The identification of pyrazolo[1,5-a]pyridines as potent p38 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.09.040

A series of pyrazolo[1,5-a]pyridine derivatives was designed and synthesized as novel potent p38 kinase inhibitors. Our approaches towards improving in vitro metabolism and in vivo pharmacokinetic properties of the series are described.

Full text of DOI:10.1016/j.bmcl.2008.09.040

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5428–5430
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The identification of pyrazolo[1,5-a]pyridines as potent p38 kinase inhibitors
*
Mui Cheung , Philip A. Harris, Jennifer G. Badiang, Gregory E. Peckham, Stanley D. Chamberlain ,
Michael J. Alberti, David K. Jung, Stephanie S. Harris, Neal H. Bramson, Andrea H. Epperly,
Stephen A. Stimpson, Michael R. Peel à
GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 July 2008
Revised 4 September 2008
Accepted 9 September 2008
Available online 12 September 2008
A series of pyrazolo[1,5-a]pyridine derivatives was designed and synthesized as novel potent p38 kinase
inhibitors. Our approaches towards improving in vitro metabolism and in vivo pharmacokinetic proper-
ties of the series are described.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Pyrazolopyridine
p38 Inhibitor
P38, also known as cytokine-suppressive anti-inflammatory
drug binding protein (CSBP), is a member of the mitogen activated
protein (MAP) kinase family that is involved in stress and inflam-
matory response signal transduction pathways.1,2 A large number
of small molecular inhibitors of p38 have been reported, and many
are undergoing clinical trials for the treatment of inflammatory
and autoimmune diseases such as rheumatoid arthritis, Crohn’s
disease, psoriasis and surgery-induced tissue injury.3–5 Recently,
the potential utility of inhibiting p38 for the treatment of ischemic
heart disease was also proposed.6 The most clinically advanced p38
MAP kinase inhibitors include SCIO-469 (talmapimod), VX-702 and
PS-540446 which are currently in phase 2 clinical trials for the
treatment of rheumatoid arthritis and additional indications.
As part of a lead optimization program, 2-(4-fluorophenyl)-3-
(4-pyridinyl)pyrazolo-[1,5-a]pyridine (1) was identified as a prom-
netic profile of 1 which possessed high in vivo clearances in both
rat and dog of 49 and 34 mL/min/kg, respectively, which would
not support a target of once daily dosing. Herein we report our ap-
proach to improve the pharmacokinetic properties of this series by
identification of a key potential site of oxidative metabolism on the
pyrazolo-[1,5-a]pyridine core and attachment of various substitu-
tions at that site to inhibit metabolism.
Our initial approach was to investigate the structure–activity
relationship (SAR) at the pyrazolopyridine heterocycle targeting
the open 4-, 5-, 6-, and 7-positions (see Fig. 1). This approach
was chemically flexible and would provide useful information
about potency and in vitro stability of the template. A parallel
strategy was to examine the potential metabolites of 1 by incuba-
tion in the S9 liver microsomes and then follow up with metabolite
identification using MS/MS and NMR spectroscopy. The aim was to
identify the potential sites of metabolism of the molecule and use
this information to design appropriate substitutions to inhibit this
process.
ising p38 kinase inhibitor with an IC50 of 0.12 l
M.7 Compound 1
inhibited TNF release from human peripheral blood mononuclear
cells (PBMC) following stimulation with LPS with an IC50 of 0.3–
1.3
l
M. In addition, 1 showed 65% inhibition of murine TNF pro-
The syntheses of pyrazolo-[1,5-a]pyridines have been described
duction in mice following an LPS challenge when dosed at
30 mg/kg orally.8 As an initial lead this was a very attractive start-
ing point. The main area for improvement was the pharmacoki-
elsewhere.8–10 As shown in Scheme 1, compounds 1–10 were pre-
F
7
N
N
6
Abbreviations: CSBP, cytokine-suppressive anti-inflammatory drug binding
protein; MAP kinase, mitogen-activated protein kinase; PBMNC, human peripheral
blood monouclear cells; SAR, structure–activity relationships.
* Corresponding author. Tel.: +1 610 270 7782; fax: +1 610 270 4490.
E-mail address: mui.h.cheung@gsk.com (M. Cheung).
5
4
N
1
Present address: Inhibitex Inc., Alpharetta, GA, USA.
Present address: Scynexis Inc., Research Triangle Park, NC, USA.
à
Figure 1. Structure of 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.040

M. Cheung et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5428–5430
5429
ping the anion of 1 with dimethyl disulfide to form the 7-S-Me
derivative, which was then oxidized with meta-chloroperoxyben-
zoic acid to yield the 7-methyl sulfoxide. Subsequent displacement
of the 7-methyl sulfoxide with alkoxide nucleophiles afforded the
7-alkoxy substituted compounds (12–14). Alternatively, com-
pounds 12–14 could be prepared by direct displacement of 7-chlo-
ride compound (11) with alkoxides.
As shown in Table 1, substitutions at the pyrazolopyridine ring
with electron-donating groups (compounds 4–7, 12–13) and elec-
tron-withdrawing groups (compounds 2–3, 8–11) are well-
tolerated.
F
R
F
1) NaOH, MeOH
reflux
+
_
N
N
CF3CO2
NH2
R
N
2) NBS, NaHCO3, DMF
1-10
DBU, CH3CN
MeO2C
3) Pd(PPh)3, toluene
reflux
SnBu
3
CO2Me
N
Scheme 1. Synthesis of 1–10.
When evaluating the metabolic stability of pyrazolopyridines in
the S9 microsomal fraction, pyrazolopyridines with electron rich
groups attached (5–7) tended to have a higher metabolic turnover
than those with electron-withdrawing groups (2 and 3). As shown
in Table 2, after 30 min of incubation in the liver S9 fractions, the
6-methylated compound (6) was highly metabolized in rat and
dog when compared to the 6-fluoro compound (3).
F
Cl
F
N
7
N
N
N
BuLi, THF, -78 oC
NCS, rt
11
1
N
N
ROH,
t-BuOK,
THF
BuLi, THF, -78 o
then MeSSMe, rt.
C
The pyrazolo[1,5-a]pyridine core is known as a site of metabo-
lism.11,12 To identify the potential metabolites, 1 was incubated in
liver S9 microsomes and then metabolite identification was
achieved using MS/MS and NMR spectroscopy. Following analysis
of samples of compound 1 in mouse, rat, dog and human liver
microsomes, dihyroxylated compounds with molecular weights
of 324, which is +34 of the parent compound 1, were identified
as the major metabolites. NMR spectroscopy identified the metab-
olite structures as compounds 15 and 16 where dihydroxylation
occurred at the 6- and 7-positions, and the 4- and 7-positions,
respectively, in a 60:40 ratio. The likely pathway is initial epoxida-
tion at the 6-/7-position, followed by epoxide-opening nucleo-
philic addition of water at either the 4- or 6-positions (Scheme
3). This in vitro metabolism pathway would support the earlier
observation that electron deficient substituents on the pyrazolo-
pyridine ring reduced S9 metabolism, as reduction of electron den-
sity at the pyridine ring will lower the propensity for the initial
epoxidation.
Based on the earlier SAR and the metabolite information, we ex-
pected that metabolism of the pyrazolopyridine core could be re-
duced by either increasing steric hindrance or incorporation
electron-withdrawing groups at the 6- or 7-positions. As shown
in Table 3, incorporation of the electron-withdrawing group at
the 6-position such as 6-CF3 (8) and bulky groups at the 7-position
such as 7-OCH2CF3 (14) and 7-O-cyclopentane (12) significantly re-
duced metabolite liability when compared to the parent compound
(1) with no observed turnover in both rat and dog S9 microsomes.
F
SMe
F
OR
N
N
N
N
1) m-CPBA, CHCl3
2) ROH, t-BuOK, THF
N
N
12-14
Scheme 2. Synthesis of 11–14.
pared by cyclization of methyl 3-(4-fluorophenyl)-2-propynoate
with the appropriately substituted N-aminopyridines to form the
pyrazolopyridine methyl ester. Hydrolysis of methyl ester followed
by in situ decarboxylation and bromination afforded the bromide
intermediate which underwent Susuki coupling with 4-(tributyl-
stannyl)pyridine to provide pyrazolopyridines 1–10. Compounds
11–14 were synthesized by functionalization of the 7-position of
1 as described in Scheme 2. Deprotonation of 1 with n-butyl lith-
ium and subsequent trapping of the resulting anion with electro-
philes such as N-chlorosuccinimide or p-toluenesulfonyl chloride
provided compound 11. Compounds 12–14 were prepared by trap-
Table 1
P38 kinase activity of pyrazolopyridines
F
N
7
R
6
N
Table 2
5
In vitro metabolic stability (% metabolized) of p38 inhibitors in liver S9 fractions in rat
and dog
4
N
F
N
7
R
6
a
N
Compound
R
p38 enzyme IC50 (lM)
1
2
3
4
5
6
7
8
H
4-F
6-F
0.12
0.24
0.16
0.28
0.16
0.15
0.15
0.20
0.14
0.15
0.15
0.17
0.05
0.07
5
4
N
4-Me
5-Me
6-Me
7-Me
6-CF3
6-CN
6-Cl
7-Cl
Compound
R
Rat S9a
Dog S9a
1
2
3
5
6
7
H
4-F
6-F
5-Me
6-Me
7-Me
27
21
1
60
99
43
81
39
23
ND
81
93
9
10
11
12
13
14
7-O-Cyclopentane
7-O-Et
7-OCH2CF3
a
Values are means of P2 incubations. Substrate concentration is 15 lM with
5 mg protein/mL. Readout is % turnover of parent compound after 30 min incuba-
a
tion in liver S9 fractions. ND, no data.
Values are means of greater than two experiments.

5430
M. Cheung et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5428–5430
Table 5
F
OH
N
In vivo pharmacokinetic properties of p38 inhibitors in male Beagle dogs
N
OH
Compound Dose (mg/
kg)
CL (mL/min/
kg)
Vdss (L/
kg)
t1/2
(h)
AUC (
mL)
l
g h/
%F
F
1
14
1 (iv)a
1 (iv)b
34
17
3.8
8.2
1.9
6.5
0.5
1.5
18
32
15
+
N
7
N
P450
mediated
N
O
6
H2O
N
1
a
n = 3; iv formulation: solution in 10% HP-b-cyclodextrin in 0.05 M acetic acid;
F
oral formulation: suspension in HPMC with Tween 80.
OH
b
n = 2.
N
N
The pharmacokinetic properties of compound 14 were evalu-
ated in the dog. As shown in Table 5, compound 14 showed a re-
duced clearance, longer half-life, and improved AUC and oral
bioavailability in the dog compared to the patent compound (1).
A series of pyrazolo[1,5-a]pyridine derivatives were identified
as potent p38 kinase inhibitors. Initial SAR indicated that the pyr-
azolopyridine heterocycle could be the site of metabolism which
was confirmed by metabolite identification of compound 1. The
improvement in the in vitro and in vivo pharmacokinetic proper-
ties of the series was achieved by reducing electron density at
the 6-position and increasing steric hindrance at the 7-position
of the pyrazolopyridine ring.
HO
N
16
Scheme 3. Potential metabolites for compound 1.
Table 3
In vitro metabolic stability (% metabolized) of p38 inhibitors in liver S9 fractions in rat
and dog
F
N
7
R
6
N
5
Acknowledgments
4
N
We thank Kirk Stevens for reviewing the manuscript. We thank
Peiyuan Lin for providing the cellular data of 1 in PBMC. We also
acknowledge Jamie Conway and Heather Pink for providing the
in vivo data of 1 in TNF release model in mice.
Compound
R
Rat S9a
Dog S9a
1
7
8
11
12
14
H
27
43
0
37
8
81
93
0
84
18
0
7-Me
6-CF3
7-Cl
References and notes
7-O-Cyclopentane
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.; Strickler, J. E.;
McLaughlin, M. M.; Siemens, I. R.; Fisher, S. M.; Livi, G. P. l.; White, J. R.; Adams,
J. L.; Young, P. R. Nature 1994, 372, 739.
7-OCH2CF3
0
a
Values are means of P2 incubations. Substrate concentration is 15 lM with
5 mg protein/mL. Readout is % turnover of parent compound after 30 min incuba-
tion in liver S9 fractions. ND, no data.
2. Lee, J. C.; Young, P. R. J. Leukocyte Biol. 1996, 59, 152.
3. Peifer, C.; Wagner, G.; Laufer, S. Curr. Top. Med. Chem. 2006, 6, 113.
4. Goldstein, D. M.; Gabriel, T. Curr. Top. Med. Chem. 2005, 5, 1017.
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Table 4
6. Clark, J. E.; Sarafraz, N.; Marber, M. S. Pharmacol. Ther. 2007, 116, 192.
In vivo pharmacokinetic properties of p38 inhibitors in male Lewis rats
7. (a) The p38
a IC50s reported in this article were determined as follows: the
peptide substrate used in the p38 assay was biotin-IPTSPITTTYFFFRRR-amide.
The p38 and MEK6 proteins were purified to homogeneity from E. coli
expression systems. The fusion proteins were tagged at the N-terminus with
glutathione-S-transferase (GST). The maximum activation was achieved by
Compound Dose (mg/
kg)
CL (mL/min/
kg)
Vdss (L/
kg)
t1/2
(h)
AUC (
mL)
lg h/
%F
1
5
5 (iv)a
1 (iv)a
1 (iv)b
1 (iv)b
49
83
34
40
3.2
2.0
5.4
5.4
1.2
1.2
2.5
2.1
1.7
0.2
0.5
0.5
50
ND
84
72
incubating 20 lL of a reaction mixture of 30 nM MEK6 protein and 120 nM p38
protein in the presence of 1.5
Hepes, pH 7.5, added to 15
l
M peptide and 10 mM Mg(CH3CO2)2 in 100 mM
L of a mixture of 1.5 M ATP with 0.08 Ci
L of inhibitor in 6% DMSO. The controls were
8
14
l
l
l
[g-33P]ATP, with or without 15
l
reactions in the presence (negative controls) or absence (positive controls) of
50 mM EDTA. Reactions were allowed to proceed for 60 min at rt and quenched
with addition of 50 lL of 250 mM EDTA and mixed with 150 lL of Streptavidin
SPA beads (Amersham) to 0.5 mg/reaction. The Dynatech Microfluor white U-
bottom plates were sealed and the beads were allowed to settle overnight. The
plates were counted in a Packard TopCount for 60 s. IC50 values were obtained
by fitting raw data to %I = 100 Ã (1 À (I À C2)/(C1 À C2)), where I was CPM of
background, C1 was positive control, and C2 was negative control. (b) Kinase
a
n = 3; iv formulation: solution in 10% HP-b-cyclodextrin in 0.05 M acetic acid;
oral formulation: suspension in HPMC with Tween 80.
b
n = 2. ND, no data.
Unexpectedly, compound with the electron-withdrawing group at
the 7-position (11) did not improve metabolite stability when
compared to parent compound (1) and compound with the elec-
tron-donating group at the 7-position (7).
The improvement in the in vitro metabolic stability also trans-
lated into increased in vivo metabolic stability. As shown in Table
4, compounds 8 and 14 with no observed turnover in rat S9
microsomes have significantly lower clearance in rats than com-
pound 5 where 60% turnover was observed. In addition, both com-
selectivity profile of 1 (all data reported as IC50 with n P 2): p38 = 0.12
GSK3b = 2.2 M, C-Raf/Mek/Erk = 5.8 M, EGFR = 6.3 M; AKT3, CDK2/CyclinA,
C-FMS, ERBB2, ERBB4, PDHK4, TIE2, and VEGFR2 >10 M.
lM,
l
l
l
l
8. Alberti, M. J.; Baldwin, I. R.; Cheung, M.; Cockerill, S.; Flack, S.; Harris, P. A.;
Jung, D. K.; Peckham, G.; Peel, M. R.; Stanford, J. B.; Stevens, K.; Veal, J. M. WO
0216359, 2002.
9. Alberti, M. J.; Chamberlain, S. D.; Cheung, M.; Gudmundsson, K.; Harris, P. A.;
Johns, B. A.; Jung, D. K.; Peel, M. R.; Stanford, J. B. WO 0278700, 2002.
10. Stevens, K. L.; Jung, D. K.; Alberti, M. J.; Badiang, J. G.; Peckham, G. E.; Veal, J. M.;
Cheung, M.; Harris, P. A.; Chamberlain, S. D.; Peel, M. R. Org. Lett. 2005, 7, 4753.
11. Nagatsu, Y.; Higuchi, T.; Hirobe, M. Chem. Pharm. Bull. 1989, 37, 1410.
12. Awano, K.; Iwase, K.; Nagatsu, Y.; Suzue, S. Chem. Pharm. Bull. 1992, 40, 639.
pounds
bioavailability in rat.
8 and 14 have improved half-lives and excellent