'Reverse' α-ketoamide-based p38 MAP kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.09.028

We have identified a second series of potent p38 inhibitors. As with our first generation series, these compounds are based on an α-ketoamide scaffold. The reversal of the ketoamide order, however, introduces more chemical flexibility and in addition resu

Full text of DOI:10.1016/j.bmcl.2008.09.028

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5456–5459
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
‘Reverse’
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, Andrew Gibbs, Jeff Kahl, Linda Kessler, Jan Lundström,
Stephen Miller, Hiroshi Nakanishi, Ed Roberts, Eddine Saiah, Robert Sullivan, Zhijun Wang and
Christopher J. Larson
Drug Discovery, Kemia, Inc., 5871 Oberlin Drive, Suite 100, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2008
Revised 6 September 2008
Accepted 8 September 2008
Available online 11 September 2008
We have identified a second series of potent p38 inhibitors. As with our first generation series, these com-
pounds are based on an
a-ketoamide scaffold. The reversal of the ketoamide order, however, introduces
more chemical flexibility and in addition results in improve potencies against p38.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
p38 MAP kinase
Antiinflammatory
SAR
Ketoamides
Cytokines
Drug-like
The mitogen-activated protein (MAP) kinase p38 has been
recognized as a highly attractive target for therapeutic intervention
due to its role in the stress-activated signal transduction pathway
leading to the release of proinflammatory cytokines such as
tumour necrosis factor-
is well established that these cytokines play an important role in
This limited number of noncompetitive, or allosteric, as compared
to competitive, or orthosteric, p38 inhibitors led us to believe that
it could be an attractive strategy to deliver a small molecule p38
therapeutic. We recently identified an indazol analogue (1) via high
throughput screening (HTS) from which we derived a new struc-
tural class of potent, at least partially noncompetitive/allosteric,
p38 inhibitors related to BIRB-796.5 Herein, we now report that
a (TNF-a
) and interleukin-1b (IL-1b).1 It
the pathogenesis of various inflammatory diseases.
After the ground breaking work on allosteric p38 inhibitors by
BI that led to BIRB-796,2 AstraZeneca3 and Astex4 followed with
their own versions of p38 inhibitors that bind to an allosteric site
(noncompetitive inhibitors).
reversing the ketoamide order of our first generation a-ketoamide
p38 inhibitors of type 2 not only leads to more diverse libraries
but also results in drug-like compounds with improve potencies
against p38. We again chose the cellular TNF-
a
inhibitory assay to
guide our SAR efforts.
N
O
O
N
O
O
N
O
N
O
O
N
N
H
N
N
H
N
N
N
N
H
N
H
N
NH
N
BIRB-796
F
O
O
AstraZeneca
Astex
Reverse ketoamides 3–5, 8 and 9 were assembled through reaction
of the corresponding acid chlorides 17 and 23, derived from
a
* Corresponding author. Tel.: +1 853 232 4672.
E-mail address: catalaba@hotmail.com (A.G. Montalban).
-ketoacids 16 and 22, with aminonaphthalene 18.2 Two different
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.028

A. G. Montalban et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5456–5459
5457
Our initial SAR survey is summarized in Table 1 and consistent
(3–7) with the trends and potencies we reported previously for our
O
first generation a
-ketoamide p38 inhibitors (2),5 for which we pos-
O
N
N
H
O
tulate that in addition to the ATP cavity of p38, these compounds
also bind to an allosteric site. In this binding mode the t-butyl
group occupies the exposed Phe169 hydrophobic pocket, 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 and the morpholino group forms a
conserved hydrogen bond with the backbone amide hydrogen of
Met109. In addition, we suggest that the improved potencies ob-
served for the oximes (2 X = NOH) are due to additional binding
interactions in which the oxime O–H appears to hydrogen bond
to one of the amide carbonyl groups of the conserve residue of
Glu71. Although, docking results suggest that the amide in 2 simul-
taneously hydrogen-bonds to the backbone amide hydrogen of
Asp168 and the carboxylate of Glu71 via the carbonyl oxygen
and N–H, respectively, this could not be unambiguously confirmed
for the new reverse ketoamide series through molecular modelling.
Inhibition of p38 phosphorylation by its upstream kinase, however,
proved supportive (see Table 3) of a similar binding mode since the
conformation in which p38 is locked when inhibited allosterically
(vide supra) is incompatible with ATP binding (Fig. 1).
N
N
N
NH
N
O
N
H
N
R
X
1
2
synthetic routes were explored for the synthesis of the requisite
ketoacids as shown below. First, copper6 catalyzed cross-coupling
reaction of pyrazole 10 with the appropriate arylboronic acid gave
the esters 11 which after reduction (12) followed by Dess–Martin
oxidation yielded the aldehydes 13. The aldehydes 13 were in turn
reacted with TMSCN to give the cyanohydrins 14 which after con-
comitant deprotection and hydrolysis (15) were oxidized to give
the pyrazol-a-ketoacids 16 (R = aryl).
The second route is exemplified for furan-a-ketoacid 22
(Scheme 2).7 Treatment of the furan derivative 19 with (cyanom-
ethylene)phosphorane in the presence of EDCI gave cyanoketo-
phosphorane 20. Dimethyldioxirane oxidation in MeOH
converted 20 into
requisite acid 22.
The oxime 6 was readily prepared as a mixture of geometrical
isomers by treating 3 with hydroxylamine in the presence of
pyridine (Scheme 1). Reduction of 4 with NaBH4, on the other
hand, gave the alcohol 7 (Scheme 1).
a-ketoester 21 which after hydrolysis gave the
Interestingly, the methylated derivative 8 (Table 1) was signifi-
cantly more potent than its first generation ketoamide counterpart
(2 R = Me, X = O, TNF-
a IC50 = 0.44 l
M).5 This was further con-
firmed by the activity of compound 9 in which the pyrazole moiety
was replaced by a furan-ring. The same chemical change proved
OEt
OTMS
CN
i
iv
N
N
H
N
N
X
N
R
N
R
O
10
14
11 X = CO2Et
12 X = CH2OH
13 X = CHO
ii
iii
v
N
O
O
O
O
viii
N
N
H
N
vi
OH
N
R
N
N
R
N
R
O
X
O
CO2H
O
N
3-5, 8
O
16 X = OH
17 X = Cl
vii
15
ix
x
18
6
7
NH2
Scheme 1. Reagents and conditions: (i) CH2Cl2, pyridine, ArB(OH)2, Cu(OAc)2, 4 Å molecular sieves, rt, 16 h; (ii) THF, DIBAL-H, rt, 1 h; (iii) CH2Cl2, Dess–Martin periodinane, rt,
30 min; (iv) THF, TMSCN, n-BuLi(cat), 0 °C to rt, 16 h; (v) MeCN, cHCl, 80 °C, 16 h then MeOH, KOH, reflux, 2 h; (vi) CH2Cl2, Dess–Martin periodinane, rt, 30 min; (vii) CH2Cl2,
DMF(cat), oxalyl chloride, rt, 1 h; (viii) EtOAc, 0.5 N NaHCO3, rt, 16 h; (ix) EtOH, NH2OHÁHCl, pyridine, 45 °C, 12 h; (x) EtOH, NaBH4, rt, 12 h.
O
Ph3P
i
ii
OH
X
O
CN
O
O
O
O
O
21 X = OMe
22 X = OH
23 X = Cl
20
iii
iv
19
Scheme 2. Reagents and conditions: (i) CH2Cl2, (cyanomethylene)-phosphorane, EDCI, DMAP, rt, 16 h, 81%; (ii) CH2Cl2/MeOH, dimethyldioxirane, rt, 30 min, 92%; (iii) THF,
1 N LiOH, 50 °C, 1 h; CH2Cl2, DMF(cat), oxalyl chloride, rt, 1 h.

5458
A. G. Montalban et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5456–5459
Table 1
ATP Binding Site
P38-a inhibition data for reverse ketoamide derivatives
Phe169 Pocket
Asp (168)
NH2
N
O
Met (109)
H2N
N
O
O
O
O
O
N
H
X
Z
N
X
Y
R
Y
R
H
O
CO2
-
Kinase Specificity
Pocket
Glu (71)
a
Compound
R
X
Y
Z
TNF-a IC50 (lM)
Figure 1. Simplified proposed binding mode of reverse a-ketoamides to p38a.
3
4
5
6
7
8
9
m-Tolyl
p-Tolyl
3,4-Difluorophenyl
m-Tolyl
p-Tolyl
Me
N
N
N
N
N
N
O
N
N
N
N
N
N
C
CO
CO
CO
CNOH
CHOH
CO
0.28
0.33
0.18
0.049
3.3
synthetically more challenging with the first generation ketoamide
arrangement. In fact, we were not able to prepare any first gener-
ation ketoamide in which the pyrazole moiety was replaced by a
furan-ring. We speculate, however, that the improved potencies
of compounds 8 and 9 could be due to the repulsive interaction be-
tween the carbonyl group directly attached to the pyrazole or furan
and the carboxylate of Glu71. As a result the carbonyl group and
heteroaromatic ring may be more inclined to achieve coplanarity
(which is less hindered by a smaller R-group) and, as our model-
ling8 suggests and in contrast to our first generation ketoamides,
adopt a conformation better suited for binding to the hydrophobic
portion of the side chain of the conserved residue of Glu71. The
oxygen in 2 (X = O), on the other hand, acts as a hydrogen-bond
acceptor for the amide hydrogen of the conserved Lys53 residue
which would otherwise form a salt-bridge with the carboxylate
of Glu71.5
0.070
0.061
Me
CO
a IC50 of LPS-stimulated TNF-
a production in 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.
Table 2
P38-
a
inhibition data for furano ketoamide derivatives
R
O
Other changes that resulted in improved potencies over our first
generation ketoamide series were replacement of the ethoxy- for
more rigid-spacers (Table 2). The first generation ketoamide 27,
N
H
O
O
for example, inhibited TNF-a production with an IC50 of 0.85 lM
whereas the related reverse ketoamide analogue 24 was 20-fold
more potent. Similar potencies were obtained with an aminopy-
rimidine spacer whereby the pyrimidine nitrogen at the 4-position
with respect to the amino linker appears to be better placed to
interact with the amide hydrogen of Met109 than at the 2-position
consistent with the docked structures of 25 and 26, respectively. In
contrast, compounds from our first generation ketoamide series
containing an aminopyrimidine spacer trended towards reduced
a
Compound
R
TNF-a IC50 (lM)
24
0.040
N
N
O
25
26
0.059
N
HN
N
TNF-a
inhibitory activity.
0.46
N
N
N
HN
N
O
O
N
N
N
a IC50 of LPS-stimulated TNF-
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.
a production in immortalized human cells of a
O
H
27
Compounds 24, 25 and 26 were prepared via palladium catalyzed
cross-coupling reaction of the common intermediate 29 with the
organometallic reagent 30,9 and the corresponding aminopyrimi-
dines, respectively (Scheme 3). Compound 29 was in turn obtained
from reaction of the acid chloride 23 with the commercially avail-
able bromo-derivative 28.
Table 3
Comparison of IC50 data of selected compounds for the biochemical-, cell-based- and
phospho-p38 assay
Compound
P38a
IC50
(lM)
TNF-a
IC50
(l
M)
PP38-a IC50 (lM)
Activity in the THP-1 whole cell assay was confirmed for com-
6
8
9
24
0.059
0.014
0.022
0.009
0.049
0.070
0.061
0.040
0.10
0.070
0.27
pounds 6, 8, 9 and 24 in a human p38a kinase assay (Table 3).
The results from the p38 kinase assay was indistinguishable from
a
0.020
the cell-based data when taking the different assay conditions and
intra-assay variability into account.
The same set of compounds (Table 3) was tested in a p38 phos-
phorylation inhibition assay and the results suggest that they
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.

A. G. Montalban et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5456–5459
5459
O
N
N
30
24
Br
SnBu3
ii
Br
N
28
O
NH2
i
H2N
N
23
N
H
O
25
26
ii
ii
O
29
N
H2N
N
Scheme 3. Reagents and conditions: (i) EtOAc, 0.5 N NaHCO3, rt, 16 h, 87%; (ii) Pd(OAc)2, BINAP, Cs2CO3, toluene, 80 °C, 16 h.
Table 4
References and notes
Single dose plasma pharmacokinetic parameters of 6 following dosing in rats
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Invest. Drugs 2003, 4, 566; (d) Kumar, S.; Boehm, J.; Lee, J. C. Nat. Rev. Drug
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therein.
iv
po
Dose (mg/kg)
Tmax (h)
10
30
1.5
4.0
2.9
26
54
Cmax
(
lg/mL)
T
½
(h)
AUC (
l
g h/mL)
F%
Cl (L/h/kg)
Vdss (L/kg)
0.61
1.5
4. Gil, A. L.; Frederickson, M.; Cleasby, A.; Woodhead, S. J.; Carr, M. G.; Woodhead,
A. J.; Walker, M. T.; Congreve, M. S.; Devine, L. A.; Tisi, D.; O’Reilly, M.; Seavers,
L. C. A.; Davis, D. J.; Curry, J.; Anthony, R.; Padova, A.; Murray, C. W.; Carr, R. A.
E.; Jhoti, H. J. Med. Chem. 2005, 48, 414.
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8. Models of p38 in the DFG-out and DFG-in conformations were generated from
the X-ray crystal structures of BIRB-796 and one of its analogues (pdb1kv1.ent
and pdb1kv2.ent) and SB203580 (pdb1au9.ent) respectively, using the docking
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caused the necessary conformational change to inhibit p38
a
phos-
phorylation by its upstream kinase (MKK3/MKK6)10 similar to our
first generation series. These results support that our p38 inhibi-
tors are likely to bind to the DFG-out conformation of the protein
and, as with our first generation ketoamide series, we propose that
these compounds inhibit at least partially via an allosteric/non-
competitive mode rather than an orthosteric/competitive mode
of binding in a manner similar to BIRB-796.2
In vitro ADME evaluation of our reverse a-ketoamide series was
consistent with drug-like characteristics. This was further con-
firmed by in vivo rat snapshot PK studies of compound 6 for which
the parameters are summarized in Table 4.
In summary, we have developed a second generation of potent
a
-ketoamide p38 inhibitors. In contrast to our first generation,
reversing the -ketoamide scaffold results in increase chemical
flexibility and improved potencies against p38. We believe, based
on computational modeling and a phospho p38 inhibition assay,
a
a
that these compounds are novel allosteric p38 inhibitors. This
series of inhibitors show potential for the development of an oral
treatment of inflammatory conditions and further optimization of
this novel scaffold will be reported in due course.