N-phenyl-N-purin-6-yl ureas: The design and synthesis of p38α MAP kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00048-9

The design, synthesis and SAR of a series of 2,6,9-trisubstituted purine inhibitors of p38α kinase is reported. Synthetic routes were devised to allow for array synthesis in which all three points of diversity could be facilely explored. The binding of this novel series to p38α kinase, which was predicted to have several key interactions in common with SB-203580, was confirmed by X-ray crystallography of 19 (p38 IC₅₀=82 nM).

Full text of DOI:10.1016/S0960-894X(03)00048-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1191–1194
N-Phenyl-N-purin-6-yl Ureas: The Design and Synthesis of
P38ꢀ MAP Kinase Inhibitors
Zehong Wan,a,* Jeffrey C. Boehm,a Michael J. Bower,b Shouki Kassis,c John C. Lee,d
Baoguang Zhaoc and Jerry L. Adamse
aDepartment of Medicinal Chemistry, Respiratory and Inflammation CEDD, GlaxoSmithKline Pharmaceuticals,
709 Swedeland Road, PO Box 1539, King of Prussia, PA 19406, USA
bDepartment of Computational, Analytical and Structural Science, Discovery Research, GlaxoSmithKline Pharmaceuticals,
709 Swedeland Road, PO Box 1539, King of Prussia, PA 19406, USA
cDepartment of Discovery Biology, Discovery Research, GlaxoSmithKline Pharmaceuticals,
709 Swedeland Road, PO Box 1539, King of Prussia, PA 19406, USA
dDepartment of High Throughput Biology, Discovery Research, GlaxoSmithKline Pharmaceuticals,
709 Swedeland Road, PO Box 1539, King of Prussia, PA 19406, USA
eDepartment of Medicinal Chemistry, Microbial, Musculoskeletal and Proliferative Diseases CEDD,
GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, PO Box 1539, King of Prussia, PA 19406, USA
Received 18 October 2002; accepted 2 December 2002
Abstract—The design, synthesis and SAR of a series of 2,6,9-trisubstituted purine inhibitors of p38a kinase is reported. Synthetic
routes were devised to allow for array synthesis in which all three points of diversity could be facilely explored. The binding of this
novel series to p38a kinase, which was predicted to have several key interactions in common with SB-203580, was confirmed by
X-ray crystallography of 19 (p38 IC50=82 nM).
# 2003 Elsevier Science Ltd. All rights reserved.
Enbrel, a soluble TNF-a receptor, and Remicade, an
antibody to TNF-a have demonstrated clinical efficacy
in the treatment of rheumatoid arthritis (RA) and
inflammatory bowel disease (IBD).1 These protein
agents, which act by blocking the action of TNF-a, are
significant therapeutic advances for the treatment of
these debilitating conditions. p38 is a member of the
intracellular family of mitogen activated protein kina-
ses, implicated in the phosphorylation cascade leading
to the release of TNF-a and other cytokines. The inhib-
ition of p38 kinase has been proposed as an alternative
small molecule approach to block the action of TNF-a.2
As a small molecule approach would enjoy the advan-
tages of more convenient oral administration, p38a
inhibition has received widespread attention as a thera-
peutic target. Several research teams have undertaken
the challenge to identify p38a inhibitors suitable for
clinical development and recently multiple p38 kinase
inhibitors have advanced into clinical trials as potential
therapies for the treatment of RA.3 These include,
SB-242235, a member of the pyridinyl-imidazole class
of selective p38 inhibitors, as well as, VX-745 and BIRB
796, compounds from alternate structural classes.4
Our group has had an on going effort utilizing database
screening and structure-based design strategies to iden-
tify novel p38a inhibitors. Having demonstrated that
the inhibition of P450 seen with SB-203580 and analo-
gues was a result of the 4-pyridyl group,5 we were par-
ticularly interested in replacing the pyridyl group with
an amide carbonyl as an alternate H-bond acceptor.
Hence, we were intrigued by the publication of a Vertex
Pharmaceuticals patent application claiming ureidopyr-
idines as p38 inhibitors.6 Molecular modeling experi-
ments performed with compound A7 suggested that the
urea carbonyl could form a H-bond donor-acceptor
interaction with the backbone amide NH of M109 that
would mimic the H-bond of the N-1 adenine of ATP
and is analogous to that seen for the 4-pyridyl nitrogen
of SB-203580.8 Additionally, in this model one of the
*Corresponding author. Fax: +1-610-270-6609; e-mail: zehong_2_
wan@gsk.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00048-9

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Z. Wan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1191–1194
terminal urea nitrogen NH groups of A could mimic the
H-bond of the N-6 amino group of adenine to the amide
carbonyl of H107 (Fig. 1).
Our point of departure for the synthesis is 2,6-dichloro-
9-methyl purine (1, Scheme 1) which was readily avail-
able from the methylation of 2,6-dichloropurine.10
Selective displacement of chlorine at C6 with sodium
methylsulfide afforded 2. Palladium mediated Suzuki
cross-coupling of the remaining chlorine with 3 and
oxidation with oxone provided sulfone 4. Substitution
with 2,6-difluoroaniline followed by treatment with tri-
phosgene and ammonium hydroxide then furnished
urea 6. This scheme allows for the introduction of dif-
ferently substituted anilines at C6 late in the synthesis
and was utilized to synthesize compounds 20–23.
This communication describes our efforts towards the
synthesis and SAR studies of a novel purine series of
p38a inhibitors that were designed based upon the
model shown in Figure 2. The purine template was
chosen in order to allow for a third point of diversity
(R3) fit into the ATP triphosphate binding site, an area
known to tolerate diverse substitutions. The preparation
of the required trisubstituted purines was accomplished
utilizing a strategy, which relied on the different reac-
tion preferences of halogen atoms at C2 and C6 of
purine.9 Schemes 1–4 summarize four strategies for the
preparation of target B analogues.
Figure 1. Structures of SB-203580 and compound A.
Scheme 2.
Figure 2. Structure of p38 Inhibitors B.
Scheme 3.
Scheme 1.
Scheme 4.

Z. Wan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1191–1194
1193
In order to optimize the series, late introduction of
substituents at C2 was achieved utilizing a synthetic
method described in Scheme 2. The preparation of urea
10 started with 6-chloro-2-iodo-9-methyl purine (7)
which was easily constructed from 2-amino-6-chloro
purine following a literature procedure.11 Selective dis-
placement of the chlorine of 7 with 2-chloroaniline
afforded 8. Palladium catalyzed Suzuki cross-coupling
of iodide 8 with 3 provided 9. Compound 9 could also
be obtained with a similar yield by carrying the reaction
sequence in the reverse order (Suzuki coupling, then
displacement with 2-chloroaniline). Treatment of 9 with
triphosgene and ammonium hydroxide then furnished
urea 10. This strategy, which permits the independent
introduction of different substituents at either C2 or C6,
was applied to prepare compounds 24–35.
unsubstituted aniline, is essentially inactive. p38 inhibi-
tion is improved with halogen substituents (6, 10, 21, 23,
and 24), but not with the substitutions of electron
donating groups (e.g., methoxy, 22). Inhibitor 10 has an
ortho chlorine, and it is eighty fold more active than 23,
indicating a strong preference for ortho versus para
substitution. Variation of R2 was also found to be
important for optimizing p38 kinase activity. The
unsubstituted analogue (25) displays modest potency
(IC50=0.51 mM) while meta fluoro substitution is not
well tolerated (27 and 30), ortho substitution is, with
every compound (26, 31, and 33) improved relative to
25. In terms of the substituent characteristics, halogen is
6-fold more potent than methyl. For the purpose of
improving physio-chemical properties of the inhibitors,
two R3 substitutions were prepared with the optimal R1
and R2. Both compounds (19 and 36) proved to be
potent p38 inhibitors, exhibiting IC50 values compar-
able to the N9 methyl group derivative (26). Note-
worthy is improved aqueous solubility of 19 (>20 mM)
versus 26 (1 mM).14 This may suggest that the N9 posi-
tion of the purine can be used to adjust physio-chemical
properties without compromising p38 potency.
Encouraged by the success of manipulating substituents
at C2 and C6, we explored the introduction of varia-
tions at N9 late in the synthetic sequence using a pro-
tection/deprotection sequence. To this end, compound
13 was prepared from 2-amino-6-chloro purine (11,
Scheme 3) employing a two-step procedure; protection
of N9 with trimethylsilylethoxymethyl chloride
(SEMCl) followed by conversion of NH2 at C2 to
iodine. Compound 13, whose structure was unambigu-
ously confirmed by X-ray crystallography shown in
Figure 3, is an versatile building block, providing access
to variable substitution at C2, C6 and N9. The chlorine
at C6 is more reactive toward nucleophilic substitutions,
while in contrast, the iodine at C2 is more reactive in
coupling reactions catalyzed by palladium(0).12
In order to confirm the binding mode of this novel class
with p38 proposed in Figure 2, the X-ray structure of 19
bound to p38a was determined from crystals soaked
with a solution of the inhibitor (Fig. 4). We were
pleased to find that the proposed model accurately pre-
dicted several key features of the crystallographically
determined binding mode. These features include: (1) an
intra-molecular hydrogen bond from the urea group to
N1 of the purine which positions the urea co-planar
The utility of 13 was demonstrated through the synth-
esis of two N-9 substitution analogues (Scheme 4). Dis-
placement of chlorine with 2,6-difluoroaniline followed
by Suzuki cross-coupling of iodine with 2-fluoro-
phenylboronic acid (14) afforded 15. The two step
sequence could also be reversed (Suzuki cross-coupling
followed by displacement of chlorine) to produce 15 in a
comparable yield. Removal of SEM (TBAF, THF,
reflux, 4A-MS) afforded disubstituted purine 16. Reac-
tion with alkyl chloride 17 furnished 18 exclusively,
which was transformed into urea 19. This synthetic
procedure was employed to construct compound 36 as
well.
Table 1. Results of SAR studies around the purine template
Compd Method
(scheme)
R1
H
R2
R3
IC50
(mM)
20
6
1
1
2
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
4
4
2-Me, 4-F
Me
Me
>16.7
Table 1 summarizes the p38 inhibition results.13 Group
R1 was the first to be investigated. Compound 20, the
2,6-di-F 2-Me, 4-F
0.128
0.123
1.4
10
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
19
2-Cl
4-F
2-OMe
4-Cl
2-Me, 4-F
2-Me, 4-F
2-Me, 4-F
2-Me, 4-F
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
>16.7
9.8
2,4,6-tri-F 2-Me, 4-F
0.163
0.51
0.036
5
1
0.026
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
2,6-di-F
H
2-F
3-F
4-F
2,4-di-F
3,5-di-F
2-Cl
>16.7
0.03
0.55
0.14
0.35
2.1
3-Cl
2-Me
3-Me
4-Me
2-F
Me
Bn
0.016
0.082
2-F
2-di-Me-amino-Et
Figure 3. Crystal structure of compound 13.

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Z. Wan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1191–1194
References and Notes
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7. Compound Ais example 410 of WO 9827098 in ref 6.
8. Wang, Z. L.; Canagarajah, B. J.; Boehm, J. C.; Kassis, S.;
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9. Reaction preferences of 2-Cl versus 6-Cl in nucleophilic
substitutions are well documented. For an example, see: Nor-
man, T. C.; Gray, N. S.; Koh, J. T.; Schultz, P. G. J. Am.
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Figure 4. Compound 19 bound in p38 MAP kinase.
with purine, (2) the hydrogen bond donor-acceptor
between the kinase backbone M109 amide NH and
H107 amide carbonyl to the urea which replicates the
interaction of adenine N1 and the C6 NH2 of ATP that
is conserved across all kinases,15 (3) the binding of the
C2 fluorophenyl ring into a hydrophobic pocket which
imparts selectivity for p38,16 and (4) the projection of
the dimethylaminoethyl group (R3) into the phosphate
binding pocket. The propeller-like arrangement of the
two aryl rings attached to the central purine deserves
comment as it may shed some light on the SAR for R1
and R2. The preference for ortho substitution of these
aryls can be partially understood to the extent it will
enhance the conformational bias towards the crystallo-
graphically observed propeller conformation. However,
for the R1 group a steric clash with the N7 of adenine
already forces this conformation and the more impor-
tant feature of ortho substitution may be that it directs
the attached residues back into the lipophilic pocket
instead of solvent. Additional notable features are acid-
base pairings of Lys53 with N-3 of the purine and
Asp168 with the dimethylamino group and the p–cation
interactions of the 2-fluorophenyl and dimethyl groups
with Lys53 and Tyr35, respectively. While the pro-
nounced electronic requirement (compare 20 to 21 and
10 to 22), is not readily understood, it may be related to
effects on the H-bonding donor–acceptor properties of
the urea.
10. Parker, C. W.; Entsch, B.; Letham, D. S. Phytochemistry
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12. For other related examples, see: (a) Legraverend, M.;
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R. S.; Gallagher, T. F.; Sheldrake, P. L.; Bradbeer, J.; Badger,
A. M.; Laydon, J. T.; Lee, J. C.; Hillegass, L. M.; Griswold,
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14. Solubility of 19 is 23.0 mM at pH 7.4. Solubility of 26 is
1 mM at pH 7.4. Solubility measurements were carried out
by dissolving a solid compound in 50 mM phosphate buffer
with 2% DMSO at pH 7.4. The resultant mixture was stir-
red at room temperature for 24 h, filtered and centrifuged.
Solubility data was then collected with fast gradient HPLC/
UV analysis.
In summary, a number of potent p38 inhibitors have
been prepared based upon this novel use of a purine
template. The proposed binding mode for this novel
series of inhibitors is consistent with the crystallography
results obtained from inhibitor 19/p38a. Additional
novel p38 inhibitors designed from this model will be
reported in due course.
15. Eyers, P. A.; Craxton, M.; Morrice, N.; Cohen, P.; Goe-
dert, M. Chem. Biol. 1998, 5, 321.
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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.
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Acknowledgements
The authors wish to thank James F. Callahan for help-
ful discussions and R. C. Haltiwanger for providing the
crystal structure of compound 13.