SAR of 2,6-diamino-3,5-difluoropyridinyl substituted heterocycles as novel p38MAP kinase inhibitors.

Article abstract of DOI:10.1016/S0960-894X(02)00336-0

2,6-Diamino-3,5-difluoropyridinyl substituted pyridinylimidazoles, -pyrroles, -oxazoles, -thiazoles and -triazoles have been identified as novel p38alpha inhibitors. Pyridinylimidazole 11 potently inhibited LPS-induced TNFalpha in mice, showed good efficacy in the established rat adjuvant (ED(50): 10 mg/kg po b.i.d.) and collagen induced arthritis (ED(50): 5 mg/kg po b.i.d.) with disease modifying properties based on histological analysis of the joints.

Full text of DOI:10.1016/S0960-894X(02)00336-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2109–2112
SAR of 2,6-Diamino-3,5-difluoropyridinyl Substituted
Heterocycles as Novel p38MAP Kinase Inhibitors
Laszlo Revesz,a,* Franco E. Di Padova,a Thomas Buhl,a Roland Feifel,a
Hermann Gram,a Peter Hiestand,a Ute Manning,a Romain Wolf a
and Alfred G. Zimmerlinb
aArthritis and Bone Research, Novartis Pharma AG, CH-4002 Basel, Switzerland
bPreclinical Safety, Novartis Pharma AG, CH-4002 Basel, Switzerland
Received 5 March 2002; revised 10 May 2002; accepted 13 May 2002
Abstract—2,6-Diamino-3,5-difluoropyridinyl substituted pyridinylimidazoles, -pyrroles, -oxazoles, -thiazoles and -triazoles have
been identified as novel p38a inhibitors. Pyridinylimidazole 11 potently inhibited LPS-induced TNFa in mice, showed good efficacy
in the established rat adjuvant (ED50: 10 mg/kg po b.i.d.) and collagen induced arthritis (ED50: 5 mg/kg po b.i.d.) with disease
modifying properties based on histological analysis of the joints. # 2002 Published by Elsevier Science Ltd.
Introduction
Inhibitors of p38 MAP kinase reduce the production of
pro-inflammatory cytokines, e.g., IL-1, TNFa, IFNg
and IL-6, whose excessive production initiates events
leading to inflammation and tissue destruction in dis-
eases such as rheumatoid arthritis (RA).1 p38 Inhibitors
not only block the synthesis but also the signal cascades
induced by these cytokines;2 in addition, p38 has been
Chemistry
implicated in the induction of COX-2, the inducible
prostaglandin cyclooxygenase.3 The interest in the
development of p38 MAP kinase inhibitors is based on
the expectations that p38 inhibiting drugs will treat the
underlying cause of chronic inflammatory diseases and
stop their progression. Previously, we described a series
of 4-hydroxypiperidine substituted heterocycles 14 as
p38 inhibitors with useful in vivo antiarthritic activities.
In search of p38 inhibitors suitable for clinical develop-
ment we now report new 2,6-diamino-3,5-difluoropyr-
idinyl substituted imidazoles, oxazoles, thiazoles,
triazoles and pyrroles 2 and their in vivo efficacies in
models of inflammation and rheumatoid arthritis.
Target compounds with 4-fluorophenyl as aryl sub-
stituent in 2 were prepared from imidazole 4,4 oxazole
5,4 thiazole 64 and bromopyrrole 164 (Scheme 1) involv-
ing deprotonation or bromine–lithium exchange with
nBuLi at À78 ꢀC and reacting the anions with penta-
fluoropyridine to yield the tetrafluoropyridines 7–9 and
17. The latter were converted with 25% aqueous NH3 at
150–170 ꢀC to the 2,6-diamino-3,5-difluoropyridines 11,
13, 14, 18 and the 2-amino-3,5,6-trifluoropyridines 10
and 12.
Triazole 21—obtained via [3+2] cycloaddition reaction
of Me3SiN3 with 4-pyridinylacetylene 206—was depro-
5
tonated with KN(TMS)2 and reacted with penta-
fluropyridine to the tetrafluoro intermediate 22, which
was transformed into 2,6-diamino-3,5-difluoropyridine
23 (Scheme 2).
A general method for the preparation of a diverse set of
aryl substituted pyridinylimidazoles was developed,
*Corresponding author. Tel.: +41-61-324-3231; fax: +41-61-324-
3273; e-mail: laszlo.revesz@pharma.novartis.com
0960-894X/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(02)00336-0

2110
L. Revesz et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2109–2112
allowing the introduction of aryl substituents at a late
stage of the synthesis (Scheme 3). SEM-protected imi-
dazole 247 was treated with nBuLi and the resulting
anion arylated with pentafluoropyridine followed by
bromination to 25. 4-Trimethylstannylpyridine8 and 25
were coupled regioselectively in a Stille reaction, yield-
ing pyridinylimidazole 26, where the remaining bromine
atom served to attach an aryl-substituent either by a
Suzuki9 or a Stille10 coupling reaction. Removing the
SEM-protecting group under acidic conditions and
heating in 25% aq NH3 at 170 ꢀC for 12 h delivered the
target compounds 27–29 in moderate yields.
the 4-fluorophenyl ring extending into a lipophilic
pocket near Thr106, (c) the salt bridge between the
COOH-group of Asp168 and the basic piperidinyl ring
of 1 and the 2,6-diamino-3,5-difluoropyridinyl ring of 2.
Without the possibility to form a salt bridge, unsub-
stituted imidazole 3, thiazole 6, pyrrole 15 and triazole 21
were 6–200-fold less potent than their 2,6-diamino-3,5-
difluoropyridinyl analogues 11, 14, 19 and 23 (Table 1).
Moreover, the stepwise transformation of the 2,3,5,6-
tetrafluoropyridinyl
into
the
2-amino-3,5,6-tri-
fluoropyridinyl and further to the 2,6-diamino-3,5-
difluoropyridinyl ring was accompanied by a gradual
increase in potency in parallel with the ability to form a
salt bridge. Most prominent example were the oxazoles
8 (IC50=100 mM), 12 (2.1 mM) and 13 (0.4 mM). Inter-
estingly, within the homogenous series of imidazole 11,
oxazole 13, thiazole 14, pyrrole 19 and triazole 23—all
with identical substituents on ring A—the IC50s differed
up to 100-fold with the ranking order of potency: pyrrole
19 (IC50=4 nM) >imidazole 11 (IC50=16 nM) > thia-
zole 14 (IC50=140 nM) > triazole 23 (IC50=210 nM)
> oxazole 13 (IC50=400 nM). Assuming, that ring A
may affect the hydrogen bridge formation to Met109 or
the salt bridge formation to Asp168 or both, salt bridge
formation may decrease by increasing out-of-plane
rotation of the 2,6-diamino-3,5-difluoropyridinyl ring,
going from pyrrole 19 to oxazole 13. While the 2,6-dia-
mino-3,5-difluoropyridinyl ring is assumed to prefer a
coplanar conformation with the pyrrole-ring due to F–
HN bonding, out-of-plane rotation may gradually
increase from thiazole 14, triazole 23 to the oxazole 13,
thereby rendering salt bridge formation more difficult.
An additional effect of ring A on the pK of the 4-pyr-
idinyl nitrogen can not be excluded. In contrast to ear-
lier findings,13 where the 4-fluorophenyl group was
shown to be optimal in size for the lipophilic binding
pocket near Thr106, the 2-benzofuryl analogue 28
(IC50=5 nM) demonstrates, that larger groups may also
be accommodated successfully.
Results and Discussion
Molecular modeling suggested,11,12 that three interac-
tions were the major contributors to the high affinity
binding of 1 and 2 to p38a: (a) the hydrogen bridge of
the 4-pyridinyl nitrogen to the NH amide of Met109, (b)
Scheme 1. (a) 4, nBuLi, À78 ꢀC, THF, pentafluoropyridine, 75% 7, 5,
6 and 16: nBuLi, À40 ꢀC, THF, pentafluoropyridine, 65% 8, 54% 9,
and 78% 17; (b) amination of 7: autoclave, 25% aq NH3, 150 ꢀC, 4 h,
44% 10 and 33% 11. Amination of 8: 25% aq NH3, 170 ꢀC, autoclave,
5 h, 18% 12 and 21% 13. Amination of 9: 25% aq NH3, 170 ꢀC,
autoclave, 17 h, 34% 14. Amination of 17: 25% aq NH3, 170 ꢀC,
autoclave, 17 h, 61% 18; (c) Bu4NF, THF, 2.5 h, 60 ꢀC, 75% 19.
A selection of compounds in Table 1 with potent inhi-
bition of p38a and TNFa release from LPS-stimulated
Scheme 3. (a) nBuLi, THF, À40 ꢀC, 10 min, À60 ꢀC, pentafluoro-
pyridine, 74%; (b) Br2, HOAc, NaOAc, 15 min, room temperature,
86%; (c) 4-trimethylstannylpyridine, PdCl2(PPh3)2, toluene, reflux
12 h, 46%; (d) 2-(tri-n-butylstannyl)furan, PdCl2(PPh3)2, xylene, reflux
3 h, 88%. Benzo[b]furan-2-boronic acid, PdCl2(PPh3)2, 1,2-dimethoxy-
ethane, Na2CO3 aq satd, 100 ꢀC, 1.25 h, 78%. 3-(Trifluoro-
methyl)phenylboronic acid, 1,2-dimethoxyethane, Na2CO3 aq satd,
100 ꢀC, 1.25 h, 92%; (e) EtOH/HCl concd 1:1, room temperature, 1 h,
78–85%; (f) 25% aq NH3, 170 ꢀC, autoclave, 12 h, 30–36%.
Scheme 2. (a) Me3SiN3, autoclave, 130 ꢀC, 48 h, 81%; (b) KN(TMS)2,
pentafluoropyridine, THF, 0 ꢀC to room temp, 20 min, 49%; (c) 25%
aq NH3, 170 ꢀC, autoclave, 17 h, 24%.

L. Revesz et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2109–2112
2111
Table 1.
Aryl
A
R1/R2
p38aa,14
TNFab,15
COX-1a,16
c
c
3
6
7
8
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
2-Furyl
Imidazole
Thiazole
Imidazole
Oxazole
Imidazole
Imidazole
Oxazole
Oxazole
Thiazole
Pyrrole
1.00
0.80
0.09
100
0.004
0.016
2.1
n.t.
n.t.
0.074
n.t.
0.044
0.044d
1.93
0.373
0.031
n.t.
n.t.
n.t.
>100
n.t.
100
>100
100
100
100
n.t.
6.3
n.t.
10
45
100
100
100
F/F
F/F
F/NH2
NH2/NH2
F/NH2
NH2/NH2
10
11
12
13
14
15
19
21
22
23
27
28
29
0.4
NH2/NH2
c
0.14
0.80
0.004
1.30
10
0.21
0.67
0.005
0.47
Pyrrole
NH2/NH2
c
0.018
n.t.
n.t.
Triazole
Triazole
Triazole
Imidazole
Imidazole
Imidazole
F/F
NH2/NH2
NH2/NH2
NH2/NH2
NH2/NH2
n.t.
0.294
0.027
0.054
2-Benzofuryl
3-Trifluoromethylphenyl
aIC50 (mM).
bIC50 (mM) of TNFa release from LPS-stimulated human peripheral blood mononuclear cells.
cNot applicable.
dIC50=0.25 mMfor TNF a release inhibition from LPS-stimulated human whole blood.
Table 2.
Histological evaluation of hind paws from CIA-rats
revealed a dose-dependent protective activity of 11 (at 3,
10, 30 mg/kg po b.i.d.) on bone apposition (À4, À44,
À64%), loss of proteoglycans (À17, À38, À42%), car-
tilage damage (À15, À44, À58%) and infiltration of
cells (À11, À37, À45%), pointing to a disease modifying
potential of 11 in rheumatoid arthritis. The compound
also showed an acceptable kinase selectivity profile,20
and satisfactory pharmacokinetic properties in the rat
(bioavailability: 49%; t1/2: 1 h; Cmax: 3.12 mM@ 10 mg/
kg po). Pyridinylimidazole inhibitors of p38a often risk
to inhibit CYP450 isoenzymes by coordinating their 4-
pyridinyl nitrogen to the heme iron. Increased liver
weight and significant elevations of hepatic P450 iso-
enzymes by SB203580—a representative of this struc-
ture class—were stated to be related to P450-
inhibition.23 Pyridinylimidazole 11 was devoid of
human P450 inhibition. [Isoenzyme: IC50 (mM);
CYP1A2: >100; CYP2C9: 65.5; CYP2D6: >100;
CYP3A4: >100].24
TNFaa
AIAb
CIA; ED50 (po)c
7
10
11
3
64
83
n.t.
71 (toxic)
ED50:10 mg/kg bid
n.t.
n.t.
5 mg/kg b.i.d.
43 mg/kg q.d.
n.t.
13
27
28
29
81
28
1.8
21
19
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
a% Inhibition of LPS induced TNFa release in mice17 at 10 mg/kg po.
b% Inhibition of swelling in adjuvant induced arthritis rats18 (AIA) at
25 mg/kg po b.i.d.
cCIA, collagen induced arthritis in rats.19 Inhibition of paw-swelling.
human peripheral blood mononuclear cells were tested
in vivo for their antiarthritic profile. Compounds with
IC50 <10 mMagainst COX-1 were excluded due to
unacceptable ulceration risks.
10, 11 and 13 (Table 2) were found to have antiarthritic
potential; they inhibited TNFa release by 60–80% at a
dose of 10 mg/kg po in the mouse17 and were further
tested in the established adjuvant induced arthritis
model in the rat (AIA)18 at a dose of 25 mg/kg po b.i.d.
Imidazole 10 inhibited paw-swelling by 71%, but was
not well tolerated. Oxazole 13 gave only a modest inhi-
bition of 19%, which was insufficient for further inves-
tigations. Imidazole 11 was the most potent analogue in
vivo with inhibition of LPS-induced TNFa in mice
(Table 2) and rats (ED50=4 mg/kg po).
In conclusion, a series of 2,6-diamino-3,5-difluoropyr-
idinyl substituted pyridinylimidazoles, -pyrroles, -oxa-
zoles, -thiazoles and -triazoles has been identified as
novel p38a inhibitors. Imidazole 11 from this series
showed potent disease modifying antiarthritic properties
in AIA and CIA models.
Acknowledgements
We would like to thank E. Blum and G. Rucklin for
excellent technical assistance, W. Prikoszovich, F. Struber
and B. Wietfeld for the large scale preparation of 11 and
Prof. W. van den Berg (Nijmwegen) for the histological
evaluation.
11 inhibited paw swelling in established models of
arthritis with ED50s of 10 mg/kg b.i.d. po (AIA18) and
5 mg/kg b.i.d. po (CIA19).

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L. Revesz et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2109–2112
16. COX-1 from ram seminal vesicles (No. 60100; Cayman
References and Notes
Chemical Company) was used and PGE2 determined by RIA.
17. 8-week old female OF1 mice were dosed perorally by
gavage with solutions of the compounds in DMSO/cornoil. 1 h
after dosing, LPS (20 mg/kg) was injected iv for stimulation of
TNF-a release into plasma. 1 h later, blood was collected and
TNFa was determined using a mouse specific ELISA.
18. AIA: Adjuvant induced arthritis. Female Wistar rats were
immunised with Mycobacterium tuberculosis at day 0 and
dosed with the compounds (2Â25 mg/kg po per day) from day
14 to day 20. Swelling of the joints was measured on day 20.
19. CIA: Collagen induced arthritis. Female (WAGxBUF/F1)
rats were immunised intradermally with bovine nasal septum
type II collagen emulsified in Freund’s incomplete adjuvant.
Swelling started ꢁ10 days after immunisation. Dosing of
compounds started on day 13, when swelling was nearly max-
imal. 11 was dosed once (q.d.) or twice (b.i.d.) daily for 10
days.
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