Design and synthesis of novel p38α MAP kinase inhibitors: Discovery of pyrazole-benzyl ureas bearing 2-molpholinopyrimidine moiety

Article abstract of DOI:10.1016/j.bmcl.2012.05.095

The discovery that pyrazole-benzyl urea derivatives bearing a 2-molpholinopyrimidine moiety are novel p38α inhibitors is described. A comparative view of the binding modes of SB-203580 and BIRB-796 by structural alignment of two X-ray co-crystal structures was utilized to identify this novel series. Modification of the benzyl group led to compound 2b, a highly potent p38α inhibitor. In in vivo studies, 2b inhibited the production of tumor necrosis factor-alpha in lipopolysaccharide-treated mouse in a dose-dependent manner. Furthermore, the results of a 5-day repeated oral dose toxicity study suggest that 2b has low hepatotoxicity.

Full text of DOI:10.1016/j.bmcl.2012.05.095

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5118–5122
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel p38a MAP kinase inhibitors: Discovery
of pyrazole-benzyl ureas bearing 2-molpholinopyrimidine moiety
⇑
Tadamasa Arai , Michihiro Ohno, Hideki Inoue, Shinnosuke Hayashi, Takumi Aoki, Hiroe Hirokawa,
Hiroyuki Meguro, Yoko Koga, Keiyu Oshida, Mie Kainoh, Kazuharu Suyama, Hideki Kawai
Pharmaceutical Research Laboratories, Toray Industries, Inc., 6-10-1 Tebiro, Kamakura, Kanagawa 248-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery that pyrazole-benzyl urea derivatives bearing a 2-molpholinopyrimidine moiety are novel
Received 11 April 2012
Revised 22 May 2012
Accepted 28 May 2012
Available online 12 June 2012
p38
structural alignment of two X-ray co-crystal structures was utilized to identify this novel series. Modifi-
cation of the benzyl group led to compound 2b, a highly potent p38 inhibitor. In in vivo studies, 2b
inhibited the production of tumor necrosis factor-alpha in lipopolysaccharide-treated mouse in a dose-
dependent manner. Furthermore, the results of a 5-day repeated oral dose toxicity study suggest that
2b has low hepatotoxicity.
a inhibitors is described. A comparative view of the binding modes of SB-203580 and BIRB-796 by
a
Keywords:
Kinase inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
p38a
Pyrazole-benzyl urea
Docking study
Hepatotoxicity
p38
a
mitogen-activated protein (MAP) kinase belongs to the
(e.g., SB-203580),7 and non-competitive or allosteric inhibitors
(e.g., BIRB-796),3 which interact with a region that is spatially
distinct from the ATP binding pocket (Fig. 1). The X-ray co-crystal
serine/threonine family of kinases and plays an important role in
regulating the production of inflammatory cytokines, including tu-
mor necrosis factor-alpha (TNF-
Biologic agents, such as infliximab, etanercept, and adalimumab,
that block TNF- function have been proved effective for the treat-
a
) and interleukin-1b (IL-1b).1
structure of human p38a MAP kinase and BIRB-796 shows that
conserved residues Asp168-Phe169-Gly170 (DFG) move to a new
a
position during binding.3 In the new conformation (DFG-out),
ment of inflammatory disease, including rheumatoid arthritis,
Crohn’s disease, and psoriasis.2 Consequently, many research
groups have turned their attention to developing orally active
ATP binding to p38a MAP kinase is inhibited.
In addition, it was reported that the dissociation rate of
BIRB-796 was much slower than that of SB-203580 due to the allo-
steric binding mode (DFG-out).3,8 In this context, we speculated
that the allosteric inhibitor would be preferred because of being
expected to be a long-acting drug. We therefore focused on the
small-molecule p38
for treating these diseases. A wide variety of p38
a
MAP kinase inhibitors as promising agents
MAP kinase
a
inhibitors (p38
a inhibitors) have entered clinical trials for the
treatment of chronic inflammatory disease.3,4 However, none of
these inhibitors have come onto the market yet. A critical problem
found in the clinical trials was that the therapeutic effect was not
discovery of novel allosteric p38a inhibitors using BIRB-796 as a
template. On the other hand, binding sites of SB-203580 and
BIRB-796 partially overlap, despite differences in their mode of
action. Accordingly, our strategy relies on a comparative view of
the binding modes of SB-203580 and BIRB-796 by structural align-
ment of two X-ray co-crystal structures ( Fig. 2). Through this
approach, we can visualize the overlap of their phenyl moieties,
the fluorine substituted phenyl group of SB-203580 and naphtha-
lene group of BIRB-796, located within the kinase specificity pock-
et, and the possible structural requirements of BIRB-796 for
allosteric binding mode.
sustained, indicating that p38
cient for the treatment of such chronic disease.4,5 In addition,
p38 inhibitors hold promise as therapeutic agents for acute
a inhibition alone might be insuffi-
a
inflammatory disease. For example, SCIO-469 was found efficacious
in treating acute post-surgical dental pain in a Phase II clinical
trial.6 Accordingly, we have made continuing efforts to discover
novel p38
a inhibitors with drug-like properties.
p38 inhibitors are classified into two types based on their
a
mode of action: ATP-competitive or orthosteric p38
a
inhibitors
Furthermore, two-dimensional (2D) p38a MAP kinase–ligand
interaction maps for SB-203580 and BIRB-796 (Fig. 3) suggest that
the kinase specificity pocket consists of hydrophobic amino acid
residues. We hypothesized that structural modification of the
⇑
Corresponding author. Tel.: +81 467 32 9643; fax: +81 467 32 9931.
E-mail address: Tadamasa_Arai@nts.toray.co.jp (T. Arai).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.095

T. Arai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5118–5122
5119
N
O
O
N
N
N
O
O
N
S
N
H
N
H
N
H
H3C
F
SB-203580
H3C
BIRB-796
Figure 1. Chemical structures of SB-203580 and BRIB-796.
Table 1
p38a inhibitory activity of the pyrazole-benzyl urea derivatives
O
OR
N
N
N
H
N
H
H3C
Compound
R
Inhibition of p38a MAP kinase IC50 (lM)
BIRB-796
1a
—
H
0.010
1.5
6
1
5
N
2
2a
0.012
N
3
N
4
O
Figure 2. Comparative view of the binding modes of SB-203580 and BIRB-796 by
structural alignment of two X-ray co-crystal structures (PDB: 1A9U and 1KV2)
depicted by MolFeat version 4.5.9 White dotted lines indicate hydrogen bonding to
side chain atoms (ꢀ3.0 Å).
N
N
3a
4a
0.013
0.020
N
N
N
inhibitor in the kinase specificity pocket would lead to highly
Cl
potent p38a inhibitors with favorable hydrophobic interactions.
Therefore, we replaced the naphthalene group of BIRB-796 with a
benzyl group, which allowed for synthetic flexibility in structural
modification. Herein, we report the discovery, synthesis, and phar-
macological and toxicological properties of pyrazole-benzyl urea
In the course of our initial screening of pyrazole-benzyl urea
derivatives, compound 1a (IC50 = 1.5
M) was identified.11,12 It
l
derivatives as novel p38a inhibitors.
Figure 3. 2D p38a
MAP kinase–ligand interaction maps made by MOE for (A) SB-203580 (PDB: 1A9U) and (B) BIRB-796 (PDB: 1KV2).10 Amino acid residues within 4.5 Å of
ligand are shown. The active site residues are represented as follows: polar residues, pink; hydrophobic residues, green; acidic residues, red contour ring; basic residues,
bluish contour ring; hydrogen bonding to side chain atoms, green arrow. Bluish ‘clouds’ on ligand atoms indicate their solvent-exposed surface area (a darker, larger cloud
means more solvent exposure). Light blue ‘halos’ around residues indicate the degree of interaction with ligand atoms (a larger, darker halo means more interaction). The gray
line indicates steric space for methyl substitution. The contour line is broken if it is close to an atom that is fully exposed.

5120
T. Arai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5118–5122
served as an ideal platform for accessing the ATP binding pocket.
We then attached a morpholine moiety to the pyrimidine ring of
1a in order to create an interaction with Met109 as in the case of
BIRB-796, whose morpholine oxygen participates in hydrogen
bonding with the backbone NH group of Met109 (Figs. 2 and 3B).
Compound 2a was prepared, and an in vitro assay revealed a dra-
matic improvement in p38 inhibitory activity (IC50 = 0.012 M).
Preliminary screening results are shown in Table 1.
To confirm the above key interaction of 2a with Met109, com-
pounds 3a and 4a, which lack the morpholine moiety as a hydrogen
accepter, were synthesized. To our surprise, 3a and 4a exhibited the
a
l
same p38
IC50 = 0.020
mechanisms of the identified p38
were compared. Figure 4 shows the optimized docking models of
a
inhibitory activity as 2a (3a: IC50 = 0.013
M). To obtain structural insight into the inhibitory
inhibitors, their binding modes
lM; 4a:
l
a
2a in the binding site crevice of p38a MAP kinase calculated with
Molegro Virtual Docker version 5.0.0.13 These studies provided the
rational explanation that the 2-molpholinopyrimidine moiety of
2a potentially formed
Met109 in two different binding modes. In a docking study of 2a
binding to p38 MAP kinase based on the X-ray structure co-
a hydrogen bonding interaction with
a
crystallized with BIRB-796 (PDB: 1KV2), the morpholine oxygen took
part in hydrogen bonding with the backbone NH group of Met109
(Fig. 4A). In a docking study using p38a MAP kinase co-crystallized
with N-(5-Indolyl)-3-fluoro-5-(4-morpholino)benzamide (5)14
developed by Astex (PDB: 1WBV), the nitrogen atom at the 1-posi-
tion of the pyrimidine ring was involved in hydrogen bonding with
the same NH group of Met109 ( Fig. 4B). Thus, 3a and 4a were
presumed to be as potent as 2a because of the latter interaction.
Since the 2-molpholinopyrimidine moiety of 2a provides a superior
scaffold for favorable hydrogen bonding, we then examined substi-
tution of a tert-butyl group and/or tolyl group. As the result, a vari-
ety of functional groups could be well tolerated, although the
combination of tert-butyl and tolyl groups was preferable in this
series of pyrazole-benzyl urea derivatives. Furthermore, our dock-
ing study also suggested that 2a binds to the allosteric site of
p38a MAP kinase similarly to BIRB-796, and that the ring of the ben-
zyl group occupied the kinase specificity pocket. Therefore, we next
turned our attention to modifying the benzyl group of 2a.
The pyrazole-benzyl urea derivatives 2a–h were prepared by a
three step sequence (Scheme 1). First, urea formation was achieved
by coupling the corresponding benzyl amine (7a–h) with carba-
mate 615, giving phenols 1a–h. Introduction of a pyrimidine moiety
to phenols 1a–h, followed by amination at the 2-position of the
pyrimidine ring with morpholine afforded the pyrazole-benzyl
urea derivatives (2a–h).
Figure 4. Comparison of two supposed complex structures by the docking studies
of 2a with two p38
bonding interactions to side chain atoms (ꢀ3.0 Å). (A) Binding mode of docked 2a
with the X-ray structure of p38 co-crystallized with BIRB-796 (PDB: 1KV2). (B)
a X-ray crystal structures. White dotted lines show hydrogen
The synthesized compounds were evaluated for their inhibitory
a
effects on recombinant human p38a activity and lipopolysaccha-
Binding mode of docked 2a with the X-ray structure of p38
a
co-crystallized with 5
ride (LPS)-stimulated TNF-
a
production in human whole blood
(PDB: 1WBV).
O
O
OH
R2
OH
N
N
Cl
Cl
R2
a
N
N
N
H
O
N
H
N
H2N
+
H
Cl
R1
R1
H3C
H3C
6
7a-h
1a-h
N
N
O
O
N
Cl
O
O
N
N
N
N
R2
c
b
R2
O
N
N
N
N
N
H
N
H
H
H
R1
R1
H3C
H3C
4a-h
2a-h
Scheme 1. Reagents and conditions: (a) iPr2NEt, CH3CN, 60 °C, 60% to quant.; (b) 2,6-dichloropyrimidine, aqueous NaOH, acetone, rt, 57% to quant.; (c) morpholine, Na2CO3,
EtOH, rt to 60 °C, 51–96%.

T. Arai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5118–5122
5121
Table 2
Modification of benzyl group of 2a
N
O
O
N
N
N
R2
O
N
N
H
N
H
R1
H3C
Compound
R1
R2
Inhibition of p38
a
MAP kinase IC50
(
lM)
Inhibition of TNF-
a production in
HWB IC50
(lM) LPS-treated mouse
Inhibition % at 50 mg/kg, p.o.
BIRB-796
—
H
F
Cl
CH3
CF3
F
—
H
H
H
H
H
F
Cl
CH3
0.010
0.012
0.004
0.005
0.013
0.035
0.003
0.011
0.014
0.60
0.72
0.23
0.19
0.97
>10
0.048
0.11
N.D.a
82
65
2a
2b
2c
2d
2e
2f
84
81b
N.D.a
N.D.a
N.D.a
N.D.a
N.D.a
2g
2h
F
F
a
Not determined.
At 100 mg/kg.
b
(HWB) (Table 2).12,16 In addition, we examined several compounds
from this series for in vivo efficacy at a single dose (50 mg/kg, p.o.)
in a mouse model of endotoxic shock.12,17 According to the 2D
interaction maps shown in Figure 3, the kinase specificity pocket
consisted of hydrophobic amino acid residues such as valine, leu-
2000
1500
1000
cine, and alanine; aiming to increase p38a inhibitory activity, we
##
introduced various hydrophobic substituents onto the ring of the
benzyl group. Fluoro derivative 2b (R1 = F) and chloro derivative
2c (R1 = Cl) showed significantly improved inhibition of both
##
##
500
0
p38
duction of a methyl group (R1 = CH3) or a trifluoromethyl group
(R1 = CF3) on the benzyl urea moiety were not effective for p38
inhibition (2d: IC50 = 0.013 M; 2e: IC50 = 0.035 M). Moreover,
compared with 2a and 2c, fluoro derivative 2b exhibited higher
inhibition of TNF- production in LPS-treated mouse. We then fur-
a and TNF-a production in HWB. On the other hand, the intro-
a
l
l
Control
10
30
50
2b
a
ther modified the benzyl group of 2b.
(mg/kg p.o.)
Although difluoro derivative 2f (R1 = R2 = F) was the most po-
Figure 5. In vivo effect of 2b on TNF-a production in LPS-treated mouse. Data are
tent inhibitor in this series (p38
a: IC50 = 0.003 lM; TNF-a in
expressed as mean S.E. values for three or six animals in each group. ##p <0.01:
Significant difference between the control group and 2b-treated group determined
by Dunnett’s test.
HWB: IC50 = 0.048 M), it was found to be less stable during hu-
l
man cytochrome P450 (CYP) metabolism (Intrinsic clearance 2f:
>0.80 mL/min/mg; 2a: 0.23 mL/min/mg). Compared with 2a
(LogP = 4.89), difluoro derivative 2f is significantly more lipophilic
(LogP = 5.91) due to the introduction of two fluorine substitu-
ents.18 Thus, this higher lipophilicity is considered a reason for
the lability of 2f during human CYP metabolism. Compound 2g
gated how blood chemistry changes in mouse treated with BIRB-
796.20 The plasma levels of AST, total bilirubin, and direct bilirubin,
which are biomarkers of hepatotoxicity, were increased in the 500
and 1000 mg/kg groups. On the other hand, ALT and AST values
were not significantly raised by 5-day repeated oral administration
of 2b in mouse even at the maximum investigated dose of
2000 mg/kg (Table 3). These results suggested that 2b was possibly
a less hepatotoxic candidate.
(R1 = F, R2 = Cl) and 2h (R1 = F, R2 = CH3) showed lower p38
itory activity (2g: IC50 = 0.011 M; 2h: IC50 = 0.014 M) relative to
2b. These results suggest that introduction of a hydrophobic group
as the R1 and/or R2 substituents contributed to high p38
inhibi-
a inhib-
l
l
a
tory activity through favorable hydrophobic interactions with the
kinase specificity pocket. However, these interactions were af-
fected by substituent size. A detailed in vivo study of fluoro deriv-
ative 2b was shown in Figure 5. This compound inhibited TNF-
production in LPS-treated mouse in a dose-dependent manner
In conclusion, we discovered pyrazole-benzyl urea derivatives to
be novel p38
MAP kinase suggested that the 2-molpholinopyrimidine moiety of
2a potentially formed hydrogen bonding interaction with
a inhibitors. Our docking models using 2a and p38a
a
a
Met109 in two different binding modes: between the morpholine
oxygen and the backbone NH group, and between the nitrogen atom
at the 1-position of the pyrimidine ring and the same NH group.
These pyrazole-benzyl urea derivatives are novel allosteric inhibi-
tors, and their benzyl group appears to occupy the kinase specificity
after a single oral administration (ED50 = 16 mg/kg).
Schreiber et al. have reported that the increases of alanine
transaminase (ALT) and aspartate aminotransferase (AST) were
slightly higher and more frequent in BIRB-796 groups than in the
placebo group in a clinical trial.19 Recently, Iwano et al. investi-

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T. Arai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5118–5122
9. MolFeat, version 4.5; FiatLux Co.; Tokyo, Japan. Available at http://
Table 3
www.fiatlux.co.jp (accessed April 2012).
Summary of blood chemistry after 5-day repeated oral administration of 2b in the
mouse21
10. Molecular Operating Environment (MOE), version 2011; Chemical Computing
Group: Montreal, Quebec, Canada. Available at http://www.chemcomp.com
(accessed April 2012).
Dose (mg/kg)
Control
24
45 11
500
1000
2000
11. Procedure for p38
1.5 nM), biotinylated activating transcription factor 2 (ATF2; upstate, 30 nM),
adenosine triphosphate (ATP; 100 M), and an investigated compound was
a MAP kinase activity assay: p38a MAP kinase (PanVera,
ALT (U/L)
AST (U/L)
4
26
42
4
8
32
47
5
4
28 10
46 15
l
incubated in kinase buffer (20 mM HEPES, 10 mM MgCl2, 1 mM dithiothreitol,
0.01% Tween 20, pH 7.0) for 1 h at room temperature. Phosphorylation of ATF2
aData are expressed as mean SD values for four or five animals in each group.
bEach group was compared with the control group by Dunnet’s multiple compari-
son test or Steel’s test.
by p38a MAP kinase was determined by using anti-phospho-ATF2 antibody
(cell signaling) and an AlphaScreen IgG detection kit (PerkinElmer).
12. Data analysis: IC50 and ED50 were calculated by nonlinear regression with
sigmoidal dose response curve fitting using Prism version 4.02 (GraphPad
Software). Statistical significance was determined by Dunnett’s test using SAS
System version 8.2 (SAS Institute).
pocket according to our docking study. Among the compounds syn-
thesized to modify the benzyl group, fluoro derivative 2b exhibited
highly potent p38
ited the production of TNF-
a
inhibitory activity (IC50 = 0.004
l
M), and inhib-
13. Thomsen, R.; Christensen, M. H. J. Med. Chem. 2006, 49, 3315.
14. Gill, 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.; Davis, D. J.; Curry, J.; Anthony, R.; Padova, A.;
Murray, C. W.; Carr, R. A. E.; Jhoti, H. J. Med. Chem. 2005, 48, 414.
15. Zang, L.-H.; Zhu, L. PCT Int. Appl. WO 01/04115, 2001.
a
in LPS-treated mouse in a dose-depen-
dent manner after a single oral administration (ED50 = 16 mg/kg).
Furthermore, the results of a 5-day repeated oral toxicity study
suggest that 2b has low hepatotoxicity. A broader study of pyra-
zole-benzyl urea derivatives is in progress and will be reported in
the future.
16. Procedure for TNF-
a production assay in HWB: Freshly drawn heparinized
HWB was dispensed into each well of a 96-well plate. A compound diluted in
human serum AB (Gemini) was then added to each well. After incubation for
1 h at 37 °C, LPS (200 ng/mL, Escherichia coli O111:B4; Sigma-Aldrich) was
then added. Upon incubation for 4 h at 37 °C, the supernatant was collected.
Acknowledgment
The human TNF-
a level in the supernatant was measured by homogeneous
time-resolved fluorescence assay (Cis Bio International).
17. Procedure for mouse model of endotoxic shock: Male BALB/c mice (Charles
River Japan, 8 weeks of age, n = 6/control, n = 3/treatment) were orally
administered 2b (10, 30, and 50 mg/kg; 10 mL/kg) or its vehicle (control, 27%
hydroxypropyl-b-cyclodextrin, 10 mL/kg). Thirty minutes later, each mouse
was injected intraperitoneally with LPS (1 mg/kg). Blood samples were
collected 60 min after LPS injection. Serum was separated and analyzed for
The authors gratefully thank Ryoji Hayashi (Toray Industries, Inc.)
for helpful suggestions during the preparation of the manuscript.
References and notes
TNF-
a level by commercial ELISA assay (R&D Systems) according to the
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body weight. The mice were housed in individual cages under controlled
lighting (12 h light/12 h dark cycle) at 19–25 °C with a relative humidity of 40–
60% and given pellet diet (CRF-1, Oriental Yeast Co., Ltd, Tokyo, Japan) and
municipal drinking water ad libitum. They were orally treated with 2b at doses
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