Novel p38 inhibitors with potent oral efficacy in several models of rheumatoid arthritis

Article abstract of DOI:10.1016/j.bmcl.2004.03.106

A library of trisubstituted oxazoles, thiazoles, imidazoles (1,2,4- and 2,4,5-substituted) and imidazo[1,2-b]pyridines was prepared and evaluated in vitro as p38α inhibitors and in vivo in several models of rheumatoid arthritis. Four structures - 32, 37,

Full text of DOI:10.1016/j.bmcl.2004.03.106

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3595–3599
Novel p38 inhibitors with potent oral efficacy in
several models of rheumatoid arthritis
Laszlo Revesz,* Ernst Blum, Franco E. Di Padova, Thomas Buhl, Roland Feifel,
Hermann Gram, Peter Hiestand, Ute Manning and Gerard Rucklin
Novartis Institutes for BioMedical Research, Arthritis & Bone Metabolism, CH-4002 Basel, Switzerland
Received 21 January 2004; revised 22 March 2004; accepted 29 March 2004
Abstract—A library of trisubstituted oxazoles, thiazoles, imidazoles (1,2,4- and 2,4,5-substituted) and imidazo[1,2-b]pyridines was
prepared and evaluated in vitro as p38a inhibitors and in vivo in several models of rheumatoid arthritis. Four structures––32, 37, 45
and 59––were identified as potent inhibitors of p38a with high efficacy in the LPS induced TNFa release model in the mouse, the
adjuvant induced arthritis and the collagen induced arthritis in the rat with ED50s between 1.0 and 9.5 mg/kg p.o.
Ó 2004 Elsevier Ltd. All rights reserved.
R3
Inhibition of p38a has become one of the major targets
in developing anti-inflammatory drugs, which is due to
N
N
Y
X
N
the prominent role in regulating inflammatory cytokines
like TNFa and IL-1.1 The benefits of treating rheuma-
toid arthritis patients with TNFa inhibitors (Enbrelâ
and Remicadeâ) or IL-1 inhibitors (Kineretâ) has raised
the desire to develop orally available medication.
Blockade of p38a is a very attractive option, since p38a
inhibitors are of low molecular weight, downregulate
production and signalling of TNFa, IL-1 and in addi-
tion inhibit COX-2 induction. The value of COX-2
inhibitors (Celebrexâ, Vioxxâ) has been proved by their
successful use in arthritic diseases.
N
R1
A
HO
F
1, 2, 3 (X=O, S, N)
R2
N
H
S
N
i
N
O
N
N
N
N
A:
N
N
l
j
h
k
N
N
NH
N
R1:
R3:
N
R2: 4-F, 3-CF3
Y: CH, N
HO
e
d
g
f
NH
NH
NH
We have previously disclosed2 the anti-inflammatory
properties of 4-hydroxypiperidine substituted pyrid-
inyloxazoles, -imidazoles and -thiazoles 1, 2, 3 (Scheme
1) as moderately potent p38a inhibitors. Here we wish to
report on the extension of this work directed towards a
series of p38a inhibitors with increased in vitro potency
and oral efficacy in several animal models of rheumatoid
arthritis. The novel series (Scheme 1) was assembled
around the heterocyclic template A, which represents a
diverse set of five scaffolds h–l. Alkylamine substituents
R3 (a–c) attached to the pyridinyl and pyrimidinyl rings
(Y ¼ CH, N) were chosen to reach into a lipophilic
binding pocket of p38a not utilized by ATP itself, while
a
b
c
Scheme 1.
the basic piperidinyl- and piperazinyl substituents R1
were expected to form a salt bridge with Asp168.3
4-Hydroxypiperidinyl substituted oxazoles, thiazoles
and imidazoles 22–26 were prepared according to
Scheme 2, starting from bromoketones4 4 and 5, which
were reacted at elevated temperature with formamide/
H2SO4 for oxazoles 6 and 10, formamide/P2S5 for thi-
azole 9 or ammonium formate/formamide/formic acid
providing imidazoles 7 and 11. SEM-protection of imi-
dazoles, deprotonation by nBuLi at )100 to )40 °C and
treatment with N-methyl-4-piperidone converted 6, 8, 9,
10 and 12 into the 4-hydroxypiperidinyl derivatives
* Corresponding author. Fax: +41-61-324-3273; e-mail: laszlo.
revesz@pharma.novartis.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.106

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L. Revesz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3595–3599
R3
R3
R3
Piperidinyl substituted oxazoles 32, 34 and thiazoles 36,
37 were obtained according to Scheme 3 by condensing
bromoketone4 4 or 5 with the amide or thioamide4 of N-
protected piperidine-4-carboxylic acid. Thiazole 28 was
available in 70% yield, while the yields for oxazoles 27
and 29 were lower with 11% and 40%. Fluorides 27, 28
and sulfoxide 30 heated with cyclohexylamine or S-())-
1-phenylethylamine provided oxazoles 31, 33 and thia-
zole 35. Removing the protecting groups generated the
piperidinyl-NH substituted oxazoles 32, 34 and thiazole
36. N-methylation of 36 rendered thiazole 37.
N
Y
N
Y
N
Y
Br
O
X
N
X
N
a) b)
c) d) e) f)
F
N
HO
F
F
4: R3=F; Y=CH
5: R3=SMe; Y=N
6: R3=F; X=O; Y=CH
7: R3=F; X=NH; Y=CH
13: R3=F; X=O; Y=CH
14: R3=F; X=N-SEM; Y=CH
15: R3=F; X=S; Y=CH
8: R3=F; X=N-SEM; Y=CH
9: R3=F; X=S; Y=CH
16: R3=SMe; X=O; Y=N
17: R3=SMe; X=N-SEM; Y=N
18: R3=S(O)Me; X=O; Y=N
19: R3=S(O)Me; X=N-SEM; Y=N
20: R3=c; X=N-SEM; Y=CH
21: R3=c; X=N-SEM; Y=N
22: R3=c; X=O; Y=CH
10: R3=SMe; X=O; Y=N
11: R3=SMe; X=NH; Y=N
12: R3=SMe; X=N-SEM; Y=N
23: R3=c; X=O; Y=N
1,2,4-Substituted imidazoles 44 and 45 were prepared
according to Scheme 4, starting from 2-aryl-4-bromo-
imidazoles5 38 and 39, which were regioselectively con-
verted into 4-imidazol-1-ylpyrimidines 40 and 42. The
corresponding sulfoxides were subsequently heated with
S-())-1-phenylethylamine and cyclopentylamine to
provide pyrimidines 41 and 43. Br/Li exchange and
treatment with N-methyl-4-piperidone at low tempera-
ture gave the desired 4-hydroxy-1-methylpiperidine
substituted imidazoles 44 and 45.
24: R3=c; X=S; Y=CH
25: R3=c; X=NH; Y=CH
26: R3=c; X=NH; Y=N
Scheme 2. Reagents and conditions: (a) Formamide, H2SO4 cat.,
150 °C, 15 min, 25–50% 6, 10. Ammonium formate, formamide, formic
acid, 165 °C, 20 min, 30–45% 7, 11. Formamide, P2S5, 30 min, 90 °C,
44% 9. (b) KN(TMS)2, THF/DMF (1:2), SEM-Cl, )78 °C to rt, 54–
59% 8, 12. (c) nBuLi, THF, N-methyl-4-piperidone, )100 or )78 or
)40 °C depending upon substrate; 20–60% 13–17. (d) mCPBA,
CH2Cl2/HOAc (1:2) 0 °C, 10 min, 60–70% 18, 19. (e) S-())-1-Phenyl-
ethylamine, 140–190 °C, 30 min to 4 h; 35–70% 20–24. (f) EtOH/HCl
concd (1:1) rt, 20 min, 80–90% 25, 26.
1,2,4-Substituted imidazoles 53 and 55 were prepared
according to Scheme 5, starting with the regioselective
pyrimidinylation of 2-phenyl-4-(4-piperidinyl)-imidaz-
oles5 46 and 47 to generate 48 and 50. The corre-
sponding sulfoxides 49 and 51 were heated with
cyclohexylamine and provided imidazol-1-yl pyrim-
idines 52 and 54. Removal of the ethylcarbamate pro-
13–17. The R3 group in 20–24 was introduced by
heating sulfoxides 18, 19 and fluorides 13–15 with S-())-
1-phenylethylamine. Removing the SEM-protecting
group from imidazoles 20 and 21 delivered 25 and 26.
R3
R3
R3
N
Y
N
Y
N
Y
Br
O
X
N
X
N
c) d) e) f)
a) b)
N
R4
N
R4
F
F
F
31: R3=b; R4=COOtBu; X=O; Y=CH
32: R3=b; R4=H; X=O, Y=CH
33: R3=b; R4=COOtBu; X=O; Y=N
34: R3=b; R4=H; X=O; Y=N
35: R3=c; R4=COOEt; X=S; Y=CH
36: R3=c; R4=H; X=S; Y=CH
37: R3=c; R4=Me; X=S; Y=CH
4: R3=F; Y=CH
5: R3=SMe; Y=N
27: R3=F; R4=COOtBu; X=O; Y=CH
28: R3=F; R4=COOEt; X=S; Y=CH
29: R3=SMe; R4=COOtBu; X=O; Y=N
30: R3=S(O)Me; R4=COOtBu; X=O; Y=N
Scheme 3. Reagents and conditions: (a) 4-Carbamoylpiperidine-1-carboxylic acid tert butyl ester, melt at 160 °C, 20 min. (ii) EtOH/HCl concd (1:1)
rt, 10 min. (iii) BOC2O, THF, rt, 1 h, 11% 27. 4-Thiocarbamoylpiperidine-1-carboxylic acid ethyl ester,4 DMF, 60 °C, 30 min, 70% 28. DMPU, 4-
carbamoylpiperidine-1-carboxylic acid tert butyl ester, 150 °C, 15 min, 40% 29. (b) mCPBA, CH2Cl2/HOAc (1:2) 0 °C, 10 min, 90% 30. (c)
(i) Cyclohexylamine, 200 °C, 70 min. (ii) BOC2O, THF, rt, 1 h, 79% 31. Cyclohexylamine, 110 °C, 1 h, 65% 33. S-())-1-Phenylethylamine, 195 °C, 5 h,
67% 35. (d) EtOH/HCl concd (1:1), 10 min, rt, 70–80% 32, 34. (e) CHCl3, TMSI, 4.5 h, 60 °C, 80% 36. (f) LiAlH4, THF, reflux, 30 min, 80% 37.
R3
R3
N
N
N
N
HN
a) b) c)
d)
Br
N
N
N
Br
N
N
HO
N
R2
R2
R2
38: R2=4-F
39: R2=3-CF3
40: R2=4-F; R3=SMe
41: R2=4-F; R3=c
42: R2=3-CF3; R3=SMe
43: R2=3-CF3; R3=a
44: R2=4-F; R3=c
45: R2=3-CF3; R3=a
Scheme 4. Reagents and conditions: (a) 4-Chloro-2-methylthio pyrimidine, toluene, KN(TMS)2, 24 h, 80 °C, 60–90% 40, 42. (b) mCPBA, CH2Cl2,
0 °C, 15 min, 90%. (c) S-())-1-Phenylethylamine, 5 min, 120 °C, 78% 41. Cyclopentylamine, 30 min, 60 °C, 80% 43. (d) nBuLi, THF, N-methyl-4-
piperidone, )100 °C (for 45) or )78 °C (for 44), 36–42%.

L. Revesz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3595–3599
3597
R3
HN
N
N
N
N
HN
N
N
N
R4
N
R4
N
R4
N
N
N
a) b)
c) d)
R2
R2
R2
46: R2=4-F; R4=COOEt
47: R2=3-CF3; R4=COOEt
48: R2=4-F; R3=SMe; R4=COOEt
52: R2=4-F; R4=COOEt
53: R2=4-F; R4=H
54: R2=3-CF3; R4=COOEt
55: R2=3-CF3; R4=H
49: R2=4-F; R3=S(O)Me; R4=COOEt
50: R2=3-CF3; R3=SMe; R4=COOEt
51: R2=3-CF3; R3=S(O)Me; R4=COOEt
Scheme 5. Reagents and conditions: (a) 4-Chloro-2-methylthio pyrimidine, DMF/toluene (2:1), KN(TMS)2, 8 h, 80 °C, 70–80% 48, 50. (b) mCPBA,
CH2Cl2/HOAc (1:2) 0 °C, 10 min, 90% 49, 51. (c) Cyclohexylamine, 1 h, 110 °C, 80% 52, 54. (d) TMSI, CHCl3, 3.5 h, 60 °C, 75% 53, 55.
tecting group by treating with TMSI yielded the NH-
piperidines 53 and 55.
of alkylamine side chains, removing the Boc-protecting
group and reductive amination gave the N-methylpip-
erazines 58 and 59.
Piperazine substituted imidazo[4,5-b]pyridines 58 and 59
were prepared according to Scheme 6, starting from 2-
amino-6-chloro-3-nitropyridine6 56. Chlorine in 56 was
substituted by mono-protected piperazine, the nitro
group hydrogenated, and the resulting 2,3-diamino-
pyridine condensed with 4-fluorobenzoic acid in po-
lyphosphoric acid (PPA) to provide 57. Regioselective
pyrimidinylation of 57 with 2-methylthio-4-iodopyrimi-
dine,7 oxidation of sulfide to sulfoxide and introduction
Table 1 summarizes the p38a8 and cellular IC50 values9
and the % inhibition of LPS induced TNFa release in
mice upon oral administration.10 Compared to the lead
structures 1–3, the novel compounds 22–59 in Table 1
showed a 45–150-fold increase in binding and cellular
activity, some with excellent oral efficacies. p38a affini-
ties showed little dependency of the central scaffold A,
which implies that scaffolds h–l are bioisosters, capable
R3
O
N
N
O
N
Cl
a) b) c) d) HN
N
N+
N
N
N
O
e) f) g) h) i)
N
N
N
O
H2N
N
N
F
F
57
56
58: R3=c
59: R3=a
Scheme 6. Reagents and conditions: (a) Piperazine-1-carboxylic acid ethyl ester, isopropanol reflux 2–3 h, 75%. (b) Pd/C, H2, EtOH, 85%.
(c) 4-Fluorobenzoic acid, PPA, 130–150 °C, 1–5 h, 55%. (d) BOC2O, EtOAc, Na2CO3, rt, 2 h, 70%. (e) KN(TMS)2, 2-methylthio-4-iodopyrimidine,
DMF, 2 h, 155 °C, 55%. (f) mCPBA, CH2Cl2/HOAc (1:1), 0 °C, 30 min, 65%. (g) S-())-1-Phenylethylamine or cyclopentylamine, 1 h, 120 °C, 70–80%.
(h) EtOH/HCl concd (1:1) rt, 1 h, 90%. (i) NaBH4, 40% aq formaldehyde, 2 h, rt, 80–90%.
Table 1
Compound
A
R1
R2
R3
Y
p38aa8
TNFa/hPBMCa;9
TNFa/mouse10 (%)
1
h
i
d
d
d
d
d
d
d
d
f
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
3-CF3
4-F
3-CF3
4-F
4-F
H
H
H
c
CH
CH
CH
CH
N
0.350
0.450
0.090
0.009
0.011
0.005
0.007
0.002
0.015
0.011
0.004
0.018
0.004
0.029
0.005
0.008
0.006
0.015
0.18
50
0
2
0.145
0.015
0.003
0.008
0.001
0.003
0.0001
0.0002
0.009
n.t.
3
j
77
21
19
94
42
83
90
48
63
88
94
94
29
77
83
94
22
23
24
25
26
32
34
36
37
44
45
53
55
58
59
h
h
i
c
c
CH
CH
N
j
c
j
c
h
h
i
b
b
c
CH
N
f
f
CH
CH
N
i
e
d
d
f
c
0.0006
n.t.
k
k
k
k
l
c
a
b
b
c
N
0.001
0.0008
0.002
0.001
0.001
N
f
N
g
g
N
l
a
N
a IC50 (lM).

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L. Revesz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3595–3599
Table 2
HN
HN
HN
HN
N
N
N
F
N
N
N
N
N
O
N
S
N
N
N
NH
N
N
N
N
HO
N
F
F
F
F
F
32
37
59
45
32
2.29
64
1.83
50.1
37
2.06
57
45
1.07
58
9.51
59
1.41
55
4.22
LPS/TNFa10 ED50 (mg/kg p.o.)
AIA11 % swelling (25 mg/kg b.i.d. p.o.)
CIA12 ED50 (mg/kg p.o. q.d.)
F (%)
7
20.3
12.5
6.0
23.2
3.5
72.8
4.1
Half life (h)
Log D (pH 7.4)
21.2
2.9
2.1
2.0
to position their aromatic, heteroaromatic and hetero-
cyclic substituents in a spacial relationship favourable to
interact with the p38a binding pocket. While the new
series are potent in vitro inhibitors (p38a IC50s: 0.02–
0.002 lM; hPBMC IC50s: 0.036–0.0001 lM) their in vivo
efficacies vary in a broad range between inactive to
highly potent upon oral administration. Among the four
oxazoles (A ¼ h) 22, 23, 32 and 34, only 32 showed
>80% inhibition in the acute TNFa mouse model.
Among the six imidazoles (A ¼ j, k) 25, 26, 44, 45, 53
and 55, only 26 and 45 showed excellent oral efficies in
spite of low bioavailability (F : 10% for 26; 23% for 45).
Among the three thiazoles (A ¼ i) 36, 37 and 24, only the
latter two showed >80% inhibition in the TNFa/mouse
model. Imidazo[1,2-b]pyridines (A ¼ l) 58 and 59 were
both potent upon oral administration.
the most selective analogue and exhibited a >160-fold
selectivity against a panel of 13 kinases,16 while 32, 37
and 59 had submicromolar affinities against kinases like
JNK2, EGFR or HER-1.16
In summary, our pilot library outlined in Scheme 1
comprising 18 compounds delivered four structurally
diverse and novel p38a inhibitors with impressive
enzymatic, cellular and in vivo activities in three models
of rheumatoid arthritis.
Acknowledgements
The kilolab is gratefully acknowledged for preparing
ample quantities of 32, 37, 45 and 59. We thank Prof.
W. van den Berg (Nijmwegen) for the histological
evaluation.
From eight compounds in Table 1 with >80% inhibition
in the acute LPS/TNFa model, five also showed good
efficacy in the subchronic adjuvant induced arthritis
(AIA)11 model in the rat with swelling inhibited by 55–
64% at a dose of 25 mg/kg b.i.d. p.o. Compounds
demonstrating low body weight increase in AIA, low
bioavailability, inhibition of cytochrome P450 isoen-
zymes13 or COX-1 inhibition14 or genotoxicity15 were
dropped from further profiling, leaving four structures
to be evaluated in the collagen induced arthritis (CIA)12
model in the rat: 32, 37, 45 and 59. Table 2 summarizes
the in vivo data and reveals the high potency of all four
compounds in three relevant models of rheumatoid
arthritis. LPS induced TNFa release in mice was
inhibited with ED50s between 1.07 and 2.29 mg/kg p.o.,
while the ED50s to inhibit swelling in rat CIA12 were
between 1.83 and 9.51 mg/kg p.o. Histological evalua-
tion of hind paws from CIA-rats (not shown here)
revealed a dose-dependent protective activity on bone
apposition, loss of proteoglycans, cartilage damage and
infiltration of cells, pointing to a disease modifying
potential of 32, 37, 45 and 59 in rheumatoid arthritis.
The range of half lives between 3.5 and 21.2 h, and oral
bioavailabilities (F ) between 20.3% and 72.8% offered
interesting pharmacokinetic profiles. 45 appeared to be
References and notes
1. Chakravarty, S.; Dugar, S. Annu. Rep. Med. Chem. 2002,
37, 177–186.
2. Revesz, L.; Di Padova, F. E.; Buhl, T.; Feifel, R.; Gram,
H.; Hiestand, P.; Manning, U.; Zimmerlin, A. G. Bioorg.
Med. Chem. Lett. 2000, 10, 1261–1264.
3. Wang, Z.; Canagarajah, B. J.; Boehm, J. C.; Kassisa, S.;
Cobb, M. H.; Young, P. R.; Abdel-Meguid, S.; Adams, J.
L.; Goldsmith, E. J. Structure 1998, 6, 1117.
4. Revesz, L. WO 0130778; Chem. Abstr. 2001, 134, 340507.
5. Revesz, L.; Schlapbach, A. WO 0063204; Chem. Abstr.
2000, 133, 321897.
6. Von Bebenburg, W.; Steinmetz, G.; Thiele, K. Chemiker-
Zeitung 1979, 103, 387–399.
7. Majeed, A. J.; Antonsen, O.; Benneche, T.; Undheim, K.
Tetrahedron 1989, 45, 993–1006.
8. A phosphorylated form of His-p38a MAP kinase (10 ng/
well) of murine origin was used and immobilized GST-
ATF-2 as substrate in the presence of 120 lM cold ATP.
9. Human peripheral blood mononuclear cells from healthy
volunteers were incubated with the inhibitor and stimu-

L. Revesz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3595–3599
3599
lated with LPS/IFN-c for 3 h. The supernatants were
collected for TNF-a determination by ELISA.
was nearly maximal. Compounds were dosed once (q.d.)
daily for 10 days.
10. Eight-week old female OF1 mice were dosed perorally by
gavage with solutions of the compounds (10 mg/kg) in
DMSO/cornoil. One hour after dosing, LPS (20 mg/kg)
was injected iv for stimulation of TNF-a release into
plasma. One hour later blood was collected and TNF-a
was determined using a mouse specific ELISA.
11. AIA: adjuvant induced arthritis. Female Wistar rats were
immunized with Mycobacterium tuberculosis at day 0 and
dosed with the compounds (2 ꢀ 25 mg/kg p.o. per day)
from day 14 to 20. Swelling of the joints was measured on
day 20.
12. CIA: Collagen induced arthritis. Female (WAGxBUF/F1)
rats were immunized intradermally with bovine nasal
septum type II collagen emulsified in Freund’s incomplete
adjuvant. Swelling started ꢁ10 days after immunization.
Dosing of compounds started on day 13, when swelling
13. Profiling continued, if IC50 > 2 lM for human P450
isoenzymes CYP1A2, CYP2C9, CYP2D6, CYP3A4.
14. Profiling continued, if IC50 > 80 lM for COX-1.
15. Profiling continued, if Ames assay and the Comet assay
(in vitro with human lymphocytes) were negative.
16. Selectivity profiles were determined in house as described.17
Kinase (IC50 (lM) for 32/37/45/59): hJNK1 (0.15/1.9/>10/
1.5); hJNK2 (0.03/0.261/4.66/0.047); CDK1 (>10/>10/>10/
>10); HER-1 (1.1/0.28/>10/>10); c-Abl (>10/>10/>10/
>10); c-Src (>10/8.0/>10/>10); Kdr (1.4/1.7/>10/>10);
c-Met (>10/>10/>10/>10); c-Kit (>10/>10/>10/>10);
IGF1R (>10/>10/>10/>10); HER-2 (2.0/1.1/>10/>10);
c-Raf (8.7/2.1/>10/>10); hEGFR (1.1/0.7/>10/8.7).
17. Revesz, L.; Di Padova, F. E.; Buhl, T.; Feifel, R.; Gram,
H.; Hiestand, P.; Manning, U.; Wolf, R.; Zimmerlin, A.
G. Bioorg. Med. Chem. Lett. 2002, 12, 2109–2112.