The development of new bicyclic pyrazole-based cytokine synthesis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.07.024

4-Aryl-5-pyrimidyl-based cytokine synthesis inhibitors of TNF-α production, which contain a novel bicyclic pyrazole heterocyclic core, are described. Many of these inhibitors showed low nanomolar activity against LPS-induced TNF-α production in a THP-1 cell-based assay and against human p38α MAP kinase in an isolated enzyme assay. The X-ray crystal structure of a bicyclic pyrazole inhibitor co-crystallized with mutated p38 (mp38) is presented. 4-Aryl-5-pyrimidyl-based cytokine synthesis inhibitors of TNF-α production, which contain a novel bicyclic pyrazole heterocyclic core, are described. Many of these inhibitors showed low nanomolar activity against LPS-induced TNF-α production in a THP-1 cell-based assay and against human p38α MAP kinase in an isolated enzyme assay. The X-ray crystal structure of a bicyclic pyrazole inhibitor co-crystallized with mutated p38 (mp38) is presented.

Full text of DOI:10.1016/j.bmcl.2004.07.024

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4945–4948
The development of new bicyclic pyrazole-based cytokine
synthesis inhibitors
Jennifer A. Townes,* Adam Golebiowski, Michael P. Clark, Matthew J. Laufersweiler,
Todd A. Brugel, Mark Sabat, Roger G. Bookland, Steve K. Laughlin, John C. VanRens,
Biswanath De, Lily C. Hsieh, Susan C. Xu, Michael J. Janusz and Richard L. Walter
Procter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd, Mason, OH 45040, USA
Received 21 May 2004; revised 12 July 2004; accepted 12 July 2004
Available online 4 August 2004
Abstract—4-Aryl-5-pyrimidyl-based cytokine synthesis inhibitors of TNF-a production, which contain a novel bicyclic pyrazole
heterocyclic core, are described. Many of these inhibitors showed low nanomolar activity against LPS-induced TNF-a production
in a THP-1 cell-based assay and against human p38a MAP kinase in an isolated enzyme assay. The X-ray crystal structure of a
bicyclic pyrazole inhibitor co-crystallized with mutated p38 (mp38) is presented.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
in the efficacy of our inhibitors against the overall cas-
cade that leads to TNF-a formation, inhibition of p38
kinase may be one possible pathway through which
the intended cellular effect is being demonstrated.
Tumor necrosis factor-a (TNF-a) is a key pro-inflam-
matory cytokine produced in response to immunostimu-
lants primarily by activated monocytes/macrophages to
help clear infections and damaged cells from injured tis-
sues. However, the overexpression of TNF-a has been
implicated in a number of serious inflammatory disor-
ders1 such as rheumatoid arthritis,2 inflammatory bowel
disease (IBD),3 osteoarthritis,4 and CrohnÕs disease.
Furthermore, TNF-a is a potent inducer of other pro-
inflammatory cytokines such as IL-1, IL-6, and IL-8.5
Accordingly, agents that inhibit TNF-a production
can diminish the levels of these proinflammatory cyto-
kines resulting in a reduction of inflammation and pre-
vention of further tissue destruction.
Various p38 inhibitors have been reported containing a
common 4-aryl-5-pyridinyl (or 5-pyridyl)-based motif
fused to a five or six-membered heterocyclic core,6 such
as SB2035807 (Fig. 1), which represents a prototypical
pyridyl imidazole-based inhibitor.
In a previous paper, we have reported the development
of a class of cytokine inhibitors, which contains a vicinal
bis-aryl five-membered heterocyclic core (Fig. 2).8 In our
continued effort towards the development of disease
modifying treatments for osteoarthritis, we began to
Our research strategy involves designing kinase inhibi-
tors that will result in the inhibition of TNF-a forma-
tion. There are multiple points at which one may
effectively inhibit TNF-a synthesis since several MAP
kinases exist in the signaling cascade. With this in mind,
our primary assay entails screening analogs for inhibi-
tion of TNF-a production in LPS-stimulated human
monocytic cells (THP-1). Although we are interested
F
O
N
S
N
H
N
SB203580
TNF-α IC50 = 72 nM
p38 kinase IC50 = 136 nM
Keywords: Bicyclic pyrazole; TNF-a; p38; Anti-inflammatory.
Corresponding author. Tel.: +1-5136224539; fax: +1-5136223681;
e-mail: townes.ja@pg.com
*
Figure 1. Imidazole-based pro-inflammatory cytokine inhibitor.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.024

4946
J. A. Townes et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4945–4948
F
O
O
F
N
O
OCH3
OH
N
N
(a)
F
OCH3
N
X
Y
N
2
5 a (X=C, Y=H)
b (X=N, Y=SMe)
1
F
TNF-α IC50 = 490 nM
O
Figure 2. Vicinal bis-aryl five-membered heterocyclic cytokine inhib-
itor.
OCH3
(b)
(c)
O
N
X
investigate other vicinal bis-aryl five-membered hetero-
cycles as potential TNF-a inhibitors and these efforts
led to the development of a new structural class of cyto-
kine inhibitors, which contain a novel bicyclic pyrazole
core. Herein we wish to report the synthesis and struc-
ture–activity relationships for this developing series.
Y
6 a (X=C, Y=H)
b (X=N, Y=SMe)
F
F
O
O
(d)
NH
X
N
N
N
H
2. Chemistry
n
N
N
X
X
Based upon the success of the triazole series (Fig. 2),8 we
began to explore similar heterocycles as potential TNF-
a inhibitors such as the pyrazolone series. The unsubsti-
tuted parent pyrazolone 7 was synthesized in four steps
from commercially available starting materials. Aldol
reaction of methyl 4-fluorophenylacetate (2, Scheme 1)
with 4-pyridine-carboxaldehyde (3) or 2-methylsulf-
anyl-pyrimidine-4-carbaldehyde (4) using LDA gives b-
hydroxyester products 5a–b. Alcohols 5a–b are oxidized
with chromium trioxide/pyridine to provide b-ketoesters
6a–b containing the desired 4-fluorophenyl and either
pyridyl or pyrimidyl functionality. The pyrazolone core
is installed through a thermal cyclization reaction be-
tween b-ketoesters 6a–b and semicarbazide hydrochlo-
ride (EtOH, 78ꢁC) to give 1,2-dihydropyrazolones 7
and 8. We envisioned further functionalization of 7 via
alkylation with dihaloalkanes to provide bicyclic pyr-
azolone 9. However, alkylation of the 1,2-dihydropyrazo-
lone 7 with 1,3-dibromopropane in the presence of NaH
gives bicyclic pyrazole 10a (Scheme 2) instead of the
anticipated bicyclic pyrazolone 9.9 Alkylation of the
1,2-dihydropyrazolone 8 with either 1,2-dibromopro-
pane or 1,2-dibromoethane in the presence of NaH gives
10b–c as either a [5,6]- or [5,5]-bicyclic pyrazole, respec-
tively. The 2-substituted pyrimidyl bicyclic pyrazole ser-
ies provides an added benefit of a second site for
functionalization. Oxidation of the thiomethylether
group in 10b–c to the corresponding sulfoxide 11 is per-
formed using mCPBA in CHCl3. Finally, displacement
of sulfoxide 11 with either amines (R1NH2, toluene,
120ꢁC) or phenol (ArOH, NaH, THF) gives the corre-
sponding amino- and phenoxy-bicyclic pyrazoles.
Y
Y
7 (X=C, Y=H)
8 (X=N, Y=SMe)
9 (X=C, Y=H, n=1)
Scheme 1. Synthesis of pyrazolones 2 and 8. Reagents and conditions:
(a) LDA, 4-pyridine-carboxaldehyde 3 or 2-methylsulfanyl-pyrim-
idine-4-carbaldehyde 4, THF, À78ꢁC, 76%; (b) CrO3, pyridine,
CH2Cl2, 43%; (c) Semicarbazide hydrochloride, EtOH, 78ꢁC, 81%;
(d) Br(CH2)nBr, NaH, DMF, rt.
F
F
O
O
N
(a)
(b)
NH
n
N
N
H
X
N
X
N
Y
Y
7 (X=C, Y=H)
10 a (X=C, Y=H, n=2)
b (X=N, Y=SMe, n=2)
c (X=N, Y=SMe, n=1)
8 (X=N, Y=SMe)
F
F
O
O
(c)
N
N
n
n
N
N
X
X
N
N
SOMe
Y
11
12a-e
n = 1-2
Y = ArO or R1NH
n = 1-2
Scheme 2. Synthesis of the bicyclic pyrazole series. Reagents and
conditions: (a) Br(CH2)nBr, NaH, DMF, rt, 44%; (b) mCPBA, CHCl3,
0ꢁC, quant.; (c) R1NH, toluene, 120ꢁC or ArOH, NaH, THF.
3. Results and discussion
tion of TNF-a in LPS-stimulated human monocytic
cells (THP-1)10 and for the inhibition of human p38
MAP kinase.13 The initial [5,6]-bicyclic pyrazole 10a
demonstrated similar effectiveness as an inhibitor of
Table 1 summarizes the results from our screening of se-
ven novel bicyclic pyrazoles for inhibiting the produc-

J. A. Townes et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4945–4948
Table 1. TNF-a inhibition data for the bicyclic pyrazole derivatives
4947
F
O
N
n
N
N
X
Y
Compound
n
X
Y
TNF-a
Human p38
IC50 (lM) MAP Kinase
IC50 (lM)
1
–– –– ––
0.49
3.1
0.31
0.32
0.46
7
10a
––
2
C
C
H
H
3.1
O
12a
12b
2
N
>10
6.6
O
1
N
0.30
2.2
NH
12c
12d
12e
1
1
1
N
N
N
>2.0
11.2
NH
1.7
0.28
0.36
MeO
Figure 3. X-ray crystal structure of bicyclic pyrazole 12b co-crystal-
lized with mutated p38 (mp38) enzyme.
NH
0.08
sec-butylamine substitution on the 2-position of the pyr-
imidine ring demonstrated a pronounced improvement
in potency against TNF-a formation (IC50 = 0.08lM)
as compared to the other analogs in this series. Presum-
ably, the improvement in activity seen with 12e is due to
the amine providing an additional interaction with the
carbonyl of Met109 as compared to 12b. Additionally,
bicyclic pyrazoles 12d and 12e proved to be as effective
as the initial pyrazolone lead 7 against human p38
MAP kinase.
both TNF-a synthesis and human p38 MAP kinase as
the unsubstituted parent pyrazolone 7. In the pyrimidyl
series, the [5,5]-bicyclic pyrazole inhibitor 12b proved to
be a more potent inhibitor of both TNF-a production
and human p38 MAP kinase than its [5,6]-bicyclic pyr-
azole counterpart 12a. The X-ray crystal structure14 of
bicyclic pyrazole 12b co-crystallized with mutated p38
(mp38) is shown in Figure 3. The mutated p38a herein
described is a double mutant (S180A, Y182F) of murine
p38a.17 The phenoxy-substituted inhibitor 12b orients
itself in the mp38 enzyme such that the 4-fluorophenyl
ring lies in the hydrophobic (specificity) pocket
(Thr106). The inhibitor has a direct interaction between
the oxygen of the pyrazole ring and Lys53 as well as
an interaction between the nitrogen of the pyrimidine
ring and the Met109 N–H. However, the oxygen of the
phenoxy-substituent suffers from a possible repulsive
The discrepancies between the human p38 MAP kinase
data and the TNF-a inhibition data suggest that the
bicyclic pyrazole series is possibly inhibiting TNF-a pro-
duction via multiple mechanisms. Recent literature re-
ports18 have described known p38 inhibitors to be
even more effective at inhibiting the RICK (Rip-like
interacting caspase-like apoptosis-regulatory protein
[CLARP] kinase/Rip2/CARDIAK) kinase, which is a
signal transducer of inflammatory responses.
interaction with the carbonyl of Met109
.
Various amine analogs were then synthesized in the
hope of obtaining a more favorable interaction between
the amine substituent and the carbonyl of the Met109
residue. However, sulfoxide displacement with a benzyl-
amine (12c) resulted in a less potent inhibitor. Smaller
alkyl amines such as 2-methoxypropyl amine (12d) re-
sulted in a modest improvement in potency against
TNF-a production. Bicyclic pyrazole 12e containing a
In conclusion, we have reported the synthesis of a series
of novel bicyclic pyrazole inhibitors of TNF-a synthesis.
Beginning from a modestly active lead 10a, we have
identified bicyclic pyrazole 12e (IC50 = 0.08lM) as a po-
tent inhibitor of TNF-a formation in an in vitro assay
against LPS-stimulated production of TNF-a. We hope
to report further progress in developing this series of
bicyclic pyrazoles in the near future.

4948
J. A. Townes et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4945–4948
11. Mohler, K. M.; Sleath, P. R.; Fitzner, J. N.; Cerretti, D.
Acknowledgements
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We are grateful to Marlene Mekel for obtaining X-ray
crystal data.
12. Barltrop, J. A.; Owen, T. C.; Cory, A. H.; Cory, J. G.
Bioorg. Med. Chem. Lett. 1991, 1, 611–614.
References and notes
13. p38 Kinase was assayed in a buffer containing 25mM
HEPES, 25mM b-glycerophosphate, 25mM MgCl2,
0.1mM Na3VO4, 2mM DTT, and 50lM ATP, in the
presence of inhibitor at concentrations of 10,000, 2000,
400, and 80nM in 96-well microtiter plates (Costar). The
substrate ATF2 was used at 50ng/reaction (coated onto
plates by overnight incubation at 4ꢁC). The reaction was
carried out at 37ꢁC for 1h. Phosphorylated ATF2 was
detected using a phosphoATF2 (Thr71) specific primary
antibody (Cell Signaling), which was then followed by
ALP-conjugated goat anti-rabbit IgG (Jackson Immune
Research). The OD was taken at 405nm with a reference
at 490nm.
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14. The mutant enzyme cannot be phosphorylated and,
therefore, it is homologous to the inactive form of murine
p38a. Protein expression and purification were carried out
as previously described for the murine enzyme.15 For
crystallization, mutated p38a was incubated overnight
(12–16h) with 1mM compound. Cocrystals were grown
by hanging drop vapor diffusion using PEG as a preci-
pitating agent and overall protocols similar to those
previously described for the human enzyme.17 Crystals
7. Badger, A. M.; Bradbeer, J. N.; Votta, B.; Lee, J. C.;
Adams, J. L.; Griswold, D. E. J. Pharmacol. Exp. Ther.
1996, 279, 1453–1461.
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˚
typically diffracted to better than 2.0A resolution and
were of the previously reported space group: P212121;
16
˚
a = 65.2A, b = 74.6A, c = 78.1A.
˚
˚
X-ray data were
collected at beamline 17-BM in the facilities of the
Industrial Macromolecular Crystallography Association
Collaborative Access Team (IMCA-CAT) at the
Advanced Photon Source. These facilities are supported
by the companies of the Industrial Macromolecular
Crystallography Association through a contract with
Illinois Institute of Technology (IIT), executed through
IITÕs Center for Synchrotron Radiation Research and
Instrumentation. Use of the Advanced Photon Source was
supported by the US Department of Energy, Basic Energy
Sciences, Office of Science, under Contract No. W-31-109-
Eng-38.
9. For the synthesis of bicyclic pyrazolones, see: 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.;
Janusz, M. J. J. Med. Chem. 2004, 47, 2724–2727.
10. The compounds were evaluated for their abilities to inhibit
TNF-a production in human monocytic cells (THP-1)
stimulated by LPS. Duplicate cultures of human mono-
cytic (THP-1)11 cells (2.0 · 105/well) were incubated for
15min in the presence of inhibitor at concentrations of
10,000, 2000, 400, and 80nM before the stimulation of
cytokine release by the addition of lipopolysaccharide
(final LPS concentration, 1lg/mL). The amount of TNF-a
released was measured 4h later using an ELISA assay
(R&D Systems, Minneapolis, MN) and the viability of the
cells was measured using an MTS assay12 (Promega Co.,
Madison, WI) following the 4h incubation period. No
inhibitor-induced cytotoxicity was observed during this
assay.
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, 2327–2332.
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, 242–245.
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