Imidazopyrimidines, potent inhibitors of p38 MAP kinase

Article abstract of DOI:10.1016/S0960-894X(02)01020-X

The MAP kinase p38 is implicated in the release of the pro-inflammatory cytokines TNF-α and IL-1β. Inhibition of cytokine release may be a useful treatment for inflammatory conditions such as rheumatoid arthritis and Crohn's disease. A novel series of imidazopyrimidines have been discovered that potently inhibit p38 and suppress the production of TNF-α in vivo.

Full text of DOI:10.1016/S0960-894X(02)01020-X

Bioorganic & MedicinalChemistry Letters 13 (2003) 347–350
Imidazopyrimidines, Potent Inhibitors of p38 MAP Kinase
Kenneth C. Rupert,* James R. Henry,*,y John H. Dodd,{ Scott A. Wadsworth,
Druie E. Cavender, Gilbert C. Olini, Bohumila Fahmy and John J. Siekierka
Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202, Raritan, NJ 08869, USA
Received 23 September 2002; revised 20 November 2002; accepted 22 November 2002
Abstract—The MAP kinase p38 is implicated in the release of the pro-inflammatory cytokines TNF-a and IL-1b. Inhibition of
cytokine release may be a useful treatment for inflammatory conditions such as rheumatoid arthritis and Crohn’s disease. A novel
series of imidazopyrimidines have been discovered that potently inhibit p38 and suppress the production of TNF-a in vivo.
# 2002 Elsevier Science Ltd. All rights reserved.
The pro-inflammatory cytokines TNF-a and IL-1b play
important roles in chronic inflammatory diseases such
as rheumatoid arthritis, Crohn’s disease, and psoriasis.1
Enzymatic activity of the mitogen-activated protein
(MAP) kinase p38 is required for the release of TNF-a
and IL-1b from monocytes and is also necessary for
signal transduction through the cell-surface receptors
for TNF-a and IL-1b.2 Therefore it is not surprising
that inhibitors of p38 (such as the prototypicalSB
203580 (1)) are effective in in vivo models of inflamma-
tory conditions.3
LPS induced TNF-a inhibition in mice.4a While com-
pound 4 had somewhat modest activity in the in vitro
assays, it showed excellent potency in the in vivo mouse
modeland compared more favorabyl in vivo with 2 and
3 (Table 1).
Given this data, a research program was launched to
optimize this chemicalseries for both in vitro potency
and in vivo efficacy.
The synthesis of 4 is straight forward (Scheme 1).
Deprotonation of 4-picoline (5) and addition of the
resulting anion to ethyl 4-fluorobenzoate (6) produces
the ketone 7. Bromination of 7 occurs readily to afford
the a-bromoketone 8. Treatment of 8 with excess 2,4-
diaminopyrimidine (9) produces 4 regiospecifically.
We have previously disclosed several different structural
classes of p38 inhibitors having improved in vitro and in
vivo activity such as the pyrrolopyridine 24 and the
pyrrolobenzimidazole 35 (Fig. 1). In our ongoing effort
to discover novel, potent inhibitors of p38, we now
report the discovery and development of a series of
imidazopyrimidines showing excellent in vitro and in
vivo potency. Our research approach was to develop
novelheterocyclic scaffolds that maintain the key binding
elements of known inhibitors such as the 4-pyridyl ring
and the 4-fluorophenylring in the appropriate spatial
relationship. In development of the next generation series
of inhibitors related to 2, we synthesized the imidazo-
pyrimidine 4.6 Our screening regime consisted of a p38a
enzyme inhibition assay,7 LPS induced TNF-a inhibition
in human peripheral blood mononuclear cells,4a and
It has been shown that incorporating substitution on
8
the 4-pyridylring can improve potency. The 4-pyridyl
ring of 4 lent itself to substitution (Scheme 2).
Commercially available 2-bromo-4-methylpyridine (10)
underwent palladium catalyzed coupling with amines to
cleanly afford the 2-substituted-4-methyl pyridines 11. It
was necessary to protect the nitrogen of 11 if primary
amines were used in the palladium amination. Deprotona-
tion of 11 and reaction of the resulting anion with ethyl
benzoates (12) gave the corresponding ketones. Depro-
tection and bromination gave the a bromoketones 13.
Cyclization of 13 with excess 2,4-diaminopyrimidine 9
gave the desired substituted pyridine analogues 14a–d.
*Corresponding author. Tel.: +1-908-704-4093; Fax: +1-908-526-
6469; e-mail: krupert@prdus.jnj.com
yCurrent address: Eli Lilly and Co., Indianapolis, IN, USA.
{Current address: Bristol-Myers Squibb Institute, Princeton, NJ,
USA.
The SAR of the substituted pyridine analogues is outlined
in Table 2. Introduction of the benzylamine substituent
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)01020-X

348
K. C. Rupert et al. / Bioorg. Med. Chem. Lett. 13 (2003) 347–350
Figure 1. p38 Kinase inhibitors.
Table 1. Data comparison of 2–4
Scheme 1. Synthesis of 4. (i) NaHMDS, THF, 100%; (ii) Br2, HBr,
AcOH, 97%; (iii) ethanol, reflux, 33%.
Compd
p38a
Enzyme
IC50
TNF-a
Inhibition
(Cells) IC50
TNF-a
Inhibition (Mouse)%
Inhibition 10 mg/kg
2
3
4
1.5 mM
22 nM
570 nM
7 nM
60 nM
40 nM
75
70
97
14a provided a profound increase in p38a enzyme
activity as well as cellular inhibition of TNF-a com-
pared to 4. Compound 14a inhibited p38a with an IC50
of 6 nM versus 570 nM for 4, and had an IC50 of 6 nM
in the cellular assay versus 40 nM for 4. We postulate
that this increase in potency may result from both a
lipophilic interaction of the benzyl group with the
enzyme as well as an additional hydrogen bonding
interaction from the amino group. Further study would
be required to verify this however. Surprisingly 14a was
less potent in vivo than 4, 42% inhibition versus 97%
inhibition at 10 mg/kg. This reduction in activity could
be due to unfavorable metabolism at the N-benzyl
group. Attempts to block metabolism by introduction
of a N-a-methylbenzyl group possessing S stereo-
chemistry gave a further increase in potency over 14a.
Compound 14b had an IC50 of 1 nM in the p38a
enzyme assay versus 6 nM for 14a, as well as increased
potency in the cellular assay 2 nM versus 6 nM. A much
l arger increase in potency was observed in whol e animal s.
Compound 14b inhibited TNF-a production by 96%;
similar to the potency originally observed for 4.
Scheme 2. Synthesis of substituted pyridine analogues. (i) Pd2(dba)3,
H2NR1, BINAP, 50–75%; (ii) (BOC)2O, tBuONa, tBuOH, 50–75%;
(iii) NaHMDS, THF, 75–100%; (iv) HCl, THF, 90%; (v) Br2, HBr,
AcOH, 70–85%; (vi) ethanol, reflux, 20–30%.
our next goalwas to ealborate the series further by
replacing the substituted pyridines with a series of sub-
stituted pyrimidines. Positive effects with this type of
structuralmodification have been seen with other inhib-
itors.8,9 A similar synthesis was developed beginning
with 2-mercapto-4-methylpyrimidine ( 15) (Scheme 3).
Alkylation of 15 with iodomethane gave 4-methyl-2-
(thiomethyl)pyrimidine (16). Deprotonation and reaction
of the anion of 16 with ethylbenzoate gave the ketone 17.
Bromination of 17 occurred smoothly to give the
a-bromoketone 18. Cyclization of 18 with 2,4-diamino-
pyrimidine 9 afforded the thiomethylpyrimidine analogue
19.
It was not necessary to incorporate substitution on the
arylring adjacent to the substituted pyridine. Compound
14c bearing an unsubstituted phenylring and ( S)-1-
methoxy-2-propylamine substitution on the pyridine
ring was a potent inhibitor of the enzyme, as well as in
the cellular and in vivo assays. Incorporation of the
chiralmethylgroup into a ring as demonstrated in 14d
did not affect the potency in the enzyme and cellular
assays, but had a profound effect on in vivo potency,
completely failing to inhibit TNF-a production in mice.
Thiomethylpyrimidine 19 proved to be a usefulinter-
mediate. The thiomethylsubstituent of 19 could be
removed reductively with Raney nickel to give the
unsubstituted pyrimidine 20 or oxidized with oxone to
the methylsulfone pyrimidine 21. The sulfone of 21
could then be displaced by both oxygen and nitrogen
nucleophiles to give compounds 22–4 (Scheme 4).
Since replacement of the pyridine of 4 with substituted
pyridines gave increases in vitro and in vivo potency,

K. C. Rupert et al. / Bioorg. Med. Chem. Lett. 13 (2003) 347–350
Table 2. SAR of substituted pyridine analogues Table 3. SAR of substituted pyrimidine analogues
349
R1
R2
p38a Enzyme TNF-a (cells) TNF-a (Mice)
Y
p38a Enzyme
IC50 (nM)
TNF-a (cells)
IC50 (nM)
TNF-a (Mice)%
inhib. 10 mg/kg
IC50 (nM)
IC50 (nM)
% Inhib.
10 mg/kg
H
20
SCH3
19
OCH3
22
990
457
210
304
28
73
23
87
4-F
6
6
42
90
4-F
H
1
2
2
96
93
37
8
2.3
0.6
66
0.5
100
H
8
3
Inactive
The data for the pyrimidine analogues is shown in Table
3. The unsubstituted pyrimidine analogue 20 was sub-
stantially weaker than 4 in the enzyme and cellular
assays, having an IC50 of 990 nM versus 570 nM in the
enzyme assay and 304 nM versus 40 nM in the cellular
assay. Pyrimidine 20 was also slightly weaker in vivo,
inhibiting TNF-a production in mice by 73% as com-
pared to 97% for 4. The intermediate 19 also displayed
activity, but only weakly inhibited TNF-a production in
vivo by 23%. The oxygen analogue 22 was slightly more
potent than 19 in the enzyme assay but significantly
more potent in vivo inhibiting TNF-a production by
87%. Compound 23 containing the (R)-a-methylbenzyl-
amino group showed a further increase in enzyme and
cellular activities 37 nM and 2.3 nM respectively, but a
slight decrease in whole animal activity to 66% inhibi-
tion. The enantiomer of 23, compound 24 resulting
from the reaction of 21 with (S)-a-methylbenzylamine
proved to be the most potent of the substituted pyr-
imidine analogues both in vitro and in vivo.
Scheme 3. Synthesis of substituted pyrimidine analogues. (i) NaOH,
CH3I, H2O, 80%; (ii) NaHMDS, THF, 90%; (iii) Br2, HBr, AcOH,
80%; (iv) ethanol, reflux, 30%.
Pyrimidine 24 inhibited p38a with and IC50 of 8 nM,
and inhibited TNF-a production in cells with an IC50 of
0.6 nM. Compound 24 was also extremely potent in
vivo, inhibiting TNF-a production in mice completely
when administered orally at 10 mg/kg. Since the (S)-a-
methylbenzylamine substituted pyridine 14b, and pyr-
imidine 24, were both extremely potent in vivo, the
administered doses were lowered to achieve differentia-
tion between the two compounds. Both compounds
were dosed orally to mice at 2 mg/kg. Pyridine 14b
inhibited TNF-a production by 34% while pyrimidine
24 was still extremely effective, inhibiting by 83%. This
data suggests that the substituted pyrimidine series of
imidazopyrimidines has greater in vivo efficacy than
that of the substituted pyridine analogues (Table 4).
In summary, a new series of potent p38 kinase inhibi-
tors having excellent enzymatic, cellular, and in vivo
activities has been developed. The synthesis is brief
and scalable, allowing for a variety of analogues to be
prepared.
Scheme 4. Synthesis of substituted pyrimidine analogues. (i) Raney
Ni, EtOH, H2O, 44%; (ii) Oxone, MeOH, H2O, 70%; (iii) Y=N,O
nucleophiles, heat, 40–60%.

350
K. C. Rupert et al. / Bioorg. Med. Chem. Lett. 13 (2003) 347–350
Table 4. Comparison of 14b and 24
Analogue
p38a Enzyme
IC50 (nM)
TNF-a (cells)
IC50 (nM)
TNF-a (Mice)%
Inhib. 10 mg/kg
TNF-a (Mice)%
Inhib. 2 mg/kg
14b
24
1
8
2
0.6
96
100
34
83
Replacement of an unsubstituted pyridine ring with a
substituted pyridine or pyrimidine ring provided inhibi-
tors having increased in vitro potency while maintaining
excellent potency in whole animals. The most potent
substituted pyridine analogue, 14b, inhibited p38a with
an IC50 of 1 nM and nearly completely inhibited TNF-a
production in mice at 10 mg/kg,while pyrimidine 24
inhibited p38a with an IC50 of 8 nM and completely
inhibited TNF-a production in mice at 10 mg/kg. This
compound remained effective at even lower doses, inhib-
iting TNF-a production in vivo by 83% at 2 mg/kg. The
excellent in vitro and in vivo potency of this series war-
rants further investigation in more advanced models of
inflammatory disease.
Cavender, D. E.; Schafer, P. H.; Siekierka, J. J. Bioorg. Med.
Chem. Lett. 1998, 8, 3335. (c) Henry, J. R.; Dodd, J. H. Tet-
rahedron Lett. 1998, 39, 8763. (d) Dodd, J. H.; Henry, J. R.;
Rupert, K. C. PCT Int. Appl. WO 98/47899.
5. Dodd, J. H.; Henry, J. R.; Rupert, K. C. U.S. Patent
6,147,096.
6. Dodd, J. H.; Henry, J. R.; Rupert, K. C. PCT Int. Appl.
WO 01/34605.
7. Recombinant, activated, 6ꢀHis-tagged mouse p38a enzyme
was purified from osmotically shocked Drosophila S2 cells in
our laboratory, using a p38a clone generously provided by Dr.
Richard Ulevitch, Scripps Research Institute, La Jolla, CA.
p38 was incubated in kinase reaction buffer (25 mM HEPES
pH 7.5, 10 mM MgCl2, 10 mM MnCl2) containing 25 mM
ATP, with 60 mg myelin basic protein (MBP) as substrate (Life
Technologies, Gaithersburg, MD) and 1mCi g-33P-ATP (3000
Ci/mmol, Amersham Life Science, Arlington Heights, IL),
with or without test compounds or vehicle (DMSO, 2% final
concentration), in a totalvoul me of 60 mL, in a round-bottom
polypropylene 96-well plate. After 30 min at 30 ꢁC, reactions
were stopped and proteins precipitated by the addition of 60 mL/
well of 50% trichloroacetic acid (TCA), and the precipitates
transferred to a 96-well Durapore membrane filterplate (Milli-
pore, Bedford, MA). Wells were filtered using a Millipore
vacuum manifold, washed 5ꢀ with 200 mL/well of 10% TCA/10
mM sodium phosphate, and briefly air-dried. Thirty mL/well of
Microscint-20 scintillant (Packard, Meriden, CT) was added,
the plate sealed with plastic film (Packard), and counted in a
Packard TopCount microplate scintillation counter.
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