Development of orally bioavailable bicyclic pyrazolones as inhibitors of tumor necrosis factor-α production

Article abstract of DOI:10.1021/jm049968m

2-Aryl-3-pyrimidinyl based tumor necrosis factor-α (TNF-α) inhibitors, which contain a novel bicyclic pyrazolone core, are described. Many showed low-nanomolar activity against lipopolysaccharide-induced TNF-α production in monocytic cells. Secondary scre

Full text of DOI:10.1021/jm049968m

2724
J . Med. Chem. 2004, 47, 2724-2727
Letters
Develop m en t of Or a lly Bioa va ila ble
Bicyclic P yr a zolon es a s In h ibitor s of
Tu m or Necr osis F a ctor -r P r od u ction
Michael P. Clark,* Steven K. Laughlin,
Matthew J . Laufersweiler, Roger G. Bookland,
Todd A. Brugel, Adam Golebiowski, Mark P. Sabat,
J ennifer A. Townes, J ohn C. VanRens,
J ane F. Djung, Michael G. Natchus, Biswanath De,
Lily C. Hsieh, Susan C. Xu, Rick L. Walter,
Marlene J . Mekel, Sandra A. Heitmeyer,
Kimberly K. Brown, Karen J uergens,
F igu r e 1. Vicinal aryl/pyridinyl (pyrimidinyl) TNF-R inhibi-
tors.
The prototypical p38 inhibitor (SB-203580)4 contain-
ing the 4-pyridinylimidazole motif was found to be
competitive with ATP and to interact with the p38 ATP
binding site. It is known that many other relevant
kinases, including the J NK and ERK MAP kinases, that
share structural and functional homology with p38 can
be modulated with ATP mimetics as inhibitors.5 Ad-
ditionally, it is estimated that 518 kinases exist in the
human genome.6 Therefore, kinase specificity could
prove to be a major hurdle in designing safe MAP kinase
inhibitors.
Our research strategy involves designing kinase
inhibitors that will result in the inhibition of TNF-R
formation. Since multiple kinases exist within the
signaling cascade, there exist several points at which
one may effectively inhibit TNF-R formation. By opti-
mization of the TNF-R inhibitory activity rather than
utilizing a single kinase assay, the end result is estab-
lishing efficacy by shutting down the overall production
of TNF-R. Therefore, our primary screening assay
entails evaluating analogues for lipopolysaccharide
(LPS) stimulated TNF-R inhibition in the THP-1 cellular
assay.7 Herein, we report a new structural class of
2-aryl-3-pyrimidinyl-based TNF-R inhibitors that con-
tain a novel bicyclic pyrazolone core. Our investigations
have yielded amino-substituted pyrimidine bicyclic pyra-
zolone analogues, such as phenylaminopyrimidine 1 and
alkylaminopyrimidine 2, as extremely potent inhibitors
of TNF-R production (Figure 1).
Synthesis of bicyclic pyrazolone (1) was accomplished
in seven steps from the known pyrazolidine 38 (Scheme
1). Removal of the Boc protecting group, acylation with
4-fluorophenylacetyl chloride, and hydrogenolysis of the
Cbz protecting group affords the amine 4. Acylation of
amine 4 with the pyrimidine acid chloride 59 and base-
induced cyclization of the resulting bisamide provide the
intermediate bicyclic pyrazolone 6. Oxidation of the
sulfide 6 to the sulfone allows for facile displacement
with 2,6-dichloroaniline to afford bicyclic pyrazolone 1.
Table 1 summarizes a preliminary survey of phenyl-
amino moieties introduced to the 2-position of the
pyrimidine ring of the bicyclic pyrazolones. Examination
of the results demonstrates that small electron-with-
drawing substituents at the ortho position of the aniline
ring tend to increase potency (1 and 7) relative to the
Yetunde O. Taiwo, and Michael J . J anusz
Health Care Research Center,
Procter and Gamble Pharmaceuticals,
8700 Mason-Montgomery Road, Mason, Ohio 45040
Received J anuary 9, 2004
Abstr a ct: 2-Aryl-3-pyrimidinyl based tumor necrosis factor-R
(TNF-R) inhibitors, which contain a novel bicyclic pyrazolone
core, are described. Many showed low-nanomolar activity
against lipopolysaccharide-induced TNF-R production in mono-
cytic cells. Secondary screening data are presented for the
pyrimidinyl bicyclic pyrazolones. Several of these analogues
showed good oral bioavailability in rat and efficacy in the rat
iodoacetate in vivo model.
In our continuing efforts toward the development of
disease-modifying treatments for osteoarthritis, we are
targeting the development of novel mitogen-activated
protein (MAP) kinase inhibitors. MAP kinases consti-
tute a part of the signal transduction pathway respon-
sible for the relay of information from the cell surface
to the nucleus. MAP kinases, such as the p38 and J NK
(c-J un amino terminal kinase) families, are activated
in response to infection, cellular stress, and the proin-
flammatory cytokines TNF-R (tumor necrosis factor) and
IL-1â (interleukin-1).1 The overexpression of these cy-
tokines has been implicated in the pathogenesis of a
number of serious inflammatory disorders such as
rheumatoid arthritis (RA),2 osteoarthritis (OA),3 and
Crohn’s disease. The effective therapeutic treatment of
inflammatory diseases such as RA with the monoclonal
anti-TNF-R antibody Remicade (infliximab), the TNF-R
receptor fusion protein Enbrel (etanercept), and the
soluble IL-1 receptor antagonist Anakinra (kineret) has
been hampered by the high cost and inconveniences
associated with these biologics. Therefore, a low molec-
ular weight, orally bioavailable molecule that sup-
presses TNF-R production remains an attractive target.
It has been established that agents that inhibit the
enzyme p38 MAP kinase can decrease levels of these
proinflammatory cytokines1 and thereby reduce inflam-
mation and prevent further tissue destruction.
* To whom correspondence should be addressed. Phone: (513) 622-
0012. Fax: (513) 622-3681. E-mail: clark.mp@pg.com.
10.1021/jm049968m CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/24/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2725
Ta ble 2. TNF-R Inhibition Values of the
Alkylaminopyrimidine Bicyclic Pyrazolone Analogues
Sch em e 1. Synthetic Scheme for
Phenylaminopyrimidine Bicyclic Pyrazolone 1a
a
Reagents and conditions: (a) SOCl2, MeOH, 90%; (b) 4-fluo-
rophenylacetyl chloride, NaOH, H2O/CH2Cl2, 96%; (c) H2, Pd/C,
methanol, 83%; (d) acid chloride 5, NaOH, H2O, CH2Cl2, 90%; (e)
NaH, DMF, 0 °C, 52%; (f) oxone, THF/MeOH/H2O; (g) NaH, 2,6-
dichloroaniline, THF, 50%, two steps.
Ta ble 1. TNF-R Inhibition Values of the
Phenylaminopyrimidine Bicyclic Pyrazolone Analogues
a
IC50 of LPS-stimulated TNF-R production in human monocytic
cells (THP-1). Standard deviation for assays is typically (30% of
the mean or less.
a
IC50 of LPS-stimulated TNF-R production in human monocytic
cells (THP-1). Standard deviation for assays is typically (30% of
the mean or less.
Table 2 summarizes a broad survey of alkylamino
bicyclic pyrazolone analogues. Examination of the more
potent inhibitors in Table 2 demonstrates a greater
tolerance for diverse substituents in the alkylamino
series not seen in the aniline appendages in Table 1.
In general, the most potent analogues possessed an
R-methyl group on the amino substituent, as exemplified
by comparison of 2 and 11-17 relative to 23. One of
the most active compounds (2) was 10-fold more potent
than its corresponding (R)-methylbenzylamino10 enan-
tiomer 17. Comparison of the [5,5]- and [5,6]-bicyclic
pyrazolone scaffolds shows more than a 10-fold decrease
meta and para positions (data not shown) and to the
unsubstituted aniline (8). Replacement of the phenyl
ring with a heterocycle, such as a pyridine or pyrimidine
ring, dramatically reduced the potency (9 and 10)
relative to the aniline (8) derivative.
The related alkylaminopyrimidine bicyclic pyrazolone
series was also synthesized from the intermediate 6.
Nucleophilic displacement of the sulfone functionality
with a wide array of alkylamines was carried out at
higher temperatures in toluene. Yields for the sulfone
displacement ranged from 30% to 65%.

2726 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Letters
Ta ble 3. Solubility, Metabolism, and Rat Oral Bioavailability
Ta ble 5. Kinase Profiling Dataa
of Aminopyrimidine Bicyclic Pyrazolone Analogues
kinase
CRAF
MEK1
ERK1
ERK2/MAPK2
MKK4/SKK1
J NK1R1
J NK2R2
J NK3
2b
14b
21b
TNF-R
solubility
(mg/mL)
metabolismb
(%)
61
99
82
80
166
52
91
95
9
108
117
105
73
55
102
96
88
90
65
8
5
72
-
compd
IC50 (nM)a
% F (rat)
2
11
14
21
23
3
2
22
50
74
0.20
0.46
1.85
0.84
0.10
50
60
29
<5
46
29
10
25
63
18
8
4
36
99
a
IC50 of LPS-stimulated TNF-R production in human monocytic
GSK3â
Lck
101
161
98
117
95
cells (THP-1). Metabolism11 measured as percent loss at 4 h in
b
141
119
101
99
111
112
rat hepatocytes.
PKBocAkt1
CDK2/cyclin A
CDK1/cyclin B
MAPK1/IKKB
MAPKAP-K2
89
108
96
Ta ble 4. Comparison of IC50 Values/Inhibition Data for 2, 14,
and 21
91
113
IC50 (nM)
a
TNF-R
TNF-R
IL-1â
All assays done in triplicate, values at 1 µM compound
b
compd
THP-1
PBMCa
PBMCb
p38R
concentration with ATP at the Km. Kinase activity expressed as
a percentage of that in control incubations. Standard deviation
for assays is typically (10%.
2
14
21
3
22
50
0.4
8
16
0.8
42
39
8
54
27
a
IC50 of LPS-stimulated TNF-R production in human PBMC.
IC50 of LPS-stimulated IL-1â production in human PBMC.
b
in potency in the [5,6]-analogues (2, 11, 14, and 21
relative to 13, 12, 15 and 22).
An early goal was to improve the aqueous solubility
of our initial, more potent alkylaminopyrimidine bicyclic
pyrazolone leads. Both 2 and 23 suffered from poor
solubility11 and moderate instability in our in vitro rat
hepatocyte metabolic stability assay12 (Table 3). Not
surprisingly, substitution of the methylbenzylamine
with smaller alkylamines provided more soluble com-
pounds such as pyrazolone 11. Unfortunately, 11, while
it is our most active compound to date, still suffered
from metabolic instability. Analysis of the major me-
tabolites led to the conclusion that the butyl side chain
of the amine was being oxidized, most likely in the
terminal position. To circumvent this oxidation, we
chose to make 21. The addition of the methoxy group
in the terminal position dramatically decreased me-
tabolism to less than 5% while increasing the solubility
to 840 µg/mL. These two factors likely contributed to
the large increase in bioavailability (63%) in the rat
model. The most solubilizing amino substituent, the
2-hydroxy-1,2-dimethylpropylamine (14), showed a sig-
nificant increase in solubility while still retaining good
potency. Compounds 2 and 14 have been screeened in
the rat iodoacetate model, and both showed positive oral
efficacy (15-25 mg/kg).13
Compounds 2, 14, and 21 were tested in a human
p38R kinase assay,14 and the observed values closely
corresponded with the THP-1 whole-cell inhibition data
(Table 4). Compound 2 was the most potent p38 inhibi-
tor at 8 nM, while 14 and 21 also showed excellent
potency at 54 and 27 nM, respectively. All three amino-
substituted pyrimidine bicyclic pyrazolone derivatives
showed excellent inhibition in peripheral blood mono-
nuclear cells (PBMC)15 of both cytokines TNF-R and
interleukin-1â (IL-1â).
F igu r e 2. Pyrazolone 14 cocrystallized in mutated p38 active
site.
J NK-2 make pyrazolone 14 currently the most selective
of the aminopyrimidine bicyclic pyrazolones.
After achieving good selectivity, solubility, and oral
efficacy in the rat IA model with 14, we then cocrystal-
lized 14 with mutated p38R16 (Figure 2). We observed
the typical vicinal bis-aryl p38 inhibitor interactions,
including binding of the fluorophenyl ring into the Thr-
106 hydrophobic pocket and two hydrogen-bonding
interactions between the Met-109 amide NH and the
nitrogen of the pyrimidine ring and between the Met-
109 carbonyl oxygen and the nitrogen of the secondary
amine. We also observed a strong hydrogen-bonding
interaction between the carbonyl oxygen of the pyraz-
olone ring and Lys-53.
A kinase profiling study on 2, 14, and 21 was then
conducted (Table 5). Compound 14 shows the greatest
selectivity versus the J NK kinases while only signifi-
cantly inhibiting ERK1 kinase. The increased selectivity
against J NK-3 and good selectivity versus J NK-1 and
In conclusion, we have developed a novel bicyclic
pyrazolone scaffold that maintains the important bind-
ing motif of the vicinal 4-fluorophenyl and pyrimidinyl
ring systems in the p38 ATP binding site. In general,

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2727
release by the addition of LPS (1 µg/mL). The amount of TNF-R
released was measured 4 h later using an ELISA (R&D Systems,
Minneapolis, MN). The viability of the cells after the 4 h of
incubation was measured using MTS assay18 (Promega Co.,
Madison, WI). Standard deviation for THP-1 cellular assays were
typically (30% of the mean or less.
the alkylaminopyrimidine bicyclic pyrazolones tended
to be more potent than the aminophenylpyrimidine
derivatives against LPS-stimulated human monocytic
cells (THP-1). Compounds 2, 14, and 21 are effective
inhibitors of TNF-R and IL-1â cytokine release from
PBMC. These three compounds show excellent potency
against human p38, good selectivity in the kinase panel,
and positive oral efficacy (15-25 mg/kg) in the rat
iodoacetate in vivo model. Further optimization of this
novel scaffold will be reported in future publications.
(8) Zhang, W.-J .; Berglund, A.; Kao, J . L.-F.; Couty, J .-P.; Ger-
shengorn, M. C.; Marshall, G. R. Impact of Azaproline on Amide
Cis-Trans Isomerism: Conformational Analyses and NMR
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control inflammatory cytokines. Patent application WO 03/
24971, 2003.
(10) Liverton, N. J .; Butcher, J . W.; Claiborne, C. F.; Claremon, D.
G.; Libby, B. E.; Nguyen, K. T.; Pitzenberger, S. M.; Selnick, H.
G.; Smith, G. R.; Tebben, A.; Vacca, J . P.; Varga, S. L.; Agarwal,
L.; Dancheck, K.; Forsyth, A. J .; Fletcher, D. S.; Frantz, B.;
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(11) Solubility assay procedure. Solubility of analogues was measured
in 50 mM phosphate buffer, pH 7.4, ionic strength 0.15 M. The
solubilities were determined by the shake flask method after
24 h of equilibration. Concentrations in aqueous solutions were
determined for the supernatant of centrifuged and filtered
samples by UV-vis spectrophotometry. Calibration solutions
were prepared in acetonitrile/buffer in a 1:1 volume ratio. The
samples were diluted with acetonitrile to obtain identical media
with calibration standards.
(12) In vitro metabolism assay procedure. The in vitro metabolic
stability of analogues in plated rat hepatocytes (Sprague-Dawley)
obtained from Cedra Corporation was determined in triplicate
using a total volume of 0.2 mL containing 0.25 µM NCE
incubated in rat hepatocyte and matrigel blank microtiter plates.
The plates were maintained at 37 °C throughout the study.
Samples were removed from wells at 0, 2, and 4 h, and NCE
samples were analyzed by HPLC/MS/MS with reverse-phase
chromatography. To improve analytical efficiency, compounds
were grouped together (postincubation) into a multicompound
assay. Samples from like time points containing the different
compounds were combined, and an internal standard (1.1 ng/
mL stock) was added. Results for each compound were expressed
as the ratio of the compound response area to the internal
standard response area. Percent loss was calculated by dividing
the 2 and 4 h ratios by the 0 h ratio.
Ack n ow led gm en t. We are grateful to A. L. Roe, D.
C. Ackley, A. M. Walter, C. R. Dietsch, and D. M. Bornes
for pharmacokinetic and metabolic stability data, and
M. Buchalova for solubility data.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for all final compounds and intermediates, including
characterization data for final compounds 1, 2, 7-29. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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J M049968M