New modifications to the area of pyrazole-naphthyl urea based p38 MAP kinase inhibitors that bind to the adenine/ATP site

Article abstract of DOI:10.1016/j.bmcl.2007.05.042

Discovery of the pyrazole-naphthyl urea class of p38 MAP kinase inhibitors typified by the clinical candidate BIRB 796 has encouraged further exploration of this particular scaffold. Modification to the part of the inhibitor that occupies the adenine/ATP

Full text of DOI:10.1016/j.bmcl.2007.05.042

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4242–4247
New modifications to the area of pyrazole-naphthyl urea based
p38 MAP kinase inhibitors that bind to the adenine/ATP site
Neil Moss,a,* Steffen Breitfelder,b Raj Betageri,a Pier F. Cirillo,a Tazmeen Fadra,a
Eugene R. Hickey,a Thomas Kirrane,a Rachel R. Kroe,a Jeffrey Madwed,a
Richard M. Nelson,a Christopher A. Pargellis,a Kevin C. Qian,a John Regan,a
Alan Swinamera and Carol Torcellinia
aDepartments of Medicinal Chemistry, Immunology and Inflammation, Cardiovascular Disease, Biologics and Biomolecular Sciences,
or Drug Discovery Support, Boehringer Ingelheim Pharmaceutical, Inc., 900 Ridgebury Road, Ridgefield, CT 0687, USA
bDepartment of Medicinal Chemistry, Boehringer Ingelheim Pharma GmbH and Co. KG, Birkendorfer Strasse 65,
D-88397 Biberach an der Riss, Germany
Received 12 March 2007; revised 10 May 2007; accepted 11 May 2007
Available online 18 May 2007
Abstract—Discovery of the pyrazole-naphthyl urea class of p38 MAP kinase inhibitors typified by the clinical candidate BIRB 796
has encouraged further exploration of this particular scaffold. Modification to the part of the inhibitor that occupies the adenine/
ATP binding site has resulted in a new way to obtain potent inhibitors that possess favorable in vitro and in vivo properties.
Ó 2007 Elsevier Ltd. All rights reserved.
Considerable research over the past several years has
been devoted to the identification of inhibitors of p38
MAP kinase.1 This enzyme acts as a key regulator in
the signaling pathways leading to the production of sev-
eral proinflammatory cytokines such as TNFa and IL-1.2
The clinical and commercial success of anti-TNFa
biologics such as EnbrelÒ, RemicadeÒ, and HumiraÒ
for arthritis alone highlights the therapeutic benefit of
proinflammatory cytokine inhibition.3 Consequently,
researchers anticipate that p38 inhibitors will display
at least similar therapeutic benefit as the anti-cytokine
biologics with the convenience of oral dosage.
available by movement of a phenylalanine found in
the conserved DFG motif of the kinase activation loop.7
Figure 1 graphically depicts key interactions made be-
tween BIRB 796 and p38. Upon considering additional
ways to expand on the potential of this pyrazole-naph-
thyl urea scaffold, we directed attention to the role and
Met109
Adenine
binding
site
NH
O
Lipophilic
(Phe169)
pocket
Phe169
N
We previously disclosed a class of p38 inhibitors based
on a pyrazole-naphthyl urea scaffold as typified by the
clinical candidate BIRB 796.4,5 A distinguishing feature
of this class of compounds is a binding mode to p38 that
involves interactions beyond those typically found with
many ATP site kinase inhibitors.6 In addition to com-
monly observed interactions in the adenine binding site
and the adjacent kinase specificity pocket, this class of
compounds also takes advantage of binding sites made
Asp168
NH
O
O
N
N
H
N
N
H
CO2-
Lipophilic
(kinase specificity)
pocket
Glu71
Keywords: p38 MAP kinase; Inhibitor.
*
Figure 1. Graphical representation of key interactions between BIRB
796 and p38.
Corresponding author. Tel.: +1 203 798 5101; fax: +1 203 791
6072; e-mail: nmoss@rdg.boehringeringleheim
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.042

N. Moss et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4242–4247
4243
Table 1. Effect of R group on potency
R
O
N
N
N
N
H
H
Compound
R
Tm (°C)a
Inhibition of TNFa from
Inhibition of TNFa from
THP-1 cells IC50 (nM)b
human whole blood IC50 (nM)c
O
H
N
BIRB 796
1
63.5
55.5
18
780
O
320
>15,000
2
3
55.5
55.5
1430
470
>15,000
>15,000
O
N
4
54.9
1350
>15,000
N
O
N
N
5
6
7
59.9
57.1
57.7
36
56
56
1100
3900
1700
O
O
O
N
N
8
60.1
61.0
63.6
62.3
55.3
104
46
9
9200
3600
230
590
—
N
9
N
N
N
10
11
12
O
O
N
26
390
N
O
O
N
13
59.4
70
6400
O
a Values are means of three experiments, standard deviation 0.1–0.5 °C.
b Values are means of 2–5 experiments, standard deviations for n > 2 experiments typically 50% of reported value.
c Values are geometric means from 8–12 donor experiments, standard deviation typically 50% of reported value.

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N. Moss et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4242–4247
CHO
N
Ar
Ar
a
b
Ar
N
Br
Br
Bu3Sn
O
O
c
d
N
Ar
5, 6, 10, 11, 13
O
Y
Br
H2N
H2N
O
N
N
X
N
OPh
H
Y
b,c
d + Y
e
(CH2)n
N
O
(CH2)n
NH2
3, 4, 7
Br
Br
n = 0 or 2
Scheme 1. Reagents and conditions: (a) Morpholine, NaHB(OAc)3, HOAc, THF, rt, 3 h, 31–91%; (b) t-BuLi, THF, Bu3SnCl, À78 °C, 42–77%; (c)
Pd(PPh3)4, dioxane, 100 °C, 24 h, 29–76%; (d) DMSO, 45 °C 12–16 h, 49–81%; (e) (BrCH2CH2)2O, i-PrNEt2, DMF, 31–90%.
contribution of the ethoxy morpholino group of BIRB
796 that occupies the adenine binding site of p38. The
perceived flexible nature and relatively small size of this
group compared to the size and flexibility of groups
from other classes of inhibitors that occupy the ade-
nine/ATP site encouraged us to look for other ways to
utilize the pyrazole-naphthyl urea scaffold.
results in an increase of both molecular and cellular
potency, while direct attachment has little positive or
negative effect. We obtained a similar positive result
through appending a morpholine group to a furan (c.f.
compounds 12 and 13). Since the overall potency values
of compound 5 trended to be best of the morpholine
analogues, it became a frame of reference for additional
modifications. Replacement of the morpholine oxygen
atom in BIRB 796 with a CH2 or NH resulted in an
8 °C drop in Tm (>250-fold drop in calculated Kd).5 This
structure–activity result supported the X-ray co-crystal
observation that the morpholine oxygen of BIRB 796
engages in a key hydrogen bond with the NH of Met
109, the same amino acid that interacts with the adenine
group of ATP. Consequently, it was initially surprising
that the dimethyl amino and the N-methyl piperidinyl
analogues of compound 5 (compounds 8 and 9) pos-
sessed essentially equal binding potency to compound
5. These results clearly indicated that the appended mor-
pholine group in compound 5 was improving binding
potency in a manner distinct from the morpholine group
in BIRB 796. While compounds 5, 8, and 9 have very
similar molecular potency, the morpholine derivative 5
possessed the best potency in an assay of LPS-induced
TNFa production in human whole blood.
The key molecules in this paper originated from the idea
of using an aromatic ring appended to the naphthyl
group of compound 1 as a template for adding function-
ality that could engage in interactions with the adenine/
ATP binding site. Compound 2 was prepared as the ini-
tial test case. Cross coupling of naphthyl bromide X
with phenylboronic acid provided a phenylnaphthyl
amine product that upon treatment with pyrazole carba-
mate Y gave compound 2. The equivalent molecular
potency of compounds 1 and 2 (Table 1) as determined
by a thermal denaturation assay (3.0–3.5 °C change in
Tm roughly equivalent to 10-fold change in binding po-
tency)8 showed that a phenyl ring was tolerated and
could potentially serve as a useful template. We previ-
ously demonstrated that the presence of the morpholine
group in BIRB 796 not only improved molecular
potency but also improved potency in cell assays of
LPS-induced TNFa production.5 Consequently, the
morpholine group became a logical first choice to ap-
pend to the phenyl ring of compound 2 using a 0–2 car-
bon linker (compounds, 3–7). Scheme 1 outlines general
methodology applicable to most of the compounds in
Table 1 and relies on the Stille coupling of a morphol-
ine-containing aryl tin derivative to naphthyl bromide
X.9 Completion of the final urea molecule involves react-
ing the resultant arylnaphthyl amine with pyrazole car-
bamate Y.
Compound 5 is significantly more lipophilic (could not
determine a logD7.4) than BIRB 796 (logD7.4 = 4.3).
One way we explored to reduce lipophilicity involved
replacement of the right hand side phenyl group in com-
pound 5 with a pyridyl (compounds 10 [logD7.4 = 5.2]
and 11). This modification resulted in a modest increase
in molecular potency, to the level of BIRB 796. More
significantly, the cellular potency, particularly of com-
pound 10 in human whole blood, proved better than
BIRB 796.
As can be seen from a comparison of compounds 2–7 in
Table 1, appending a morpholine group to the phenyl
ring of compound 2 via one or two methylene groups
The successful outcome of introducing a pyridyl group
encouraged an investigation of nitrogen heterocycles

N. Moss et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4242–4247
4245
Table 2. Effect of R group on potency
N
N
O
O
N
N
N
N
H
H
R
Compound
R
logD7.4 Tm (°C)a
Inhibition of TNF-a from
THP-1 cells IC50 (nM)b Human whole blood IC50 (nM)c LPS treated mice at dose (mg/kg)
10
14
5.2
3.9
63.6
59.6
9
230
950
74% at 10
NSd at 3
32
NS at 10
15
4.2
65.1
11
200
86% at 10
50% at 3
N
16
17
4.0
3.9
63.7
64.3
14
11
160
95% at 10
85% at 3
N
N
90
73% at 3
N
N
a Values are means of three experiments, standard deviation 0.1–0.5 °C.
b Values are means of 2–5 experiments, standard deviations for n > 2 experiments typically 50% of reported value.
c Values are geometric means from 8–12 donor experiments, standard deviation typically 50% of reported value.
d Not statistically significant inhibition.
on the other end of the molecule. Given the still subop-
timal physicochemical properties of compound 10, one
intention was to investigate the consequences of further
decreasing overall lipophilicity (Table 2). The left hand
side fragments of compounds 14 and 15 were prepared
as previously reported.5 The pyrimidine and pyrazole
containing left hand sides required for the preparation
of analogues 16 and 17 relied on the vinamidinium
chemistry outlined in Scheme 2.
improve potency in the human whole blood assay. The
impact of these heterocycles becomes more substantial
when comparing the potency in a mouse model of LPS-in-
duced TNFa production. In this well-established model,
mice are dosed 30 min prior to LPS challenge, and plasma
TNFa levels are determined 1 h after LPS challenge.10
While BIRB 796 inhibits 63% of TNFa at a dose of
10 mg/kg,5 the heterocyclic compounds 16 and 17 in
particular trend to better efficacy at 3-fold lower dose.
The reason for this improvement in in vivo potency is
not immediately clear. The protein binding for BIRB
796, 10, and 16 are all greater than 97% as determined
by equilibrium dialysis. A determination of compound
plasma concentrations at 90 min post dosing revealslevels
of compounds 16 and 17 that are at least 10-fold lower
than those observed for either BIRB 796 or compound
10. One might assume the concentrations of these
compounds in the tissues most responsible for TNFa
The decrease in potency observed by replacing the lipo-
philic tolyl group in 10 with a methyl group (14) confirms
the importance of a bulkier moiety at this position as pre-
viously observed with BIRB 796.5 Curiously the drop in
Tm in this series is significantly less (4.0 °C) than for BIRB
796 (9.0 °C). In contrast, decreasing lipophilicity through
introduction of nitrogen heterocycles at this position has
little impact on molecular potency while trending to

4246
N. Moss et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4242–4247
production to be significantly different than those seen in
plasma.
The kinase selectivity profile of compound 10 appears
similar to BIRB 796.11 While compound 10 does not in-
hibit over 40 kinases at a test concentration of 3 lM, it
does inhibit Jnk 2 (IC50 2–5 nM), cRaf (IC50 400 nM),
and Src, Lyn, and Abl (IC50 1–2 lM).
The differential SAR observed between the portions of
BIRB 796 and compound 10 that occupy the adenine
binding site of p38 encouraged us to obtain an X-ray
co-crystal structure of compound 10.12 Unlike the mor-
pholine group of BIRB 796, the morpholine group of
compound 10 does not appear to be involved in any
obvious hydrogen bonding interactions in the adenine
binding site (see Fig. 2). This is consistent with the sim-
ilar Tms observed for compounds 5, 8, and 9. However,
the combination of the pyridyl and the morpholine to-
gether does occupy more of the adenine/ATP binding
site than does the ethoxy morpholine group of BIRB
796. Since the morpholine and other dialkyl amino
groups in this new series of compounds do appear
important for good binding potency, this may be a
consequence of van der Waals interactions and/or
desolvation within the adenine/ATP binding site. An
additional notable feature is a water mediated hydrogen
bond between the pyridyl nitrogen and the NH of Met
109 and carbonyl of His 107. This indirect interaction
may contribute to the increased potency of compound
10 over compound 5.
The results discussed herein highlight an extension to
our previously reported class of potent p38 kinase inhib-
itors. These new compounds take advantage of interac-
tions in the adenine binding site of p38 unique from
BIRB 796. Investigations to decrease the overall lipo-
a
b
c
N
d
CO2Et
CO2H
Figure 2. Comparison of compound 10 (top, yellow) and BIRB 796
(bottom, white) in the adenine binding site of p38.
N
N
CO2Et
O
N
H
philicity of this class of compounds resulted in com-
pounds with substantially improved potency in the
mouse model of LPS-induced TNFa production, and
thus further highlight the therapeutic potential of this
class of compounds.
N
N
O
N
f or g
CO2H
H2N
e
N
R
N
CO2Et
N
16, 17
h
References and notes
+
NMe2
-
Me2N
R =
PF6
N
N
N
N
1. (a) Dominguez, C.; Powers, D. A.; Tamayo, N. Curr.
Opin. Drug Disc. Dev. 2005, 8, 421; (b) Nikas, S. N.;
Drosos, A. A. Curr. Opin. Invest. Drugs 2004, 5, 1205; (c)
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Scheme 2. Reagents and condition: (a) LiHMDS, THF, diethyl
oxalate; (b) H2NNH2, HOAc, 90 °C, 66% two steps; (c) KOtBu,
DMSO, t-butyl bromoacetate, 70%; (d) TFA, 94%; (e) POCl3, DMF,
NaPF6, H2O, 78%; (f) acetamidine hydrochloride, NaOEt, EtOH,
60%; (g) N-methylhydrazine, EtOH, LiOH, MeOH, 78%; (h) NEt3,
PhH, DPPA, naphthylamine X, 50–75%.

N. Moss et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4242–4247
4247
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H. J. Med. Chem. 2003, 46, 4675, Tm values can be
converted to an approximate Ka using the formula
Tm = 3.08(logKa) + 31.2.
9. Certain experimental details can be found in: Betageri, R.;
Breitfelder, S.; Cirillo, P. F.; Gilmore, T. A.; Hickey, E.
R.; Kirrane, T. M.; Moriak, M. H.; Moss, N.; Patel, U.
R.; Proudfoot, J. R.; Regan, J. R.; Sharma, R.; Sun, S.;
Swinamer, A.; Takahashi, H. U.S. Patent 6,660,732, 2003.
10. Experimental details for this model can be found in Ref. 5.
11. Additional perspective on the kinase selectivity profile of
BIRB 796 can be found in the supplementary information
of the following publication. Compound 10 was not
screened against many of the kinases in this publication so
the provided selectivity information should be regarded as
a limited indication of overall selectivity. Fabian, M. A.;
Biggs, W. H.; Treiber, D. K.; Atteridge, C. E.; Azimioara,
M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen,
P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.;
Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M.
A.; Lai, A. G.; Lelias, J.-M.; Mehta, S. A.; Milanov, Z. V.;
Velasco, A.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P.
P.; Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329.
12. The coordinates of compound 10 bound to p38 have been
deposited with the RCSB Protein Data Bank (access code
2PUU).