1-Phenyl-5-pyrazolyl ureas: Potent and selective p38 kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00272-9

Inhibitors of the MAP kinase p38 are potentially useful for the treatment of arthritis and osteoporosis. Several 2,3-dichlorophenyl ureas were identified as small-molecule inhibitors of p38 by a combinatorial chemistry effort. Optimization for cellular potency led to the discovery of a new class of potent and selective p38 kinase inhibitors, exemplified by the 1-phenyl-5-pyrazolyl urea 7 (IC₅₀ = 13 nM). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00272-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2051±2054
1-Phenyl-5-pyrazolyl Ureas: Potent and Selective p38
Kinase Inhibitors
Jacques Dumas,a,* Holia Hatoum-Mokdad,a Robert Sibley,a Bernd Riedl,a
William J. Scott,a Mary Katherine Monahan,a Timothy B. Lowinger,a
Catherine Brennan,a Reina Natero,a Ti€any Turner,a Je€rey S. Johnson,a
Robert Schoenleber,a Ajay Bhargava,b Scott M. Wilhelm,b
Timothy J. Housley,b Gerald E. Rangesb 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
AbstractÐInhibitors of the MAP kinase p38 are potentially useful for the treatment of arthritis and osteoporosis. Several 2,3-
dichlorophenyl ureas were identi®ed as small-molecule inhibitors of p38 by a combinatorial chemistry e€ort. Optimization for cel-
lular potency led to the discovery of a new class of potent and selective p38 kinase inhibitors, exempli®ed by the 1-phenyl-5-pyr-
azolyl urea 7 (IC50=13 nM). # 2000 Elsevier Science Ltd. All rights reserved.
Inhibitors of the MAP kinase p381,2 such as SB203580
(1, Fig. 1) provide novel approaches for the treatment of
osteoporosis and in¯ammatory disorders.3 As part of a
combinatorial chemistry program, pyrazole 2 was iden-
ti®ed as a new lead structure4 (see preceding paper). This
compound, as well as 1, also inhibits TNF and IL-1
induced IL-6 production in SW1353 cells (human
chondro-sarcoma).4
Chemistry
1-Phenyl-5-pyrazolyl ureas, such as 3, are easily accessible
by the reaction of 1-phenyl-5-aminopyrazole 4 with 2,3-
dichlorophenyl isocyanate. This reaction usually proceeds
in very good yields (Fig. 2). The requisite 1-phenyl-5-
aminopyrazoles are prepared by the condensation of
phenylhydrazines with the commercially available keto-
nitrile 5.
Figure 1. p38 Kinase inhibitors.
This communication describes our e€orts in explor-
ing the substitution of 2, leading to the more potent
1-phenyl-5-pyrazolyl urea series, as well as optimizing
the potency in the cellular functional assay.
Figure 2. General synthesis of phenylpyrazolyl ureas.
Reduction and acylation of the analogously formed 1-
(3-nitrophenyl)pyrazole urea 6 leads to 7 and 8 respec-
*Corresponding author. Tel.: +1-203-812-5204; fax: +1-203-812-2650;
e-mail: jacques.dumas.b@bayer.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00272-9

2052
J. Dumas et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2051±2054
Figure 4. Preparation of phenylthienyl ureas.
Table 1. Variation of the phenyl substituent
Compound R1 R2
R3 R4 % Inhibition p38 a2 SW 13534
(500 nM) IC50 (nM) IC50 (nM)
Figure 3. Synthesis of substituted pyrazolyl ureas.
2
Cl Cl
H
CH3
H
CF3
H
H
H
H
H
H
Br
53
820
18
19
20
21
22
23
24
25
26
27
H
H
H
CF3
H
H
H
H
H
H
Br
H
22a
30a
47
tively (Fig. 3). The para-substituted aniline 9 can be
prepared in a similar fashion. The 2-pyrazolylacetates
12±14 are prepared by derivatization of the ester 10,
which is prepared from 11.
30
H
325
290
CO2Bu H
CH3 CH3
H
H
H
H
H
H
H
H
24
33
10
H
H
H
Br
CF3
Ph
The analogue in which the pyrazole ring is replaced by
a thiophene is prepared by a di€erent protocol (Fig. 4).
Phenylthiophene 15 results from the palladium-
catalyzed coupling of the corresponding bromide 16,
which is obtained by direct bromination of thienyl urea
17 (see preceding paper).
797
aPercent inhibition measured at 5 mM.
Table 2. Variation of the pyrazole substituent
Results and Discussion
The right-hand side of 2 consists of a phenyl ring with
two lipophilic substituents. A small set of analogues
focusing on the substitution of this ring is shown in
Table 1. Of these, only 22 and 23 retain nanomolar
potency, albeit weaker than that of the lead. Pyrazole 2
is used as our reference compound for the variation of
the pyrazole substituent (Table 2). Deletion of the methyl
group of 2 (as in 28) results in an equipotent compound
in the kinase assay, but a dramatic loss of activity in the
functional assay, suggesting a cellular permeability issue.
Compound
R
% Inhibition
(500 nM)
p38 a2
SW 13534
IC50 (nM) IC50 (nM)
2
CH3
H
CH2CH2CN
CH2CF3
Ph
CH2CO2Et
CH2CO2H
CH2C(O)NH2
CH2C(O)NHCH3
CH2CH2OH
53
44
180
87
30
105
820
28
29
30
3
10
12
13
14
31
91
81
91
97
76
0
48
61
75
Inactivea
Inactivea
Inactivea
70
Inactivea
The 2-position of the pyrazole can accommodate a
variety of substituents. Based on the hypothesis that this
position of the pyrazole could interact with the sugar-
binding pocket of the enzyme, we introduced a series of
hydrogen bonding donors and acceptors. This hypoth-
esis turned out to be unlikely, since potency decreases as
hydrophilicity increases (compounds 10 and 12±14).
However, replacement of the methyl by a phenyl as in 3
results in a signi®cant increase in biochemical and cel-
lular potency. The substitution of the phenyl ring of 3 is
further examined in Table 3.
250
130
Inactivea
aLess than 20% inhibition at 2.5 mM.
With the exception of the acetamide 8 (in which the
introduction of a hydrophilic group tends to decrease
potency), all analogues are potent inhibitors of p38. The
more potent compounds of this set are the 3-nitro and
the 3-amino analogues, 6 and 7. These compounds are

J. Dumas et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2051±2054
Table 3. Structure±activity relationships of phenylpyrazolyl ureas
2053
selected for further pharmacological characterization.
The selectivity of pyrazole 7 has been assessed against
several cytosolic signaling kinases.7 With the exception
of the other p38 isoform, p38 b1 (IC50=52 nM), 7 is
only moderately active against JNK-1 (IC50=850 nM)
and abl (IC50=2.7 mM), and inactive against ERK-1
(0% inhibition at 5 mM). Additional data obtained from
MDS Panlabs con®rms this result.8
In conclusion, we wish to report a new class of highly
potent and selective p38 kinase inhibitors.9 Replacement
of the methyl group of 2 by a phenyl group leads to an
improvement of the cellular potency. Results of phar-
macological studies with the most promising analogue 7
will be reported in due course.
Compound
X
Y
Z
R1
R2
p38 a2
IC50 (nM) IC50 (nM)
SW 13534
3
15
6
7
8
32
33
34
35
36
37
38
39
40
41
9
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
N
C
N
S
H
H
NO2
NH2
NHAc
H
H
H
H
H
H
i-Pr
OCH3
H
30
130
11
70
142
23
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
13
42
>500
110
53
Inactivea
2000
209
Acknowledgements
H
CF3
OCH3
H
H
H
F
H
H
H
56
35
39
32
42
33
120
43
H
60
25
67
324
We would like to thank Christopher Browe and Robert
Dreyer (cloning and puri®cation of p38 a2 kinase),
Teresa Lopes and Du-Shieng Chien (solubility mea-
surements) for their support. We are also grateful to
Charles Maniglia, David Brittelli, Martin Osterhout,
Thuy Chau, and Robert Carlson for helpful discussions.
NO2
SO2CH3
Cl
H
H
H
NH2
49
Inactivea
912
148
CCH3
CH
29
aLess than 20% inhibition at 2.5 mM.
References and Notes
also very potent in the functional cellular assay,
although there is no good correlation between the
activity against p38 and cellular potency (33 vs 35, 6 vs
36). In general, analogues with an electron-withdrawing
group in the para position of the phenyl moiety (such as
pyrazolyl ureas 36 and 37) show more activity in the
functional assay. Finally, two analogues in which the
phenylpyrazole conformation is modi®ed, such as 2-
pyridylpyrazolyl urea 40 (¯at structure due to a strong
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.;
Sheldrake, 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. 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.
3. Badger, A. M.; Bradbeer, J.; Votta, B.; Lee, J. C.; Adams,
J. L.; Griswold, D. E. J. Pharm. Exper. Ther. 1996, 279, 1453.
4. For p38 a2 assay and SW1353 cell assay conditions, see
preceding paper (Bioorg. Med. Chem. Lett. 2000, 10, 2047).
p38 a2 Assay and SW 1353 cell assay results reported herein
re¯ect puri®ed (HPLC or LC, > 95% purity) and character-
ized (1H NMR, MS) samples.
1
internal hydrogen bond, as observed by H NMR), or
the non-planar ortho-substituted analogue 41, are still
active in the kinase assay, but very weak in cells. The
biopharmaceutical properties of the best phenylpyrazole
analogues are depicted in Table 4.
While molecular weights lie mostly within acceptable
ranges for favorable oral availability, lipophilicities and
aqueous solubilities appear to be suboptimal for many
members of this new class.5 Because of its superior
aqueous solubility, 3-aminophenylpyrazolyl urea 7 6 was
Table 4. Biopharmaceutical properties of selected phenylpyrazolyl
ureas
5. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
J. Adv. Drug Delivery Rev. 1997, 23, 3.
Compound
p38 a2
IC50 (nM) IC50 (nM) (g/mol) (daylight) solubility
SW 1353
MW
cLog P
Aqueous
6. Preparation of 6 and 7: A mixture of 4,4-dimethyl-3-oxo-
pentanenitrile 5 (32.8 g, 0.26 mol), 3-nitrophenyl hydrazine
hydrochloride (50 g, 1 equiv) and AcOH (6 mL) in EtOH (525
mL) is heated at the re¯ux temperature overnight, then cooled
down to rt and concentrated under reduced pressure. The solid
residue is washed with Et2O, suspended in EtOAc, and treated
with 300 mL of 1 M aq NaHCO3 solution. The organic layer is
separated, washed with brine, dried (MgSO4), and con-
centrated. The solid residue is washed with 30% Et2O in hex-
anes to a€ord 59.4 g (87% yield) of 2-(3-nitrophenyl)-3-
amino-5-tert-butyl pyrazole as a yellow solid: 1H NMR
(mg/mL,
pH 7.5)
3
6
7
35
36
37
39
30
11
13
35
39
32
33
70
23
42
60
25
67
49
403
448
418
433
448
481
421
5.96
6.06
4.76
5.99
6.06
4.74
6.27
24
18
594
46
66
79
17

2054
J. Dumas et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2051±2054
(DMSO-d6) d 1.22 (s, 9H), 5.46 (s, 1H), 5.49 (bs, 2H), 7.72 (t,
1H, J=8 Hz), 8.08 (m, 2H), 8.44 (t, 1H, J=2 Hz). A mixture
of the above material (13.07 g, 50 mmol) and 2,3-dichloro-
phenyl isocyanate (9.8 g, 1.04 equiv) in dry toluene (300 mL) is
heated at 70 ꢀC overnight, and cooled to rt. The reaction mix-
ture is diluted with EtOAc, washed with water, brine, dried
(MgSO4), and concentrated. The solid residue is washed with
toluene (100 mL) then with Et2O/hexanes to a€ord 6 (16 g,
(DMSO-d6) d 1.25 (s, 9H), 5.40 (bs, 2H), 6.34 (s, 1H), 6.58 (m,
2H), 6.68 (bs, 1H), 7.13 (t, 1H, J=8 Hz), 7.30 (m, 2H), 8.09
(m, 2H), 8.86 (bs, 1H), 9.23 (bs, 1H); MS (FAB) m/z 418
(M+H+, 70%). Anal. calcd C, 57.42; H, 5.06; N 16.74.
Found: C, 57.39; H, 5.10; N, 16.53.
7. His-tagged JNK-1 was expressed in Sf9 cells and puri®ed
on imidazole sepharose. Abl kinase was purchased from Cal-
biochem. Activated ERK-1 was purchased from Upstate Bio-
technology.
1
71% yield) as a white solid: H NMR (DMSO-d6) d 1.29 (s,
9H), 6.44 (s, 1H), 7.30 (m, 2H), 7.81 (t, 1H, J=8 Hz), 7.97 (m,
1H), 8.05 (bd, 1H, J=8 Hz), 8.22 (bd, 1H, J=8 Hz), 8.36 (t,
1H, J=2 Hz), 8.76 (bs, 1H), 9.33 (bs, 1H); MS (FAB) m/z 448
(M+H+, 12%). Anal. calcd C, 53.58; H, 4.27; N, 15.62.
Found: C, 53.44; H, 4.35; N, 15.35. Iron powder (18.7 g) is
added to a mixture of 6 (31.4 g, 70 mmol), AcOH (700 mL)
and water (8 mL). The reaction mixture is stirred at rt over-
night, then diluted with EtOAc and water. The pH is adjusted
to 4 by slow addition of a 1 N aq NaOH solution. The organic
layer is separated, washed with brine, dried (MgSO4), and
concentrated. The residue is puri®ed by chromatography
(SiO2) (35% EtOAc/hexanes) to a€ord 7 (25.9 g, 88% yield) as
a white solid (recrystallized from Et2O/hexanes). 1H NMR
8. Testing of 7 against a panel of kinases at MDS Panlabs
(Bothell, WA) showed modest potency against HER-2 (8.6
mM), p56lck (2.7 mM), and little e€ect against PKA, PKCa,
PKCb, PKCg, EGF receptor kinase, and p59fyn (less than
50% inhibition at 10 mM).
9. Dumas, J.; Khire, U.; Lowinger, T. B.; Riedl, B.; Scott, W.
J.; Smith, R. A.; Wood, J. E.; Hatoum-Mokdad, H.; Johnson,
J.; Redman, A.; Sibley, R. PCT Int. Appl. WO 99 32110;
Chem. Abstr. 1999, 131, 73649. Dumas, J.; Khire, U.; Low-
inger, 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.