We have identified a novel series of potent p38 MAP kinase inhibitors through structure-based design which due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME and in vivo PK studies show these compounds to have drug-like characteristics which could result in the development of an oral treatment for inflammatory conditions.

Full text of DOI:10.1016/j.bmcl.2008.02.033

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1772–1777
The design and synthesis of novel a-ketoamide-based
p38 MAP kinase inhibitors
Antonio Garrido Montalban,* Erik Boman, Chau-Dung Chang, Susana Conde Ceide,
Russell Dahl, David Dalesandro, Nancy G. J. Delaet, Eric Erb, Justin T. Ernst,
Andrew Gibbs, Jeffrey Kahl, Linda Kessler, Jan Lundstro¨m, Stephen Miller,
Hiroshi Nakanishi, Edward Roberts, Eddine Saiah, Robert Sullivan,
Zhijun Wang and Christopher J. Larson
Drug Discovery, Ke´mia, Inc., 5871 Oberlin Drive, Suite 100, San Diego, CA 92121, USA
Received 19 December 2007; revised 9 February 2008; accepted 13 February 2008
Available online 16 February 2008
Abstract—We have identified a novel series of potent p38 MAP kinase inhibitors through structure-based design which due to their
extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME and in vivo PK studies
show these compounds to have drug-like characteristics which could result in the development of an oral treatment for inflammatory
conditions.
Ó 2008 Elsevier Ltd. All rights reserved.
Since the mitogen-activated protein (MAP) kinase p38
was first reported in 1993,1 it has received an extraordi-
nary level of attention by the pharmaceutical industry2
and medicinal chemistry community.3 This notion stems
from the fact that p38 has been recognized as a highly
attractive target for therapeutic intervention. It is well
established that the proinflammatory cytokines, tumor
necrosis factor-a (TNF-a) and interleukin-1b (IL-1b),
play an important role in the pathogenesis of various
inflammatory diseases and that the stress-activated sig-
nal transduction pathway leading to these cytokines is
in part regulated by p38. Blockade of TNF-a by the
biologics etanercept (Enbrel) and infliximab (Remi-
cade), for example, is clinically proven to be effective
in the treatment of rheumatoid arthritis, Crohn’s dis-
ease, and psoriasis.4 In addition, clinical validation of
the MAP kinase pathway in rheumatoid arthritis has
been achieved with VX-7455 and BIRB-796.6 The differ-
ent safety profiles reported7 for these two distinct struc-
tural classes and others suggest that toxicity is probably
more structure- than mechanism-based, but despite all
efforts, a small-molecule therapeutic utilizing this mech-
anism of action still remains elusive.7
After the ground breaking work by SmithKline Beecham
(initially followed by RWJ8 and Merck9) that led
to SB203580,10 numerous other ATP-competitive or
orthosteric p38 inhibitors have been described.11 On the
other hand, only a few p38 inhibitors that bind to an allo-
steric site (noncompetitive inhibitors) have been reported
so far. In fact, at the time this work was initiated, the only
known p38 inhibitors of this new class were related to
BIRB-796.12 More recently, AstraZeneca13 and Astex14
have also reported p38 inhibitors that bind to an allosteric
site. In contrast to pure ATP mimics, these compounds
interact, in addition to the active site, with a region on
the kinase that is spatially distinct from the ATP pocket.
Furthermore, they inhibit p38 MAP kinase via a confor-
mation (DFG-out) that is incompatible with ATP bind-
ing. Thus, stabilization of the inactive conformation
should, in principle, lead to improved selectivity across
the kinome due to the inactive or ‘off’ state between ki-
nases being conformationally more different than the acti-
vated or ‘on’ state.15 In addition, selectivity for ATP-
competitive binders under physiological conditions may
be quite different from what is observed in vitro due to
ATP having different affinities for kinases and being pres-
ent at high concentrations.16
Keywords: p38 MAP kinase; Antiinflammatory; SAR; Ketoamides;
Cytokines; Drug-like.
*
Corresponding author. Tel.: +1 619 519 1787; fax: +1 858 538
0946; e-mail: catalaba@hotmail.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.033

A. G. Montalban et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1772–1777
1773
N
O
F
N
O
N
S
H
N
O
N
N
O
N
H
N
S
N
H
O
F
N
N
Cl
Cl
BIRB-796
SB203580
VX-745
F
The above considerations, and the limited number of
noncompetitive or allosteric, as compared to competi-
tive or orthosteric, p38 inhibitors studied thus far, led
us to believe that it could be an attractive strategy to de-
liver a small molecule p38 therapeutic. We chose the cel-
lular TNF-a inhibitory assay to guide our SAR efforts
since it has been previously used to successfully identify
potent p38a inhibitors.11b During the screening of our
compound collection, the indazol derivative 1 (TNF-a
IC50 = 2.9 lM) was identified. We noticed the partial
structural overlap to BIRB-796, and herein we report
how 1 evolved to a potent new structural class of alloste-
ric p38 inhibitors based on an a-ketoamide scaffold
using BIRB-796 as a template.
the backbone amide hydrogen of Met109 (Fig. 1). The
prototype a-ketoamide 2 was prepared, tested in vitro
and found to have a TNF-a IC50 of 440 nM. Table 1
summarizes the results of our subsequent SAR survey.
O
O
O
N
N
N
N
H
N
H
N
H
N
N
N
N
NH
O
O
1
III
II
Ketoamides 2–10 were synthesized, as shown in Scheme 1,
through reaction of the corresponding pyrazolamines 16
with the acid chloride (20) derived from naphthol ether
17. The pyrazole precursors were prepared via conden-
sation of the correspondingly substituted hydrazines
and 4,4-dimethyl-3-oxopentanenitrile in refluxing
toluene.12 Acid chloride 20, on the other hand, was syn-
thesized through the acylation of naphthol ether 17,20
followed by the hydrolysis of the resulting ester 18 and
the treatment of acid 19 with oxalyl chloride.
Molecular modeling17 suggests that, similar to BIRB-
796,12 compound 1 binds to the allosteric site of p38
whereby the tert-butyl group occupies the exposed
Phe169 hydrophobic pocket and the amide simulta-
neously hydrogen bonds via the carbonyl with the back-
bone amide hydrogen of Asp168 and through the NAH
with the carboxylate of Glu71. The nitrogen at position
two of the indazole, on the other hand, acts as a hydro-
gen-bond acceptor for the amide hydrogen of the con-
served Lys53 residue which would otherwise form a
salt-bridge with the carboxylate of Glu71, whereas the
nitrogen at position one does not seem to form any con-
tributing binding interactions. In contrast to BIRB-
796,12 the rigidity of the indazol system does not place
the phenyl ring favorably within the kinase specificity
pocket and, therefore, does not appear to form a good
edge-to-face interaction with the phenyl of the displaced
Phe169 side chain. By analogy with the BIRB-796
series12 and the goal of increasing the flexibility of the
phenyl ring while retaining the beneficial hydrogen-bond
acceptor atom at position two, the bicyclic structure was
replaced by a monocyclic system. Indeed, modeling of
a-ketoamide II showed it to be a better fit, with the phe-
nyl ring interacting with the specificity pocket similar to
SB203580-type inhibitors.18 This interaction was further
accentuated in the naphthyl analogue III in which the
second ring of the naphthyl moiety resides deep within
the kinase specificity pocket while the first one maintains
the ideal edge-to-face interaction with Phe169 in a sim-
ilar manner to BIRB-796.12 We then decided to attach
the ethoxy morpholino group to the 4-position of the
naphthalene in order to reach into the ATP binding re-
gion of p38. Again, docking results19 showed that the
extended molecular architecture of compound 2 binds
to the DFG-out conformation of p38, allowing it to
simultaneously interact with the active site and the allo-
steric pocket as is the case with BIRB-796.12 Thus, in
addition to all the above binding interactions, the mor-
pholino group forms a conserved hydrogen bond with
The synthesis of compound 11 started with the BOC-
protection (22) of commercially available amino-naph-
thol 21 followed by alkylation with 2-chloroethylmorpholine
to give 23, deprotection (24), subsequent Sandmeyer
reaction (25), and acylation to give ester 26. The
reaction of ester 26 with the anion derived from
Figure 1. Docked structure of compound 2 in a thick tube drawing to
p38a using the induced fit docking protocol based on the X-ray
structure of p38a cocrystallized with BIRB-796 (pdb1kv2.ent). Only
˚
residues within 5 A from compound 2 are shown for clarity. Hydrogen
bonds between the inhibitor and residues Glu71, Lys53, and Met109
are shown in green line.

1774
A. G. Montalban et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1772–1777
By analogy with BIRB-79612 and as our modeling sug-
gested, the group at position one of the pyrazole ring is
hypothesized to be in close proximity and could, there-
fore, favorably interact with the hydrophobic portion
of the side chain of the conserved residue of Glu71. While
the lack of substitution (3) or a plain phenyl (4) resulted
in a decreased potency, the m-substituted phenyl deriva-
tives 5 and 6 and the 3,4-disubstituted phenyl derivative 7
showed similar activities to 2. The o-tolyl derivative 8, on
the other hand, appeared again to be less potent, suggest-
ing that lack of either rotational freedom or steric bulk
around the ketoamide NH decreased the binding energy.
The combination of both a 3-fluoro- and a 4-methyl-
group as in 9 resulted in a favorable potency trend and
perhaps hinting to a preferred phenyl substitution pat-
tern. Interestingly, the methyl analogue 10 was as potent
as the parent compound 2. Next, we turned our attention
to the naphthyl ring-system. Our modeling suggested
that, in addition to the favorable pAp edge-to-face inter-
action with the phenyl ring of Phe169, substitution at the
6- and/or 7-positions of the naphthalene could further
strengthen the lipophilic interaction with the kinase spec-
ificity pocket. The 6-chloro analogue 11 was prepared but
led only to a decrease in potency, suggesting a tight fit of
the second naphthyl ring to the specificity pocket, as did
the replacement of the naphthyl- by a phenyl-ring (data
not shown) that is consistent with the calculated binding
energies of II, III, and related BIRB-796 analogues.12
Other changes and combinations (data not shown) inves-
tigated that also trended to less TNF-a inhibitory activity
include modifications of the tert-butyl group, substitu-
tion at the 4-position of the pyrazole ring, alkylation of
the amide nitrogen, removal or changing the position of
the ethoxymorpholino group, homologation of the ethyl
linker, substitution at the 3- and/or 5-positions, and the
replacement of the morpholino group for other heterocy-
clic- and ring-open-analogues. These data are generally
consistent with the related BIRB-796 series12 and suggest
that (a) the tert-butyl group has about the optimal size for
the Phe169 hydrophobic pocket; (b) a substituent adja-
cent (4-position of pyrazole) to it is not well tolerated,
probably for conformational or steric reasons; (c) the
hydrogen bond interaction between the amide NAH
Table 1. TNF-a inhibition data for pyrazole a-ketoamide derivatives
N
O
Y
O
O
X
N
N
H
N
R
Compound
R
X
Y
TNF-a
IC50 (lM)
a
BIRB-796
2
p-Tolyl
p-Tolyl
H
NH
CO
CO
CO
CO
CO
H
H
H
H
H
H
H
H
H
H
0.013
0.44
1.5
3
4
Phenyl
m-Tolyl
3-Fluorophenyl
2.1
5
0.34
0.44
0.45
1.5
6
7
3,4-Dimethylphenyl CO
o-Tolyl CO
3-F-4-Methylphenyl CO
8
9
0.22
0.44
10
11
12
13
14
15
Me
CO
CO
p-Tolyl
p-Tolyl
p-Tolyl
p-Tolyl
p-Tolyl
Cl 2.6
CNOH
CNOMe
CH2
H
H
H
H
0.023
1.5
1.4
0.97
CHOH
a IC50 of LPS-stimulated TNF-a production in the immortalized
human cells of a monocytic lineage (THP-1). IC50’s given represent
the means of a minimum of two, and generally three or more,
independent experiments. Standard deviation for assays typically
30% of the mean or less.
3-amino-5-tert-butyl-2-tolyl-2H-pyrazole under micro-
wave irradiation gave the above final compound
(Scheme 2).
Oxime 12 (X = C@NOH) and methyloxime 13
(X = C@NOMe) were readily prepared as a mixture of
geometrical isomers by treating 2 with hydroxylamine
and O-methylhydroxylamine, respectively. The reduc-
tion of 2, on the other hand, gave alcohol 15
(X = CHOH) whereas 14 was prepared from acid chlo-
ride 30 (X = CH2), which was in turn synthesized from
a-ketoester 18 as shown in Scheme 3.
RNHNH2
CN
N
NH2
toluene
reflux
O
N
R
16
N
O
O
O
O
O
N
N
N
N
H
iv
N
R
O
O
O
2-10
i
O
17
O
R'
18 R' = OMe
19 R' = OH
ii
iii
20
R' = Cl
Scheme 1. Reagents and conditions: (i) CH2Cl2, AlCl3, rt then methyl oxalyl chloride, 16 h, 97%; (ii) 6 N HCl, 80 °C, 16 h, 59%; (iii) CH2Cl2, DMF
(cat), oxalyl chloride, rt, 2 h; (iv) EtOAc, 0.5 N NaHCO3, 60 °C, 16 h.

A. G. Montalban et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1772–1777
1775
O
O
N
N
N
O
O
O
OH
O
O
N
ii
v
N
H
vi
N
O
Y
Y
Cl
Cl
11
O
OMe
26
O
21 Y = NH2
22 Y = NHBOC
23 Y = NHBOC
24 Y = NH2
i
iii
iv
25
Y = Cl
Scheme 2. Reagents and conditions: (i) EtOAc, 0.5 N NaHCO3, BOC2O, 60 °C, 16 h, quantitative; (ii) MeCN, K2CO3, N-(2-chloroethyl)-
morpholine hydrochloride, 75 °C, 16 h, 4.4%; (iii) CH2Cl2, TFA, Et3SiH, rt, 60 h, quantitative; (iv) 6 N HCl, NaNO2, 0 °C, 1 h then CuCl, 100 °C,
1 h, 11%; (v) CH2Cl2, AlCl3, rt then methyl oxalyl chloride, 16 h, 83%; (vi) 3-amino-5-tert-butyl-2-tolyl-2H-pyrazole, dioxane/DMF, BuLi, 150 °C,
microwave, 5 min, 12%.
Table 2. Comparison of IC50 data of selected compounds for the
biochemical-, cell-based-, and phospho-p38 assay
O
N
O
X
i
12
13
15
Compound
p38a IC50
(lM)
TNF-a IC50
(lM)
PP38-a IC50
(lM)
v iii
ii
14
2
BIRB-796
SB203580
0.044
0.039
0.029
0.32
0.013
0.16
0.017
6.0
5.3
iii
O
R'
18 X = CO,
27 X = CHOH, R' = OMe
VX-745
2
0.039
0.44
0.22
R' = OMe
iv
v
v i
v ii
0.31
0.30
0.15
28
X = CH2
29 X = CH2
30
,
,
,
R' = OMe
R' = OH
R' = Cl
10
12
0.15
0.023
X = CH2
0.023
IC50’s given represent the means of a minimum of two, and generally
three or more, independent experiments. Standard deviation for assays
typically 30% of the mean or less.
Scheme 3. Reagents and conditions: (i) NH2OHÆHCl, EtOH, pyridine,
45 °C, 12 h, 58%; (ii) NH2OMeÆHCl, EtOH, pyridine, 45 °C, 12 h, 76%;
(iii) MeOH, NaBH4, rt, 1 h, 12%;(iv)MeOH, Pd/C(10%wt), H2, rt, 16 h;
(v) CH2Cl2, TFA, Et3SiH, rt, 16 h; (vi) MeOH/THF, 2 N NaOH, rt, 4 h;
(vii) CH2Cl2, DMF (cat), oxalyl chloride, rt, 4 h; (viii) 3-amino-5-tert-
butyl-2-tolyl-2H-pyrazole, EtOAc, 0.5 N NaHCO3, 60 °C, 16 h.
was indistinguishable from the cell-based data when tak-
ing the different assay conditions and intra-assay vari-
ability into account.
and Glu71 is essential for binding; (d) the naphthalene 1,4
substitution pattern is ideal for orienting the groups
interacting with the Phe-out pocket and ATP binding re-
gion; and (e) with the ethoxy linker providing optimally
spaced binding moieties. Molecular modelling, on the
other hand, suggests that the alpha-keto group forms a
hydrogen bond interaction with the amide hydrogen of
the conserved Lys53 residue (vide supra). We reasoned,
therefore, that chemical modifications of the a-ketoa-
mide scaffold could probe this hypothesis and also result
in additional binding interactions not seeing with the
urea moiety of the BIRB-796 series.12 The conversion
of 2 to its corresponding oxime 12, for example, resulted
in a TNF-a inhibitory activity of 23 nM. This improve-
ment in potency may be due to the hydrogen bond inter-
action observed in the docked structure for the oxime
OAH with one of the amide carbonyl groups of the con-
serve residue of Glu71. In contrast, the urea hydrogen of
BIRB-796 in the same region forms a hydrogen bond to
the carboxylate oxygen of Glu71.12 Consequently, oxime
ether 13 and deoxygenated analogue 14, lacking either
one or both of these important hydrogen-bond donor/
acceptor moieties (e.g. carbonyl or oxime), were much
less potent. Reduction to alcohol 15 also resulted in
decreased potency.
The conformational change (DFG-in ! DFG-out) pre-
vents the activating upstream kinase (MKK3/MKK6)21
from phosphorylating p38. This conformational change
can be indirectly measured by a phosphorylation inhi-
bition assay. The same set of compounds (Table 2)
was tested in this assay and the results suggest that
they caused the necessary conformational change to in-
hibit p38 phosphorylation similar to BIRB-796. In con-
trast, the orthosteric/competitive p38 inhibitors
SB203580, and VX-745 did not prevent p38a from
being phosphorylated by its upstream kinase. BIRB-
796, SB203580, and VX-745 were measured under the
same conditions as our compounds in all three assays
and the data are included in Table 2 for comparison.
Thus, these results support that our p38 inhibitors
are likely to bind to the DFG-out conformation of
the protein. Kinetic studies have not been conducted
to confirm a formal noncompetitive versus competitive
mechanism of enzyme inhibition for compounds of this
class, but based on the reasoning from molecular mod-
eling data, similarity of the pharmacophore to that in
the structurally characterized BIRB796-p38 cocrystal
structure, and the above results for inhibition of the
flexible DFG activation loop, we propose that these
compounds inhibit at least partially via an allosteric/
noncompetitive mode rather than an orthosteric/com-
petitive mode of binding.
Activity in the THP-1 whole cell assay was confirmed
for compounds 2, 10, and 12 in a human p38a kinase as-
say (Table 2). The results from the p38a kinase assay

1776
A. G. Montalban et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1772–1777
Table 3. Single dose plasma pharmacokinetic parameters of 12
following dosing in rats
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. J. Med. Chem.
1999, 42, 2180.
iv
po
Dose (mg/kg)
Tmax (h)
10
30
10. Boehm, J. C.; Smietana, J. M.; Sorenson, M. E.; Garigi-
pati, R. S.; Gallagher, T. F.; Sheldrake, P. L.; Bradbeer, J.;
Badger, A. M.; Laydon, J. T.; Lee, J. C.; Hillegass, L. M.;
Griswold, D. E.; Breton, J. J.; Chabot-Fletcher, M. C.;
Adams, J. L. J. Med. Chem. 1996, 39, 3929.
1.0
4.5
1.4
36
Cmax (lg/mL)
T1/2 (h)
AUC (lg h/mL)
po F%
100
11. Merck: (a) Colletti, S. L.; Frie, J. L.; Dixon, E. C.; Singh,
S. B.; Choi, B. K.; Scapin, G.; Fitzgerald, C. E.; Kumar,
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G.; Samuel, K.; Schmatz, D. M.; Schwartz, C. D.; Shoop,
W. L.; Thompson, C. M.; Thompson, J. E.; Wang, R.;
Woods, A.; Zaller, D. M.; Doherty, J. B. J. Med. Chem.
2003, 46, 349, and references therein; Procter and Gamble:
(b) Laughlin, S. K.; Clark, M. P.; Djung, J. F.; Gole-
biowski, A.; Brugel, T. A.; Sabat, M.; Bookland, R. G.;
Laufersweiler, M. J.; VanRens, J. C.; Townes, J. A.; De,
B.; Hsieh, L. C.; Xu, S. C.; Walter, R. L.; Mekel, M. J.;
Janusz, M. J. Bioorg. Med. Chem. Lett. 2005, 15, 2399,
and references therein; BMS/Pharmacopeia: (c) Liu, C.;
Wrobleski, S. T.; Lin, J.; Ahmed, G.; Metzger, A.; Wityak,
J.; Gillooly, K. M.; Shuster, D. J.; McIntyre, K. W.; Pitt,
S.; Shen, D. R.; Zhang, R. F.; Zhang, H.; Doweyko, A.
M.; Diller, D.; Henderson, I.; Barrish, J. C.; Dodd, J. H.;
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6261, and references therein; Takeda: (d) Miwatashi, S.;
Arikawa, Y.; Kotani, E.; Miyamoto, M.; Naruo, K.-I.;
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Chem. 2005, 48, 5966, and references therein; Pfizer: (e)
McClure, K. F.; Abramov, Y. A.; Laird, E. R.; Barberia,
J. T.; Cai, W.; Carty, T. J.; Cortina, S. R.; Danley, D. E.;
Dipesa, A. J.; Donahue, K. M.; Dombroski, M. A.;
Elliott, N. C.; Gabel, C. A.; Han, S.; Hynes, T. R.;
LeMotte, P. K.; Mansour, M. N.; Marr, E. S.; Letavic, M.
A.; Pandit, J.; Ripin, D. B.; Sweeney, F. J.; Tan, D.; Tao,
Y. J. Med. Chem. 2005, 48, 5728; Locus: (f) Michelotti, E.
L.; Moffett, K. K.; Nguyen, D.; Kelly, M. J.; Shetty, R.;
Chai, X.; Northrop, K.; Namboodiri, V.; Campbell, B.;
Flynn, G. A.; Fujimoto, T.; Hollinger, F. P.; Bukhtiyar-
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Alfredson, T.; Bertrand, J.; Browner, M. F.; Clifford, K.;
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Labadie, S. S.; La Fargue, J.; Lapierre, J. M.; Larrabee, S.;
Li, F.; Papp, E.; McWeeney, D.; Ramesha, C.; Roberts,
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Cl (L/h/kg)
Vdss (L/kg)
0.90
1.9
In vitro ADME evaluation of our a-ketoamide series
was consistent with drug-like characteristics. This was
further confirmed by in vivo rat snapshot PK studies
of compound 12 for which the parameters are summa-
rized in Table 3.
In summary, we have developed a novel series of potent
p38 inhibitors using an a-ketoamide scaffold. Despite
limited enzyme inhibition data, we established SAR
trends that are consistent with the related BIRB-796
series and achieved potencies in the double digit nano-
molar range. We further believe, based on computa-
tional modeling and a phospho-p38a inhibition assay,
that these compounds are novel allosteric p38 inhibitors.
This series of inhibitors shows potential for the develop-
ment of an oral treatment for inflammatory conditions
and further optimization of this novel scaffold will be
reported in due course.
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17. Models of p38 in the DFG-out and DFG-in conforma-
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BIRB-796 and one of its analogues (pdb1kv1.ent and
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using the docking Software GLIDE (v4.0, Schro¨dinger,
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the PRIME comparative modeling software package
(v.1.2, Schro¨dinger, LLC, New York, USA) using the
above X-ray crystal structure as a template. Compound 2
was then docked using the induced fit protocol (GLIDE
v4.0 and PRIME v1.5, Schro¨dinger, LLC, New York,
USA); (b) Sherman, W.; Day, T.; Jacobson, M. P.;
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