Discovery of a new class of p38 kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00270-5

The MAP kinase p38 has been implicated in cytokine signaling, and its inhibitors are potentially useful for the treatment of arthritis and osteoporosis. Novel small-molecule inhibitors of p38 kinase were derived from a combinatorial chemistry effort and e

Full text of DOI:10.1016/S0960-894X(00)00270-5

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2047±2050
Discovery of a New Class of p38 Kinase Inhibitors
Jacques Dumas,a,* Robert Sibley,a Bernd Riedl,a Mary Katherine Monahan,a
Wendy Lee,a Timothy B. Lowinger,a Aniko M. Redman,a Je€rey S. Johnson,a
Jill Kingery-Wood,a William J. Scott,a Roger A. Smith,a Mark Bobko,a
Robert Schoenleber,a Gerald E. Ranges,b Timothy J. Housley,b Ajay Bhargava,b
Scott M. Wilhelmb and Alka Shrikhandeb
aDepartment of Chemistry Research, Bayer Research Center, 400 Morgan Lane, West Haven, CT 06516, USA
bDepartment of Cancer and Osteoporosis Research, Bayer Research Center, 400 Morgan Lane, West Haven, CT 06516, USA
Received 16 March 2000; accepted 2 May 2000
AbstractÐThe MAP kinase p38 has been implicated in cytokine signaling, and its inhibitors are potentially useful for the treatment
of arthritis and osteoporosis. Novel small-molecule inhibitors of p38 kinase were derived from a combinatorial chemistry e€ort and
exhibit activity in the nanomolar range. Very steep structure±activity relationships are observed within this class. # 2000 Elsevier
Science Ltd. All rights reserved.
p38 Kinase belongs to the family of MAP kinases, and
has been implicated in cytokine signaling.1 p38 Inhibi-
tors from the pyridyl-imidazole class,2,3 such as
SB203580 (1, Fig. 1), can disrupt cytokine signaling in
cells, and are e€ective in in vivo models of endotoxin
shock and arthritis.4 As part of a combinatorial chem-
istry e€ort the novel pyrazole 2 was identi®ed as a
reversible p38 inhibitor.5
At the outset of this program, urea substructures were
relatively unknown in the ®eld of kinase inhibition, as
most of the patent activity was concentrated on other
structural types.6 Only one urea example could be found
in the literature, describing broad tyrosine kinase inhi-
bitory activity with 1-phenyl-3-thienyl ureas.7 More
recently, Vertex has also reported aryl and bis-aryl ureas
such as 3 and 4 as inhibitors of p38 kinase.8
Chemistry
The goal of this program was to develop SARs around
the screening hit 2. Pyrazolyl ureas are easily accessible
in moderate to good yields by the reaction of commer-
cially available 2-methyl-3-amino-5-tert-butylpyrazole (6)
with isocyanate 59 (Fig. 2). The corresponding iso-
xazolyl ureas, such as 7, are obtained from 3-tert-butyl-
5-amino isoxazole (8) in the same way.
Figure 1. p38 Kinase inhibitors.
*Corresponding author. Tel.: +1-203-812-5204; fax: +1-203-812-
2650; e-mail: jacques.dumas.b@bayer.com
Figure 2. Synthesis of isoxazolyl and pyrazolyl ureas.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00270-5

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J. Dumas et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2047±2050
Figure 3. 2-Aminoisoxazoles and 2-aminothiadiazoles.
This method can also be used for the preparation of the
isomeric isoxazolyl ureas (from commercially available
3-amino-5-tert-butylisoxazole), and thiadiazolyl ureas.
Isoxazole 8 (Fig. 3) results from hydroxylamine con-
densation with commercially available 4,4-dimethyl-3-
oxo-pentanenitrile. This method is also very ¯exible,
and allows variation of the isoxazole substituent by the
use of other cyanoketones. 2-Amino-5-tert-butylthia-
diazole (9) can be obtained according to previously
published procedures.10
Figure 4. Synthesis of thienyl ureas.
with signi®cant potency losses when one carbon atom is
either added, as in isoxazoles 19 and 24, or removed
(entry 21). The same trend is observed when the geo-
metry of the tert-butyl group is modi®ed, keeping the
number of atoms constant as in entries 17 and 20. The
4-position of the isoxazole (R2 in Table 2) does not tol-
erate substitution very well, as even the addition of a
methyl group results in weaker compounds (entries 22,
23, and 25). The phenyl group substitution of ureas 15
(Type I) and 7 (Type II) is explored in Table 3. The
observed trends are quite consistent across the two
structural types.
Preparation of analogues in which the pyrazole ring is
replaced by a thiophene is depicted in Fig. 4. The known
methyl 3-amino-5-tert-butyl-2-thiophene-carboxylate (10)
is saponi®ed with sodium hydroxide in ethanol, then
decarboxylated upon HCl treatment. The resulting 3-
amino-5-tert-butylthiophene HCl salt 11 can be con-
verted to the urea 12 without puri®cation. The isomeric
thienyl urea 13 is obtained by Curtius rearrangement of
2-carboxy-5-tert-butylthiophene 14, followed by in situ
treatment with 2,3-dichloroaniline.
Deletion of the two chlorine atoms, exempli®ed in 27,
results in inactive compounds. Replacement of the
chlorine atoms by methyl groups (similar sizes and
lipophilicities, but di€erent electronic e€ects) results in
weaker compounds (35 and 42). Introduction of polar,
hydrogen bonding groups in the meta and para posi-
tions does not seem to be tolerated (compounds 30, 31
and 40, 41). Clearly, halogens and tri¯uoromethyl sub-
stituents properly attached to the phenyl ring, which
Rapid generation of urea libraries was achieved in our
combinatorial chemistry e€ort by conducting the amine-
isocyanate reaction in anhydrous DMF (80±95 ꢀC, 18 h).
A modular parallel-synthesis work station, incorporat-
ing Gilson 215 robotic liquid handling and a J-KEM
reaction block,11 allowed the preparation of approxi-
mately 1000 analogues, typically as 10Â10 reaction
matrices.12
Results and Discussion
Table 1. Variation of the heterocyle
Table 1 summarizes the results of the heterocycle varia-
tions developed with the original 2,3-dichlorophenyl
urea substituent.
The pyrazole unit of 2 can be successfully replaced by
an isoxazole (the two possible isomers 7 and 15 are
active in the same range) or by a thiophene (the two
prepared isomers 12 and 13 display similar, but slightly
lower potency in the p38 a2 assay). The thiadiazole
analogue 16 is inactive.
Compound
X
Y
Z
% Inhibition
(500 nM)
p38 a2
IC50 (nM)
15
7
2
16
13
12
CH
CH
CH
S
S
CH
N
O
NCH3
N
CH
CH
O
N
N
N
CH
S
88
93
58
36
53
1
82
76
For the variation of the tert-butyl group, the isoxazole
urea 7 was used as a reference compound (Table 2).
Very steep structure±activity relationships are observed,
120
160

J. Dumas et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2047±2050
2049
Table 2. Isoxazole alkyl substituent
lead compound 2 broadens the scope of this discovery
to isoxazolyl and thienyl ureas. Very steep structure±
activity relationships are observed on the bulky alkyl
group of the ®ve-membered ring heterocycle, as well as
on the phenyl moiety. The original pyrazole hit (2) and
its isoxazole analogue (15) are active in a functional
assay of cellular cytokine signaling (TNF and IL-1
induced IL-6 production in SW1353 cells) in the sub-
micromolar range, making this series a very promising
starting point for future programs.
Compound
R1
R2
% Inhibition
(500 nM)
p38 a2
IC50 (nM)
7
tert-Bu
Cyclobutyl
Adamantyl
2,2-Dimethylpropyl
n-Bu
i-Pr
tert-Bu
i-Pr
1,1-Dimethylpropyl
H
H
H
H
H
93
57
9
36
370
17
18
19
20
21
22
23
24
25
26
81
33
62
18
34
83
18
4
120
1100
290
Acknowledgements
H
CH3
CH3
H
We would like to thank Christopher Browe, Michael
Bombara, Robert Dreyer, David Osterman, Judith Cos-
grove, David Brittelli, Charles Maniglia, Robert Carlson,
and Thuy Chau for support and helpful discussions.
920
120
1,1-Dimethylpropyl CH3
CH3
H
Table 3. Substitution of the phenyl moiety
References and Notes
1. Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T.
F.; Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J.;
Heys, J. R.; Landvatter, S. W.; Stricker, J. E.; McLaughlin,
M. M.; Siemens, I. R.; Fisher, S. M.; Livi, G. P.; White, J. R.;
Adams, J. L.; Yound, P. R. Nature 1994, 372, 739. Han, J.;
Lee, J.-D.; Bibbs, L.; Ulevitch, R. J. Science 1994, 265, 808.
2. Gallagher, T. F.; Fier-Thompson, S. M.; Garigipati, R. S.;
Sorenson, M. E.; Smietana, J. M.; Lee, D.; Bender, P. E.; Lee, J.
C.; Laydon, J. T.; Chabot-Fletcher, M. C.; Breton, J. J.;
Adams, J. L. Bioorg. Med. Chem. Lett. 1995, 5, 1171. Boehm, J.
C.; Smietana, J. M.; Sorenson, M. E.; Gallagher, T. F.; Shel-
drake, P. L.; Bradbeer, J.; Badger, A. M.; Laydon, J. T.; Lee, J.
C.; Hillegrass, L. M.; Griswold, D. E.; Breton, J. J.; Chabot-
Fletcher, M. C.; Adams, J. L. J. Med. Chem. 1996, 39, 3929.
3. Wilson, K.; McCa€rey, P.; Hsiao, K.; Pazhanisamy, S.;
Galullo, V.; Bemis, G.; Fitzgibbon, J.; Caron, P.; Murcko, M.;
Su, M. Chem. Biol. 1997, 4, 423.
Compound Type R1
R2
R3
R4 % Inhibition p38 a2
(500 nM)
IC50 (nM)
27
28
29
30
31
32
33
34
15
35
36
37
38
39
7
I
I
I
I
I
I
I
I
I
H
CF3
H
H
H
H
CF3
H
H
H
Br
NHAc
H
H
H
H
Cl
H
Cl
H
H
NHAc
F
H
CF3
H
H
F
Cl
Cl
CF3
H
H
NHAc
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
33a
9
710
190
58
18
14
74
36
39
88
54
41
62
68
77
93
11
20
49
Cl
4. Badger, A. M.; Bradbeer, J. N.; Votta, B.; Lee, J. C.;
Adams, J. L.; Griswold, D. E. J. Pharm. Exper. Ther. 1996,
279, 1453.
I
CH3 CH3
II
II
II
II
II
II
II
H
H
H
H
Cl
H
H
H
Cl
H
H
Cl
NHAc
H
220
260
200
36
5. Puri®ed and His-tagged p38 a2 (expressed in E. coli) was
activated in vitro by MMK-6 to a high speci®c activity. Using
a microtiter format, all reactions were conducted in 100 mL
volumes with reagents diluted to yield 0.05 mg/well of activated
p38 a2 and 10 mg/well of myelin basic protein in assay bu€er
(25 mM HEPES 7.4, 20 mM MgCl2, 150 mM NaCl). Test
compounds (5 mL of a 10% DMSO solution in water) were
prepared and diluted into the assay to cover a ®nal con-
centration range from 5 nM to 2.5 mM. The kinase assay was
initiated by addition of 25 mL of an ATP cocktail to give a ®nal
concentration of 10 mM cold ATP and 0.2 mCi [g-33P] ATP per
well (200±400 dpm/pmol of ATP). The plate was incubated at
32 ꢀC for 35 min and the reaction quenched with 7 mL of a 1 N
aq HCl solution. The samples were harvested onto a P30 Fil-
termat (Wallac, Inc.) using a TomTec 1295 Harvester (Wallac,
Inc.), and counted in a LKB 1205 Betaplate Liquid Scintilla-
tion Counter (Wallac, Inc.). Negative controls included sub-
strate plus ATP alone. SmithKline Beecham's SB203580 (1)
was used as a standard (IC50=20 nM). At least two indepen-
dent IC50 determinations were performed on each compound,
and the mean value is reported. In all cases, standard devia-
tions were less than 50% of the mean IC50 value.
40
41
42
II CH3 CH3
combine lipophilicity and electron withdrawing e€ects,
yield potent p38 inhibition within this series. The 2,3-
dichlorophenyl ureas 2 and 15 were selected for further
in vitro characterization. Cytokine induction of IL-6 in
chondrocytes is believed to proceed through a signal
transduction pathway, which includes p38 kinase.3
Ureas 2 and 15, as well as SB203580 (1) inhibit IL-6
production in SW1353 cells treated with cytokines IL-1
and TNF.13 A dose-responsive inhibition was observed
with all three agents (IC50=0.05 mM, 0.82 mM and 1.2
mM, respectively, for 1, 2, and 15), although 2 and 15
were much less potent than 1.
In conclusion, a novel series of highly potent p38 kinase
inhibitors has been identi®ed by a combinatorial chem-
istry e€ort.14 Replacement of the pyrazole ring of the
6. For recent reviews, see: Boehm, J. C.; Adams, J. L. Exp.
Opin. Ther. Pat. 2000, 10, 25. Henry, J. R.; Cavender, D. E.;
Wadworth, S. A. Drugs Future 1999, 24, 1345.

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J. Dumas et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2047±2050
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by using a robotic liquid handler (Gilson 215, custom pro-
gram). Into each reaction vial was added a stock solution of
the amine in anhydrous DMF (0.1 M, 1.0 mL, 0.1 mmol) and
a solution of the isocyanate in anhydrous DMF (0.2 M, 0.5
mL, 0.1 mmol). The reaction vial was capped, and the reaction
mixture was heated at 80±95 ꢀC with orbital shaking for 18 h.
The reaction mixture was cooled to room temperature, and any
potential unreacted isocyanate was quenched by the addition
of methanol (1.0 mL). After 30 min, solvents were evaporated
by using a multiple sample evaporator (Savant SpeedVac).
Products were characterized for purity and identity by HPLC/
UV/MS. p38 a2 assay and SW 1353 cell assay results reported
herein re¯ect puri®ed (HPLC or LC, >95% purity) and char-
acterized (1H NMR, MS) samples.
13. SW1353 cells (human chondrosarcoma, ATCC, Bethesda,
MD) are seeded (1000 cells/100 mL DMEM 10% FCS/well)
into 96-well plates and incubated overnight. After medium
replacement, cells are exposed to test compounds for 1 h at
37ꢀC, at which time human IL-1 (1 ng/mL, Endogen, Woburn,
WA) and recombinant human TNFa (10 ng/mL) are added.
Cultures are incubated for 48 h at 37 ꢀC, then supernatant IL-
6 values are determined by ELISA (Endogen, Woburn, WA).
14. Dumas, J.; Khire, U.; Lowinger, T. B.; Paulsen, H.; Riedl,
B.; Scott, W. J.; Smith, R. A.; Wood, J. E.; Hatoum-Mokdad,
H.; Johnson, J.; Lee, W.; Redman, A. PCT Int. Appl. WO 99
32111; Chem. Abstr. 1999, 131, 87909.
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Int. Appl. WO 99 00357; Chem. Abstr. 1999, 130, 66491. Sali-
turo, F. G.; Galullo, V.; Bellon, S.; Bemis, G.; Cochan, J. PCT
Int. Appl. WO 99 58502; Chem. Abstr. 1999, 131, 336949.
9. Preparation of pyrazolyl urea 2: A mixture of 2,3-dichloro-
phenylisocyanate (1.9 g) and 2-methyl-3-amino-5-tert-butyl-
pyrazole (1.43 g, 9.33 mmol) in dry toluene was re¯uxed for 18
h, then cooled to room temp., and concentrated. The residue
was puri®ed by chromatography on silica gel, eluting with 20±
60% EtOAc in hexanes to a€ord 2.14 g (67%) of urea 2 (white
solid, recrystallized from Et2O/hexanes). 1H NMR (300 MHz,
DMSO-d6) d 1.2 (s, 9H), 3.62 (s, 3H), 6.09 (s, 1H), 7.3 (m, 2H),
8.12 (dd, 1H, J=3 and 7 Hz), 8.68 (s, 1H), 9.28 (s, 1H); MS
(FAB) m/z 341 (M+H+, 100%). Anal. calcd C, 52.80; H,
5.32; N, 16.42. Found: C, 52.88; H, 5.30; N, 16.43.
10. Matti, J.; Ledoux, C.; Kesler, E. Bull. Soc. Chim. Fr. 1959,
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11. (a) Gilson, Inc., Middleton, Wisconsin, USA. (b) A dry-
block heater that holds 100 sample vials and provides orbital
shaking was obtained from J-KEM Scienti®c, Inc., St. Louis,
Missouri, USA.
12. General procedure for the parallel synthesis of ureas:
Reagent solutions were typically dispensed into reaction vials