Development of N-2,4-pyrimidine-N-phenyl-N′-phenyl ureas as inhibitors of tumor necrosis factor alpha (TNF-α) synthesis. Part 1

Article abstract of DOI:10.1016/j.bmcl.2006.03.095

A new class of tumor necrosis factor alpha (TNF-α) synthesis inhibitors based on an N-2,4-pyrimidine-N-phenyl-N′-phenyl urea scaffold is described. Many of these compounds showed low-nanomolar activity against lipopolysaccharide stimulated TNF-α productio

Full text of DOI:10.1016/j.bmcl.2006.03.095

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3510–3513
Development of N-2,4-pyrimidine-N-phenyl-N0-phenyl ureas as
inhibitors of tumor necrosis factor alpha (TNF-a) synthesis. Part 1
Todd A. Brugel,* Jennifer A. Maier, Michael P. Clark, Mark Sabat, Adam Golebiowski,
Roger G. Bookland, Matthew J. Laufersweiler, Steven K. Laughlin, John C. VanRens,
Biswanath De, Lily C. Hsieh, Marlene J. Mekel and Michael J. Janusz
Procter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd. Mason, OH 45040, USA
Received 10 March 2006; revised 27 March 2006; accepted 28 March 2006
Available online 2 May 2006
Abstract—A new class of tumor necrosis factor alpha (TNF-a) synthesis inhibitors based on an N-2,4-pyrimidine-N-phenyl-N0-
phenyl urea scaffold is described. Many of these compounds showed low-nanomolar activity against lipopolysaccharide stimulated
TNF-a production. X-ray co-crystallization studies with mutated p38a showed that these trisubstituted ureas interact with the ATP-
binding pocket in a pseudo-bicyclic conformation brought about by the presence of an intramolecular hydrogen bonding
interaction.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The over-expression of cytokines, such as tumor necro-
sis factor alpha (TNF-a) and interleukin-1b (IL-1b), has
been implicated in a number of serious inflammatory
disorders.1 Consequently, agents that inhibit the prolif-
eration of these proinflammatory cytokines could reduce
inflammation and prevent further tissue destruction in
diseases such as rheumatoid arthritis (RA), osteoarthri-
tis (OA), and Crohn’s disease. Several biological agents
such as the soluble TNF-a receptor etanercept (Enbrel)
and the TNF-a antibody infliximab (Remicade) have
been shown to be a clinically effective treatment for these
disorders.2 However, universal acceptance of these
medications has been limited by their high cost and
need for intravenous or subcutaneous administration.
heteroindoles, ureas, and various fused bicyclic hetero-
cycles) containing a variety of functionality have been
reported to inhibit cytokine activity.4 Several companies
have progressed p38 inhibitors into clinical development
for treatment of chronic inflammatory diseases, with
varying degrees of success.5 BIRB-7966 (Fig. 1) was pro-
gressed into phase II clinical trials before development
was halted due to reported liver toxicity.7
Recently we reported the synthesis and biological
activity of a series of bicyclic pyrazolone-based inhibi-
tors of TNF-a production exemplified by 1 (TNF-a
IC50 = 22 nM, Fig. 1).8 Pyrazolone 1 was a potent inhib-
itor of p38a (IC50 = 54 nM), while showing a good over-
all profile of kinase selectivity. Herein we wish to report
a new structural class of trisubstituted ureas as cytokine
inhibitors.
It has been well documented that potent inhibitors of
p38 mitogen-activated protein (MAP) kinase attenuate
TNF-a expression in vitro as well as in vivo. Many small
molecule TNF-a production inhibitors that function by
competitively binding to the ATP-binding pocket of p38
have been reported. SB2035803(Fig. 1) represents a
prototypical 4-aryl-5-pyridylimidazole-based inhibitor,
although numerous other structural classes (e.g.,
pyrroles, pyrimidines, pyridines, pyrimidones, indoles,
Synthesis of the described pyrimidine-ureas was accom-
plished in three steps from known starting materials.
A representative procedure for this synthesis is shown
in Scheme 1. Regioselective addition of variably substi-
tuted anilines to 2,4-dichloropyrimidine 2 gave the
corresponding 6-anilino-4-chloropyrimidine 3. A second
nucleophilic addition of various alkyl amine derivatives
to 3 gave the desired disubstituted pyrimidine 4.
Reaction of 4 with an appropriate isocyanate in dichlo-
roethane (DCE) gave the corresponding trisubstituted
ureas 5–6.9
Keywords: Cytokine synthesis inhibitors; p38a kinase; Urea.
*
Corresponding author. Tel.: +1 513 622 4498; fax: +1 513 622
3681; e-mail: brugel.ta@pg.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.095

T. A. Brugel et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3510–3513
3511
Table 1. TNF-a data for urea analogs containing an N-4-fluorophenyl
F
group and an N0-substituted phenyl group
O
O
N
N
O
N
R
O
N
N
N
S
H
H
R1
N
H
N
N
HN
N
N
O
SB203580
BIRB796
TNF-α IC50 = 72 nM
p38α IC50 = 136 nM
TNF-α IC50 = 16 nM
p38α IC50 = 64 nM
F
F
O
Compound
R
R1
TNF-a IC50 (lM)
a
N
HO HN
HO HN
HO HN
MeO HN
MeO HN
MeO HN
MeO HN
N
5a
2-Cl
0.122
0.176
63%b
3.10
3.65
2.88
>5
1
N
TNF-α IC50 = 22 nM
p38α IC50 = 54 nM
N
NH
OH
5b
5c
5d
5e
5f
5g
5h
5i
2-Me
Figure 1. Small molecule proinflammatory cytokine inhibitors.
2,6-Di-Cl
2-Cl
N
N
R1
Cl
N
NH
N
N
NH
a
b
N
H
Cl
N
Cl
2-Me
2-F
2
R
R
4
3
Cl
HN
R2
O
N
HN
N
4-CF3
2-Cl
R1
HO
N
N
N
c
N
N
N
O
H
H
0.843
>5
N
5a
H
R
5 - 6
F
Scheme 1. Reagents and conditions: (a) EtOH, Na2CO3, ArNH2; (b)
i
NMP, Pr2NEt, R1NH2, 135 ꢁC; (c) DCE, R2NCO, rt to 80 ꢁC.
2-Cl
N
H
H
N
5j
2-Cl
0.539
3.61
1.42
The urea analogs synthesized in this study were all tested
for inhibition of TNF-a in an LPS stimulated human
monocytic (THP-1) whole cell-based assay.10
O
H
N
5k
5l
2-Cl
N
EtO2C
PrO2S
H
N
When 2-chlorophenyl urea 5a (Fig. 1) was tested for
inhibition of TNF-a, the compound showed good
potency (IC50 = 122 nM, Table 1). When the 2-chloro
group was substituted with a 2-methyl substituent, the
resulting analog 5b proved to be virtually equipotent
(IC50 = 176 nM). The 2,6-dichlorophenyl derivative 5c
showed somewhat reduced potency relative to the
monosubstituted phenyl analogs. However, when the
2-pyrimidyl group was changed from (1S)-2-hydroxy-
1,2-dimethylpropylamino to (1S)-2-methoxy-1-methyl-
ethylamino, the corresponding phenyl ureas 5d, 5e,
and 5f showed a greater than 10-fold loss in potency.
The 4-substituted phenyl derivative 5g was virtually
inactive in the assay, indicating the need for 2-substitu-
tion on the N0-phenyl ring to maintain activity.
2-Cl
N
N
5m
5n
2-Cl
2-Cl
>5
>5
N
Me
N
O
a Standard deviation for the assay was 30% of mean or less.
b Percentage inhibition at 1 lM.
substitution. The most active analog of this group was
the tetrahydropyran-4-ylamino derivative 5j which
was nearly 5-fold less potent than 5a. The chiral
A series of other 2-pyrimidyl urea analogs (5h–n)
were synthesized containing the N0-2-chlorophenyl

3512
T. A. Brugel et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3510–3513
Table 2. TNF-a data for analogs of N0-phenyl-substituted ureas
containing various N-substitutions
a-methylbenzylamino analog 5h also showed sub-nano-
molar activity, whereas the simple benzylamino analog
5i produced an IC50 greater than 10 lM which suggests
that alkyl branching adjacent to the amino linkage is key
to the potency of these compounds. Additionally, the
substituted aminopiperidine derivatives 5k and 5l both
proved to be only low micromolar inhibitors of TNF-
a production. Finally, the 2-pyrimidyl tertiary amino
analogs 5m–n were very poor inhibitors (IC50 > 10 lM),
suggesting a need for the amino NH in maintaining
activity.
R
N
N
HN
R2
N
R1
O
Compound
R
R1
R2
TNF-a
IC50 (lM)
a
HO HN
HO HN
HO HN
MeO HN
MeO HN
6a
6b
6c
6d
6e
6f
4-MeO-Ph
4-MeO-Ph
Ph
2-Cl
2-Me
2-Cl
2-Me
0.092
0.135
0.718
1.00
Unfortunately, this class of phenyl ureas proved to be
chemically unstable. Although the DMSO stock solu-
tions of inhibitors for biological testing were kept at
4 ꢁC and showed no degradation over several weeks’
storage, solutions (in methanol or DMSO) of com-
pounds stored at room temperature would slowly
decompose over several days. The decomposition was
composed exclusively of hydrolysis back to the aniline
precursor 4. To address this issue, we first investigated
adding electron density into the urea system by modify-
ing the electron-withdrawing 4-fluorophenyl group. This
work produced the series of phenyl urea analogs listed in
Table 2. The first group of 4-methoxyphenyl ureas 6a
and 6b were found to be nearly equipotent with the
corresponding 4-fluorophenyl analogs 5a and 5b. These
4-methoxyphenyl analogs only proved to be slightly
more stable than the 4-fluorophenyl derivatives, decom-
posing more slowly to the pyrimidyl aniline precursor.
Replacement by the unsubstituted phenyl ring as in 6c
further reduced the potency by 6-fold versus 5a. A
similar loss in potency was observed when the
dimethylhydroxypropylamino group was substituted
with the methoxypropylamino group as in 6d, in which
this compound was nearly 3-fold less potent than 5e.
The 4-N,N-dimethylaminophenyl analogs 6e and 6f were
even less active (IC50 > 10 lM).
Ph
4-Me2N-Ph 2-Cl
>5
4-Me2N-Ph 2-Me >5
N
H
6g
6h
6i
3-F-Ph
NH2
Me
2-Cl
2-Cl
6.16
N
H
MeO HN
>5
MeO HN
2-Me >5
a Standard deviation for the assay was 30% of mean or less.
Other modifications to the 4-fluorophenyl group were
made to investigate not only effects on stability, but also
potency. When the fluoro substituent was moved from
the para position to the meta position to give compound
6g, a near 10-fold loss of potency was observed com-
pared to 5h. The substitution of a phenyl group for an
N-amino group as in 6h resulted in complete loss of
activity. Finally, replacement of the N-phenyl substitu-
ent with a methyl group (6i) also produced an inactive
analog.
functionality of the inhibitor and the Met-109 residue of
the backbone of ATP-binding pocket. Additionally, the
4-MeO-phenyl group positions itself into the well-
defined hydrophobic pocket defined by the ‘gatekeeper’
Thr-106 residue. Most notably, the conformation of the
urea allows for the urea NH to form an intramolecular
˚
hydrogen bond with N-3 of the pyrimidine ring (2.69 A
N–N distance). This interaction creates a pseudo-bicy-
clic structure for this compound, which adds rigidity
to this class of inhibitors. The 2-chlorophenyl group
is positioned toward the exposed solvent pocket of
the enzyme. As seen by the overlap of bicyclic pyrazo-
lone 1, the carbonyl of urea 6a does not interact with
the amino group of Lys-53, as was seen with the pyraz-
olone carbonyl.8 This may explain the somewhat re-
duced potency of 5a against TNF-a production versus
pyrazolone 1.
Due to an inability to significantly improve chemical
stability while maintaining potency of the series, we were
unable to progress these compounds into further
pharmacokinetic or in vivo studies. However, we were
still interested in the binding mode of these compounds
in p38a. An X-ray crystal structure was obtained and
solved for the co-crystal formed between the
2-chlorophenyl urea inhibitor 6a and mutated p38a13
(Fig. 2) to obtain additional information concerning
the possible mechanism for TNF-a inhibition. This
compound contains several of the necessary interactions
for classical p38 inhibitors. Primary among these is the
hydrogen bonding motif between the 2-aminopyrimidine
We have developed a new class of trisubstituted ureas as
inhibitors of TNF-a production. First generation N-
phenyl ureas showed good potency for inhibition of
TNF-a, but proved to be chemically unstable. Attempts

T. A. Brugel et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3510–3513
3513
References and notes
1. For a recent review, see: Adams, J. L.; Badger, A. M.;
Kumar, S.; Lee, J. C. In Progress in Medicinal Chemistry;
King, F. D., Oxford, A. W., Eds.; Elsevier: Amsterdam,
2001; Vol. 39, pp 1–60.
´
2. Bolos, J. Mini-Rev. Med. Chem. 2005, 5, 857.
3. Badger, A. M.; Bradbeer, J. N.; Votta, B.; Lee, J. C.;
Adams, J. L.; Griswold, D. E. J. Pharmacol. Exp. Ther.
1996, 279, 1453.
4. Boehm, J. C.; Adams, J. L. Expert Opin. Ther. Patents
2000, 10, 25.
5. Goldstein, D. M.; Gabriel, T. Curr. Top. Med. Chem.
2005, 5, 1017.
6. Pargellis, C. A.; Tong, L.; Churchill, L.; Cirillo, P.;
Gilmore, T.; Graham, A. G.; Grob, P. M.; Hickey, E. R.;
Moss, N.; Pav, S.; Regan, J. Nat. Struct. Biol. 2002, 9, 268.
7. Lee, M. R.; Dominguez, C. Curr. Med. Chem. 2005, 12,
2979.
8. Clark, M. P.; Laughlin, S. K.; Laufersweiler, M. J.;
Bookland, R. G.; Brugel, T. A.; Golebiowski, A.; Sabat,
M.; Townes, J. A.; VanRens, J. C.; Djung, J. F.; Natchus,
M. G.; De, B.; Hsieh, L. C.; Xu, S. C.; Walter, R. L.;
Mekel, M. J.; Heitmeyer, S. A.; Brown, K. K.; Juergens,
K.; Taiwo, Y. O.; Janusz, M. J. 2004, 47, 2724.
Thr106
O
O
His107
NH
NH
HO
N
Leu108
O
O
N
Å
O
9. All final compounds were characterized by 1H NMR,
HRMS, and HPLC analysis. Purity was assessed as >95%.
10. Duplicate cultures of human monocytic cells (THP-1)11
(2.0 · 105/well) were incubated for 15 min in the presence
or absence of various concentrations of inhibitor before
the stimulation of cytokine release by the addition of
lipopolysaccharide (final LPS concentration, 1 lg/ml). The
amount of TNF-a released was measured 4 h later using
an ELISA (R&D Systems, Minneapolis, MN). The
viability of the cells after the 4 h incubation was measured
using MTS assay12 (Promega Co., Madison, WI).
11. Mohler, K. M.; Sleath, P. R.; Fitzner, J. N.; Cerretti, D.
P.; Alderson, M.; Kerwar, S. S.; Torrance, D. S.; Otten-
Evans, C.; Greenstreet, T.; Weerawarna, K.; Kronhelm, S.
R.; Petersen, M.; Gerhart, M.; Kozlosky, C. J.; March, C.
J.; Black, R. A. Nature 1994, 370, 218.
2
1
.
3
N
Cl
N
N
H
H
N
H
Å
Met109
O
6
9
.
2
OH
Figure 2. Co-crystal X-ray structure of 5b (cyan) overlaid with 1
(magenta) in the ATP-binding site of mutated p38a.
to improve chemical stability by modifying the electron-
ic properties of the various substitutions generally led to
loss of potency with no significant improvement in sta-
bility. X-ray crystallography studies with mutated
p38a showed a mode of binding for this class of urea
inhibitors that mimics that of traditional vicinal bis-aryl
MAP kinase inhibitors. Additionally, we observed that
the urea adopts a pseudo-bicyclic orientation due to
the compound’s ability to form an internal hydrogen
bond. Additional studies to improve chemical stability
and test analogs from this class for in vivo anti-
inflammatory activity will be reported in a separate
communication.
12. Barltrop, J. A.; Owen, T. C.; Cory, A. H.; Cory, J. G.
Bioorg. Med. Chem. Lett. 1991, 1, 611.
13. The X-ray coordinates have been deposited with the
Protein Data Bank, www.rcsb.org, as entry 2GHL.
Mutated p38 co-crystallization/X-ray crystallography
was conducted as follows: The mutated p38a herein
described is a double mutant (S180A, Y182F) of murine
p38a.14 The mutant enzyme cannot be phosphorylated
and, therefore, it is not competent for activation. Protein
expression and purification were carried out as previously
described for the murine enzyme.15 For crystallization,
mutated p38a was incubated overnight (12–16 h) with
1 mM compound. Co-crystals were grown by hanging
drop vapor diffusion using PEG as a precipitating agent
and overall protocols similar to those previously
described for the human enzyme.16 Crystals typically
Acknowledgments
We are grateful to M. Buchalova for supporting chem-
ical stability studies, and R. L. Walter for X-ray
co-crystallization studies. We thank A. G. Evdokimov
and M. E. Pokross for cloning, expression, and
purification of mp38 protein. We would like to
acknowledge that X-ray data were collected at
Southeast Regional Collaborative Access Team (SER-
CAT) 22-ID (or 22-BM) beamline at the Advanced
Photon Source, Argonne National Laboratory. Sup-
porting institutions may be found at www.ser-cat.org/
members.html. Use of the Advanced Photon Source
was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences,
under Contract No. W-31-109-Eng-38.
˚
diffracted to 1.9 A resolution and were of the previously
˚
reported space group: P212121; a = 65.2 A, b = 74.6 A,
˚
0
˚
c = 78.1 A (4 ).
14. Han, J.; Lee, J. D.; Bibbs, L.; Ulevitch, R. J. Science 1994,
265, 808.
15. Wang, Z.; Harkins, P. C.; Ulevitch, R. J.; Han, J.; Cobb,
M. H.; Goldsmith, E. J. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 2327.
16. Pav, S.; Whit, D. M.; Rogers, S.; Crane, K. M.;
Cywin, C. L.; Davidson, W.; Hopkins, J.; Brown, M.
L.; Pargellis, C. A.; Tong, L. Protein Sci. 1997, 6,
242.