Pyrazoloheteroaryls: Novel p38α MAP kinase inhibiting scaffolds with oral activity

Article abstract of DOI:10.1016/j.bmcl.2005.10.015

A test library with three novel p38α inhibitory scaffolds and a narrow set of substituents was prepared. Appropriate combination of substituent and scaffold generated potent p38α inhibitors, for example, pyrazolo[3,4-b]pyridine 9, pyrazolo[3,4-d]pyrimidin

Full text of DOI:10.1016/j.bmcl.2005.10.015

Bioorganic & Medicinal Chemistry Letters 16 (2006) 262–266
Pyrazoloheteroaryls: Novel p38a MAP kinase inhibiting
scaffolds with oral activity
Laszlo Revesz,* Ernst Blum, Franco E. Di Padova, Thomas Buhl, Roland Feifel,
Hermann Gram, Peter Hiestand, Ute Manning, Ulf Neumann and Gerard Rucklin
Novartis Institutes for BioMedical Research, Global Discovery Chemistry, CH-4002 Basel, Switzerland
Received 13 July 2005; revised 30 September 2005; accepted 5 October 2005
Available online 24 October 2005
Abstract—A test library with three novel p38a inhibitory scaffolds and a narrow set of substituents was prepared. Appropriate com-
bination of substituent and scaffold generated potent p38a inhibitors, for example, pyrazolo[3,4-b]pyridine 9, pyrazolo[3,4-d]pyrim-
idine 18a and pyrazolo[3,4-b]pyrazine 23b with potent in vivo activity upon oral administration in animal models of rheumatoid
arthritis.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Inhibition of p38a has become one of the major targets
in developing anti-inflammatory drugs, which is due to
R
Cl
O
Cl
its prominent role in regulating inflammatory cytokines
such as TNFa and IL-1.1 The benefits of treating rheu-
matoid arthritis patients with TNFa inhibitors (Enbrelꢁ
and Remicadeꢁ) or IL-1 inhibitors (Kineretꢁ) have
raised the desire to develop oral treatments. Blockade
of p38a is a very attractive option: p38a inhibitors
downregulate production and signalling of TNFa,
IL-1 and in addition inhibit COX-2 induction. The
value of COX-2 inhibitors (Celebrexꢁ, Vioxxꢁ) has
been proved by their use in arthritic diseases.
F
F
Y
N
F
O
N
X
N
H N
N
N
N
N
N
N
N
2
H
H
H
H
F
F
F
3 (X,Y=C)
2
1
4 (X=N, Y=C)
5 (X=C, Y=N)
zines 5, three novel p38a inhibiting scaffolds. A narrow
set of substituents R was chosen, aiming at potential
interactions with Asp164.
Since the discovery of pyridinylimidazoles,2 several
novel structural classes of p38 inhibitors have been
reported.3 Our search for p38 inhibitors unrelated to
pyridinylimidazoles resulted in pyrido[2,3-d]pyrimidine
1, a modest p38a inhibitor (IC50 = 5.8 lM), while its
ring opened analogue 2 was 10 times more potent.
The synthesis of pyrazolo[3,4-b]pyridines 9–14 is de-
scribed in Schemes 1 and 2. 2,6-Dichloro-3-lithio-
pyridine (Scheme 1) reacted with 5-bromo-2-
methoxybenzaldehyde to provide the alcohol, which
upon Jones5 oxidation gave ketone 6. The latter was
treated with hydrazine and furnished pyrazolo[3,4-
b]pyridine 7. Sonogashira6 coupling conditions were ap-
plied to attach the 1,1-dimethylpropynylamine side
chain in 8, followed by a Buchwald7 reaction to substi-
tute the Cl-atom by 2,4-difluoroaniline in 9. Hydrogena-
tion yielded the saturated analogue 10.
Further modifications gave rise to benzoylpyridines,
benzophenones4 and the pyrazoloheteroaryls 3–5, some
with subnanomolar IC50s. Here, we wish to report on
the in vitro and in vivo SAR of pyrazolo[3,4-b]pyridines
3, pyrazolo[3,4-d]pyrimidines 4 and pyrazolo[3,4-b]pyra-
Synthesis of pyrazolo[3,4-b]pyridines 13a–c and 14a,b
(Scheme 1) started by SEM protection of 7, followed
by Br–Li exchange and CO2 treatment to render acid
11. 2,4-Difluoroaniline was introduced under Buchwald7
conditions to provide 12a, which was first coupled
with 4-aminotetrahydropyran, cyclopentylamine and
Keywords: p38 inhibition; MAP kinase inhibition; Rheumatoid arthri-
tis; Antiarthritic; Anti-inflammatory; Collagen-induced arthritis;
Adjuvant-induced arthritis.
*
Corresponding author. Tel.: +41 61 324 3231; fax: +41 61 324
3273; e-mail: laszlo.revesz@novartis.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.015

L. Revesz et al. / Bioorg. Med. Chem. Lett. 16 (2006) 262–266
263
NH2
Br
Br
Br
a) b)
d)
c)
O
O
O
O
O
O
N
N
Cl
N
Cl
N
N
Cl
N
N
Cl
H
H
7
6
8
NH2
NH2
e)
f)
O
O
F
F
N
N
N
N
N
N
N
N
H
H
H
H
F
F
9
10
Scheme 1. Reagents and conditions: (a) 2,6-Dichloropyridine, LDA, ꢀ78 ꢂC, 80%. (b) CrO3, H2SO4, acetone, water, 79%. (c) H2NNH2 Æ H2O,
EtOH/1-BuOH (1:1) reflux, 1 h, 70%. (d) PdCl2(PPh3)2, CuI, Cs2CO3, 1,1-dimethylpropynylamine, reflux in DIPEA, 39%. (e) Pd(OAc)2, Cs2CO3,
R-(+)-BINAP, 2,4-difluoroaniline, 1,4-dioxane, reflux 2 h, 22%. (f) EtOH, Pd/C, H2, 30 min, 85%.
O
O
O
Br
N
OH
R
OH
O
O
O
O
F
F
e) f)
N
a) b)
N
N
c)
N
N
H
Cl
N
R
N
N
N
N
N
H
N
N
Cl
H
H
F
F
SEM
12a (R=SEM)
12b (R=H)
13a-c
11
7
d)
R
a
c
O
g)
b
F
O
N
R
N
N
H
N
NH
N
N
N
H
H
H
F
14a,b
Scheme 2. Reagents and conditions: (a) KN(TMS)2, SEM-Cl, THF, ꢀ78 ꢂC, 72% (2 regioisomers). (b) n-BuLi, ꢀ78 ꢂC, THF, CO2, 63%.
(c) Pd(OAc)2, PPh3, NaOtBu, R-(+)-BINAP, 2,4-difluoroaniline, 1,4-dioxane, reflux 15 min, 96%. (d) EtOH, HCl concn 1:1, 2 min, 60 ꢂC, 50%.
(e) 1,10-Carbonyldiimidazole, amine, THF, reflux 10 min, 60–80%. (f) 13a–c: EtOH, HCl concn 1:1, 15 min, 60–70%. (g) DIBAL-H, THF, reflux,
10 min, 70%.
1-Boc-piperazine and then deprotected to deliver amides
13a-c. DIBAL-H reduction yielded amines 14a and 14b.
Jones5 oxidation of the resulting alcohol delivered ke-
tone 20, which upon treatment with hydrazine furnished
pyrazolo[3,4-b]pyrazine 21. The remaining Cl-atom in
21 was substituted by 2,4-difluoroaniline by heating to
175 ꢂC, giving rise to 22; Sonogashira6 coupling intro-
duced the propargyl side chain in 23a,b. Hydrogenation
of the triple bond gave 24a,b.
Pyrazolo[3,4-d]pyrimidines 18 and 19 (Scheme 3) were
prepared by reacting 5-lithio-2,4-dichloropyrimidine
with 5-bromo-2-methoxybenzaldehyde and oxidising
the resulting alcohol to ketone 15. Hydrazine at low
temperature converted 15 into pyrazolo[3,4-d]pyrimi-
dine 16, which—combined for 15 min with 2,4-difluoro-
aniline at 150 ꢂC—gave 17 in high yield. Sonogashira6
coupling of 17 with a series of alkynes afforded 18a–d
in moderate yields. Hydrogenation gave rise to the
saturated analogues 19a–d.
The 1,1-dimethylpropynylamino group common to pyr-
azolo[3,4-b]pyridine 9, pyrazolo[3,4-d]pyrimidine 18a
and pyrazolo[3,4-b]pyrazine 23a (IC50s = 20, 12 and
10 nM) permitted us to compare the p38a inhibiting effi-
cacies of the three pyrazoloheteroaryl scaffolds (Table
1). The inhibitory potencies of the three scaffolds proved
to be very similar, with the tendency of the pyrazolo[3,4-
b]pyrazine analogues to be slightly superior to those of
pyrazolopyrimidines and pyrazolopyridines. An interac-
Pyrazolo[3,4-b]pyrazines 23a,b and 24a,b were prepared
(Scheme 4) by combining 2,6-dichloro-3-lithiopyrazine
at ꢀ90 ꢂC with 5-bromo-2-methoxybenzaldehyde.

264
L. Revesz et al. / Bioorg. Med. Chem. Lett. 16 (2006) 262–266
Br
N
Br
Br
Br
O
O
O
a) b)
c)
d)
O
F
N
N
N
O
N
N
O
N
N
H
Cl
N
Cl
N
N
Cl
H
H
F
16
17
15
R
R
R
NH2
OH
O
O
e)
f)
F
a
F
b
N
N
N
N
N
N
N
N
N
N
H
H
H
H
F
F
c
N
19a-d
18a-d
N
d
N
Scheme 3. Reagents and conditions: (a) 2,4-Dichloropyrimidine, LDA, ꢀ78 ꢂC, 34%. (b) CrO3, H2SO4, acetone, water, 88%. (c) H2NNH2 Æ H2O (2
equiv), MeOH, ꢀ50 to 0 ꢂC, 67%. (d) 2,4-Difluoroaniline (solvent), 15 min, 150 ꢂC, 90%. (e) 18a: PdCl2(PPh3)2, CuI, Cs2CO3, 1,1-
dimethylpropynylamine (10 equiv), DIPEA, autoclave, 130 ꢂC, 1 h, 30%. 18b: PdCl2(PPh3)2, CuI, NEt3, DMF, 1,1-dimethylpropynol (4 equiv),
1 h, 100 ꢂC, 52%. 18c: Pd(OAc)2, R-(+)-BINAP, CuI, PPh3, NaOtBu, diglyme, N,N-dimethylamino-2-propyne, autoclave, 1 h, 135 ꢂC, 65%. 18d:
Pd(OAc)2, R-(+)-BINAP, CuI, PPh3, NaOtBu, 1-methyl-4-prop-2-ynylpiperazine, diglyme, 40 min, 145 ꢂC, 75%. (f) EtOH, Pd/C, H2, 1 h, 55–80%.
Br
Br
Br
Br
O
O
O
a) b)
c)
d)
O
N
N
F
N
N
N
N
N
O
N
O
N
N
Cl
Cl
N
Cl
H
H
H
F
20
21
22
R
R
O
O
e)
f)
N
F
N
N
F
N
N
N
N
N
N
N
H
H
H
H
F
F
23a (R=NH2)
23b (R=OH)
24a (R=NH2)
24b (R=OH)
Scheme 4. Reagents and conditions: (a) LiTMP, ꢀ90 ꢂC, 2,6-dichloropyrazine, 38%. (b) CrO3, H2SO4, acetone, water, 88%. (c) H2NNH2 Æ H2O (2
equiv), MeOH, 30 min, rt, 36%. (d) 2,4-Difluoroaniline, 1 h, 175 ꢂC, 91%. (e) 23a: Pd(OAc)2, R-(+)-BINAP, CuI, NaOtBu, 1,1-dimethylpropy-
nylamine, 1 h, 140 ꢂC, 35%. 23b: PdCl2(PPh3)2, CuI, NEt3, 1,1-dimethylpropynol, 1.5 h, 85 ꢂC, 50%. (f) EtOH, Pd/C, H2, 1 h, 70%.
tion of the 1,1-dimethylpropynylamino group of 9, 18a
and 23a with Asp164 seemed unlikely, since 1,1-dimeth-
yl propynols 18b and 23b had similar potencies against
p38a (IC50s = 19, 7 nM). The saturated 1,1-dimethyl-
propylamines 10, 19a and 24a (IC50s = 33, 43 and
29 nM) and 1,1-dimethylpropanols 19b, 24b (IC50s =
47 and 21 nM) were weaker than their propynyl precur-
sors 9, 18a,b and 23a,b, underlying the preference of rig-
id substituents for high affinity. Acid 12b was not
favoured, nor its piperazine amide 13c. However, pri-
mary amides 13a and 13b were potent p38a inhibitors
(IC50s = 0.7 and 5 nM). Compared to the unsubstituted
pyrazolo[3,4-b]pyridine (R = H; not shown in Table 1),
13a was 100-fold more potent, revealing the important
contribution of R to p38a inhibition. The reduced ana-
logues 14a and 14b were considerably weaker than 13a
(IC50s = 94 and 58 nM), demonstrating again the nega-
tive influence of increasing conformational freedom in
the side chain on p38a inhibition. With the N,N-dimeth-
ylamino-2-propyne side chain in 18c and its piperazine
analogue 18d, two further substituents with subnano-
molar potency were identified (IC50s = 0.6 and
0.9 nM). One may speculate that their basic amines
interact favourably with the acidic Asp168 of p38a.8
As expected, the saturated analogues 19c and 19d
showed higher IC50s (11 and 14 nM). The 7-fold differ-
ence between 13a and 13b may de due to a unique
water-mediated hydrogen bond interaction of the pyran
ring oxygen with Asp168.
With the exception of acid 12b, all compounds under-
went an in vivo screen for oral efficacy in the acute

L. Revesz et al. / Bioorg. Med. Chem. Lett. 16 (2006) 262–266
Table 1. Inhibition of p38a and LPS-induced TNFa release in mice
265
activities with >45% inhibition of paw swelling. In spite
of their high potency against p38a, both amides 13a
and 13b were weak in AIA, possibly due to their short
half-lives (1.4 and 1.6 h), low plasma concentrations
(Cmax = 6 nM at 1 mg/kg po) and modest bioavailabili-
ties (BAV = 22% and 26%). In contrast to its 1,1-di-
Compound
p38aa
TNFab
Pyrazolo[3,4-b]pyridine
9
10
20
33
98
78
n.t.
97
97
3
12b
13a
13b
13c
14a
14b
200
0.7
5
methylpropynylamine
analogue
9
(BAV = 95%;
Cmax = 52 nM), the saturated 1,1-dimethylpropylamine
10 showed low BAV (20%), low plasma levels
(Cmax = 11 nM) and was inactive in AIA. In the pyraz-
olo[3,4-d]pyrimidine series, the 1,1-dimethylpropynyl-
amine substituted 18a showed the highest efficacy in
AIA (69% inhibition of swelling). The closely related
1,1-dimethylpropynol substituted pyrazolo[3,4-d]pyrim-
idine 18b and its saturated analogue 19b were weaker.
In the pyrazolo[3,4-b]pyrazine series, the 1,1-dimethyl-
propynylamine substituted 23a and its alcohol analogue
23b were nearly equipotent inhibitors of AIA, in spite
of the low oral BAV (9%) and short half-life (2.6 h)
of 23a.
280
94
80
38
58
Pyrazolo[3,4-d]pyrimidine
18a
18b
18c
18d
19a
19b
19c
19d
12
19
0.6
0.9
43
47
11
14
85
98
96
96
68
98
93
73
Pyrazolo[3,4-b]pyrazine
Compound 23b demonstrated an improved BAV (22%)
and higher plasma levels (Cmax: 160 nM at 1 mg/kg po).
From the SAR above one may conclude that the side
chains determine the degree of p38a inhibition, but that
the in vivo potency with a given substituent is scaffold-
dependent. For example, the three pyrazoloheteroaryls
9, 18a and 23a—sharing the same 1,1-dimethylpropynyl-
amine substituent—had similar potencies on p38a, but
largely differed in oral BAV (95%, 16% and 9%). The po-
tent in vivo effects of 18a appear to be in contrast to its
modest BAV, but can be explained by its high AUC.12
In order to confirm AIA results in a different disease
model, 9, 14a, 18a and 23b were tested in the collagen-
induced arthritis (CIA)13 model in rats. Compounds 9,
18a and 23b showed potent inhibition of swelling in
CIA (Table 2). Compounds 9 and 23b were >1000-fold,
18a >100-fold selective against a panel of 13 kinases.14
No adverse inhibition of cytochrome P450 isoenzymes
was observed.
23a
23b
24a
24b
10
7
98
98
0
29
21
94
n.t., not tested.
a IC50 (nM).10
b % inhibition of LPS-induced TNFa release in mice at 20 mg/kg po.9
LPS-induced TNFa release in the mouse9 at a standard
dose of 20 mg/kg po (Table 1). Most compounds potent-
ly inhibited the release of TNFa.
Selected compounds with TNFa inhibition >80% were
further tested in the subchronic adjuvant-induced
arthritis (AIA)11 model in the rat at 25 mg/kg po
b.i.d. (Table 2). In the pyrazolo[3,4-b]pyridine series,
the 1,1-dimethylpropynylamine and the 4-aminopyra-
nylmethyl analogues 9 and 14a showed appreciable
Work is in progress to characterise, synthesize and
optimise further analogues of the three novel p38a
inhibitory scaffolds.
Table 2. Effects on adjuvant- and collagen-induced arthritis in rats
Compound
AIAa
CIAb
Pyrazolo[3,4-b]pyridine
Acknowledgment
9
10
49
9
85
n.t.
n.t.
n.t.
10
The kinase screening data by the Novartis oncology
group are gratefully acknowledged.
13a
13b
14a
28
34
46
Pyrazolo[3,4-d]pyrimidine
References and notes
18a
18b
19b
69
34
36
75
n.t.
n.t.
1. Chakravarty, S.; Dugar, S. Annu. Rep. Med. Chem. 2002,
37, 177.
2. Jackson, P. F.; Bullington, J. L. Curr. Top. Med. Chem.
2002, 2, 1011.
Pyrazolo[3,4-b]pyrazine
23a
23b
37
41
n.t.
57
3. Cirillo, P. F.; Pargellis, C.; Regan, J. Curr. Top. Med.
Chem. 2002, 2, 1021; Dominguez, C.; Powers, D. A.;
Tamayo, N. Curr. Opin. Drug Disc. Dev. 2005, 8, 421.
4. Revesz, L.; Blum, E.; Di Padova, F. E.; Buhl, T.; Feifel,
R.; Gram, H.; Hiestand, P.; Manning, U.; Rucklin, G.
Bioorg. Med. Chem. Lett. 2004, 14, 3601.
a AIA11: adjuvant-induced arthritis in rats at 25 mg/kg po b.i.d.;
% inhibition of swelling.
b CIA13
% inhibition of swelling.
: collagen-induced arthritis in rats at 10 mg/kg po q.d.;

266
L. Revesz et al. / Bioorg. Med. Chem. Lett. 16 (2006) 262–266
5. Bowden, K.; Heilbron, I. M.; Jones, E. R. H.; Weedon, B.
C. L. J. Chem. Soc. 1946, 39.
6. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 15, 4467.
7. Muci, A. R.; Buchwald, S. L. Top. Curr. Chem. 2002, 219,
131.
8. Proposed binding mode of 18c to p38a:
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.
10. A phosphorylated form of His-p38a MAP kinase (10 ng/
well) of murine origin was used and immobilised
GST-ATF-2 as substrate in the presence of 120 lM cold
ATP.
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 po per day) from
day 14 to 20. Swelling of the joints was measured on day
20.
Lys53
Thr106
H
N
HN
OH
O
F
O
His107
NH
O
12. The area under the curve (AUC) for 18a: 512 [ng h mlꢀ1];
9: 422 [ng h mlꢀ1].
F
H2N
HO
HN
Leu108
NH
H
N
O
N
13. CIA: Collagen-induced arthritis. Female (WAGxBUF/F1)
rats were immunized intradermally with bovine nasal
septum type II collagen emulsified in Freundꢀs incomplete
adjuvant. Swelling started ꢁ10 days after immunization.
Dosing of compounds started on day 13, when swelling
was nearly maximal. Compounds were dosed po once
(q.d.) daily for 10 days.
14. Selectivity profiles were determined in-house as described.4
Kinase (IC50 or % inhibition at 10 lM for 9/18a/23b): JNK2
(100 lM/8.3 lM/>100 lM); CDK1: (0%/ꢀ16%/ꢀ13%);
HER-1 (ꢀ43%/5.2 lM/0%); c-Abl (ꢀ23%/ꢀ58%/ꢀ42%);
c-Src (ꢀ17%/ꢀ68%/ꢀ37%); Kdr (ꢀ12%/ꢀ55%/ꢀ12%);
c-Met (ꢀ38%/1.5 lM/ꢀ17%); FGFR (ꢀ27%/10 lM/
ꢀ5%); c-Kit (ꢀ31%/ꢀ37%/0%); IGF1R (0%/0%/ꢀ23%);
HER-2 (ꢀ45%/5.0 lM/ꢀ0%); and c-Raf (n.t./0%/ꢀ14%).
N
N
O
H
N
Met109
O
O
Asp168
HN
N
The proposed binding mode of pyrazoloheteroaryls to
p38a implies that the pyrazolo nitrogen forms a crucial H-
bridge to the NH of Met109 and the difluorophenyl ring
accommodates in the specificity pocket near Thr106. Rigid
basic substituents such as the 1-dimethylamino-2-propyne
side chain in 18c increase binding affinity by forming a salt
bridge to Asp168.
9. Eight-week-old female OF1 mice were dosed orally by
gavage with solutions of the compounds in DMSO/
cornoil. One hour after dosing, LPS (20 mg/kg) was