Pyrazolopyridines as potent PDE4B inhibitors: 5-Heterocycle SAR

Article abstract of DOI:10.1016/j.bmcl.2010.07.136

Following the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b] pyridine-5-carboxamides as potent and selective phosphodiesterase 4B inhibitors, [Hamblin, J. N.; Angell, T.; Ballentine, S., et al. Bioorg. Med. Chem. Lett. 2008, 18, 4237] the SAR of the 5-position was investigated further. A range of substituted heterocycles showed good potencies against PDE4. Optimisation using X-ray crystallography and computational modelling led to the discovery of 16, with sub-nM inhibition of LPS-induced TNF-α production from isolated human peripheral blood mononuclear cells.

Full text of DOI:10.1016/j.bmcl.2010.07.136

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5803–5806
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazolopyridines as potent PDE4B inhibitors: 5-Heterocycle SAR
*
Charlotte J. Mitchell , Stuart P. Ballantine, Diane M. Coe, Caroline M. Cook, Christopher J. Delves,
Mike D. Dowle, Chris D. Edlin , J. Nicole Hamblin, Stuart Holman, Martin R. Johnson, Paul S. Jones,
Sue E. Keeling, Michael Kranz, Mika Lindvall à, Fiona S. Lucas, Margarete Neu, Yemisi E. Solanke,
Don O. Somers, Naimisha A. Trivedi, Joanne O. Wiseman
GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Following the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as
potent and selective phosphodiesterase 4B inhibitors, [Hamblin, J. N.; Angell, T.; Ballentine, S., et al.
Bioorg. Med. Chem. Lett. 2008, 18, 4237] the SAR of the 5-position was investigated further. A range of
substituted heterocycles showed good potencies against PDE4. Optimisation using X-ray crystallography
Received 16 June 2010
Revised 28 July 2010
Accepted 30 July 2010
Available online 5 August 2010
and computational modelling led to the discovery of 16, with sub-nM inhibition of LPS-induced TNF-
a
production from isolated human peripheral blood mononuclear cells.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
PDE4 inhibitor
Pyrazolopyridine
SAR
Phosphodiesterase 4 (PDE4) is an enzyme responsible for the
inactivation of cyclic adenosine monophosphate (cAMP), via
hydrolysis to 50-AMP. Inhibition of PDE4 results in an elevation of
cAMP, which in turn downregulates the inflammatory response.
PDE4 is the predominant phosphodiesterase enzyme present in
inflammatory cells (e.g., neutrophils, monocytes, macrophages)2
and in addition is a key regulator of cAMP metabolism in airway
epithelial cells.3 It is therefore established as an interesting target
for the treatment of respiratory diseases such as chronic obstruc-
tive pulmonary disease (COPD).4
PDE4 inhibitors such as Roflumilast 2 (Fig. 1) have shown improved
side effect profiles in clinical trials, the maximum dose is still lim-
ited by adverse events.6
Our aim was therefore to identify a third generation of PDE4
inhibitors, which could provide a greater therapeutic window be-
tween efficacy in COPD treatment and emetic side effects.
We have previously described the identification of pyrazolo-
pyridine (PZP) 31 (Fig. 2), which showed inhibition of isolated
PDE4B7 and lipopolysaccharide (LPS) induced tumour necrosis fac-
tor alpha (TNF-a) production in human peripheral blood mononu-
First generation PDE4 inhibitors exemplified by Rolipram 1
(Fig. 1), demonstrated severe side effects of nausea and emesis at
effective anti-inflammatory doses.5 Whilst second generation
clear cells (PBMCs).8
Further work to explore the structure–activity relationship
(SAR) around the PZP template led us to investigate the replace-
ment of the 5-amide group with a range of heterocycles, initially
1,3,4- and 1,2,4-oxadiazoles.
In each case the heterocyclic rings were synthesized from key
intermediate 4. For the 1,3,4-oxadiazoles, the ester was first
Cl
O
O
O
N
O
O
N
NH
H
Cl
O
O
F
F
1
2
NH O
N
N
Figure 1. Structures of Rolipram and Roflumilast.
H
N
N
* Corresponding author. Tel.: +44 1438 762542; fax +44 1438 7683022.
E-mail address: charlotte.j.mitchell@gsk.com (C.J. Mitchell).
Present address: iThemba Pharmaceuticals, Pinelands Site, Ardeer Road, Building
T5, Modderfontein 1609, Gauteng, South Africa.
3
PDE4B pIC50 8.5
PBMC pIC50 7.7
à
Present address: Novartis Institute for Biomedical Research, 4560 Horton Street,
Emeryville, CA 74607, USA.
Figure 2. Pyrazolopyridine 5-carboxamide.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.136

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C. J. Mitchell et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5803–5806
R2
N
converted to hydrazide 5 by hydrolysis, coupling with BOC-hydra-
zine and deprotection under acidic conditions. Subsequent acyla-
tion under standard conditions to give the diacyl hydrazide 6,
followed by cyclisation with Burgess reagent,9 gave the desired tar-
gets 7a–f (Scheme 1).
In cases where the carboxylic acid required for conversion of 5
to 6 was not available, the targets were synthesized from key acid
and amine intermediates 8 and 9 (Scheme 2).
For the 1,2,4-oxadiazoles 10a–f, intermediate 4 was reacted in a
one pot procedure with a range of amidoximes to give the desired
products directly. Alternatively 4 was first hydrolysed to carboxylic
acid 11, coupled with the amidoxime and cyclised with DBU
(Scheme 3). Where the appropriate amidoximes were not available
these could be synthesized from the corresponding nitrile, by reac-
tion with hydroxylamine.10
O
O
NH O
NH N
a
O
O
N
N
N
N
N
N
10a-f
4
b
c, d
O
NH O
OH
N
N
N
11
Scheme 3. Reagents and conditions: (a) R2C(NOH)NH2, NaOEt, molecular sieves,
75 °C, 18 h; (b) NaOH, EtOH/H2O, 18 h (99%); (c) R2C(NOH)NH2, TBTU, HOBt, DIPEA,
DMF, 20 h; (d) DBU, DMF, 85 °C, 0.75 h.
As with the 1,3,4- series, some analogues were prepared via a
key amine intermediate 12 (Scheme 4).
Preliminary structure–activity studies were conducted against
isolated PDE4B,11 the predominant PDE4 sub-type in inflammatory
cells of interest.12 Results indicated that both oxadiazole isomers
were tolerated at the 5-position (Table 1). Different oxadiazole
substituents showed optimum potency in each series but no gen-
eral trend to rank one isomer above the other was observed.
For example, benzyl substituted analogues 7b/10b and pyrroli-
dine amides 7d/10d showed similar potencies in both series,
O
O
NH2
NH
O
NH
N
a, b, c
N
O
OH
N
N
N
N
N
N
11
12
e, f
d
O
N
O
N
10c
10e
NH O
NH O
a, b, c
NH2
Scheme 4. Reagents and conditions: (a) tBuOC(O)CH2C(NOH)NH2, TBTU, HOBt,
DIPEA, DMF, 2 h; (b) DBU, DMF, 85 °C, 3.5 h (89% over two steps); (c) TFA, DCM, 0 °C,
2 h (99%); (d) MeCOCl, DIPEA, DCM, 16 h (96%); (e) 4-chlorobutanoyl chloride,
DIPEA, MeCN, 16 h; (f) NaH, DMF, 16 h (83% over two-steps).
O
N
H
N
N
N
N
4
5
d
O
N
O
Table 1
SAR of 1,3,4-oxadiazoles 7a–f and 1,2,4-oxadiazoles 10a–f
N
O
NH N
NH O
e
H
R1
R1
N
N
O
N
H
O
N
N
N
NH N
R1
N
N
O
6
7a-f
N
N
N
Scheme 1. Reagents and conditions: (a) NaOH, EtOH/H2O, 18 h (99%); (b)
NH2NHCO2tBu, EDC, HOBt, DMF, 18 h (82%); (c) 4 M HCl, 1,4-dioxane, 2 h (100%);
(d) R1COOH, TBTU, DIPEA, DMF, 18 h; (e) Burgess reagent, THF, 75 °C, 2 h.
R2
N
O
NH N
O
N
N
N
O
NH O
H
R1
R1
PDE 4B
pIC50
PBMC
pIC50
R2
PDE 4B
pIC50
PBMC
pIC50
N
N
a
a
a
a
N
H
O
N
N
6
F
7a
7b
7c
8.9b
8.0b
7.3
NT
8.3
NT
10a
10b
10c
8.0b
8.2b
8.1b
NT
7.5
NT
t
t
R1 = CH2NHCO2 Bu
R1 = CH2CO2 Bu
*
*
a, b
*
*
O
O
O
N
O
N
N
N
N
NH
N
N
NH
N
N
H
N
H
*
*
O
O
NH2
OH
N
N
O
N
N
7d
8.7b
NT
10d
9.1b
NT
*
*
*
*
8
9
O
N
O
N
d, e
8.4b
7.3
7.0
6.9
10e
10f
9.4b
7.8b
8.5
NT
c
7e
7f
O
O
N
N
*
*
7d
7e
N
N
NT Compound was not progressed to PBMC assay.
Scheme 2. Reagents and conditions: (a) Burgess reagent, THF, 75 °C, 2 h (70%); (b)
TFA, DCM, 4 h (100%); (c) Pyrrolidine, pybop, DIPEA, DMF, 18 h (43%); (d) 4-
chlorobutanoyl chloride, DIPEA, MeCN, 1 h; (e) NaH, DMF, 2 h (43% over two-steps).
a
Values are mean of P2 experiments in all tables unless otherwise stated.
b
Value is a single experiment.

C. J. Mitchell et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5803–5806
5805
whereas, the pyrrolidinone substituted 1,2,4-oxadiazole 10e
showed ꢀ10-fold improvement over 1,3,4-oxazdiazole 7e.
For both isomers a range of functionalities gave good potencies
suggesting manipulation of the substituent might offer a useful
means of varying physicochemical properties.
For selected analogues, inhibition of LPS-induced TNF-
a pro-
duction in isolated human PBMCs was also measured (Table 1).
Generally both series of oxadiazole showed similar or improved
potency in the PBMC assay to 5-amide 3.
Computational modelling in the PDE4 active site suggested that
the binding mode of the oxadiazoles would vary between isomers,
rationalizing the observed divergent SAR. The five-membered het-
erocycles were suggested to be coplanar with the PZP ring system,
fixed by an intramolecular H-bond to the NH of the 4-aminotetra-
hydropyran substituent. Their conformational preferences are
determined by the superior acceptor strength of the oxadiazole
nitrogen over that of the oxygen13 (Fig. 3).
Figure 5. Overlay of X-ray crystal structures of 5-amide 3 (orange) and 1,2,4-
oxadiazole 10d (magenta) (only Glu443 shown for clarity).
oxazoles were expected to mimic, respectively, the 1,2,4- and
1,3,4-oxadiazoles already synthesized, di-substituted oxazoles
had the potential to fill both pockets with the aim of increasing
PDE4B potency even further (Fig. 6).
Whilst the 1,3,4-oxadiazole R1 substituents were predicted to
occupy the same pocket as the N substituent in the 5-amide series
(Fig. 4), the 1,2,4-oxadiazole R2 substituents were expected to ac-
cess a different pocket, previously unfilled.
Using SAR from the two oxadiazole series, di-substituted oxa-
zole 16 was designed. A benzyl group was chosen as the R1 substi-
tuent (well tolerated in the 1,3,4-oxadiazole 7b) and a pyrrolidine
amide was introduced at R2 (cf. 10d in the 1,2,4-oxadiazole series).
Synthesis proceeded from key intermediate 13. Amide coupling
followed by a Dakin–West reaction14 with the appropriate anhy-
dride led to desired intermediate 14, which was cyclised using
phosphorous oxychloride as a dehydrating agent. Basic hydrolysis
of the ester, followed by amide formation gave the desired product
16 (Scheme 5).
For completeness, mono-substituted oxazole 18 was also syn-
thesized. Amide coupling of key pyrazolopyridine intermediate
11 with 1-amino-3-phenyl-2-propanol, followed by oxidation of
the alcohol gave intermediate 17. Subsequent cyclisation with Bur-
gess reagent gave the desired 5-substituted oxazole 18 (Scheme 6).
The binding mode of the 1,2,4-isomer was confirmed by X-ray
crystallography of 10d (Fig. 5) which clearly revealed the direction
of the oxadiazole substituent.1 It is wedged between Ser-282 and
Met-347 and also in van der Waals contact with the PZP’s 4-THP.
The pyrrolidine amide can adopt two conformations, only one of
which is shown in Figure 5, the other one occupies a similar region
with an 180° amide rotation leading to a close contact between the
pyrrolidine ring and Cys-432 as well as Phe-414.
Using this information we decided to investigate oxazoles as an
alternative heterocyclic core. Whilst 4- and 5-mono-substituted
O
O
R2
N
H
H
N
N
N
N
N
N
N
R1
O
O
R2
O
O
H
H
N
N
N
N
N
N
N
N
N
R1
N
O
O
N
N
N
N
1,3,4-Oxadiazole
1,2,4-Oxadiazole
5-Mono-substituted Oxazole
4-Mono-substituted Oxazole
Figure 3. Orientation of 1,3,4- and 1,2,4-oxadiazoles relative to PZP core (red lines
represent H-bonding interactions).
O
R2
H
N
N
R1
O
N
N
N
4,5-Disubstituted Oxazole
Figure 6. Orientation of oxazoles.
O
O
CO2Me
O
NH O
N
NH O
N
a, b
Cl
N
N
N
H
N
N
13
14
c
N
O
O
CO2Me
O
NH N
NH N
d, e
O
O
N
N
N
N
N
N
16
15
Figure 4. (a) X-ray crystal structure of 5-amide 3 in PDE4 active site (with and
without protein surface); (b) X-ray crystal structure of 5-amide 3 rotated 90° (only
Glu443 shown for clarity); (c) X-ray crystal structure of 5-amide 3 (orange) overlaid
with computational model of 1,3,4-oxadiazole 7b (grey).
Scheme 5. Reagents and conditions: (a) (S)-NH2C(CO2H)CH2CO2Me, py, 7 h; (b)
(PhCH2CO)2O, reflux, 5 h; (c) POCl3, toluene, 120 °C, 23 h; (d) LiOH, THF/H2O, 40 °C,
1 h; (e) pyrrolidine, HATU, DIPEA, MeCN, 8 h.

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C. J. Mitchell et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5803–5806
O
N
O
NH O
NH O
a, b
N
OH
N
O
N
N
N
N
11
17
c
O
NH N
O
N
N
N
18
Scheme 6. Reagents and conditions: (a) PhCH2C(OH)CH2NH2, HATU, DIPEA, MeCN,
12 h (100%); (b) py.SO3H, Et3N, DMSO, 24 h (58%); (c) Burgess reagent, THF, 75 °C,
3 h (50%).
Figure 7. Overlay of X-ray crystal structures of 5-amide
di-substituted oxazole 16 (cyan) with protein surface shown.
3 (orange) and
The di-substituted oxazole 16 demonstrated sub-nM activity in
the isolated enzyme and PBMC assay (Table 2) and gave at least 10-
fold increase in potency over 1,3,4-oxadiazole 7b, 1,2,4-oxadiazole
10d (Table 1) and 5-amide 3. Comparison with the mono-subsitut-
ed oxazole 18 also showed an increase in potency on introduction
of a second substituent.
An X-ray crystal structure of 16 soaked into a PDE4B crystal was
obtained and confirmed the proposed binding mode with both
pockets occupied, rationalizing the improved potencies observed
(Fig. 7).1 The pyrrolidine amide can again adopt two conforma-
tions. The one shown in Figure 7 is the same as for the 1,2,4-oxa-
diazole in Figure 5, the other one involves an 180° flip of the
amide so the carbonyl is pointing in the opposite direction but
the pyrrolidone ring remains in the same position.
In summary, exploration of the SAR around lead pyrazolopyri-
dine 3, led us to 5-heteorcyclic compounds exemplified by 7b
and 10d. Following promising PDE4 potency shown with these ini-
tial analogues, computational modelling and X-ray crystallography
were employed to fully optimise ligand–protein interactions. The
design of 4,5-disubstituted oxazoles gave 16 which occupied two
key pockets in the active site, resulting in a significant increase
in potency.
The success of these ligands in achieving an improved therapeu-
tic index in vivo will be the subject of further papers.
Acknowledgements
The authors thank Damien Charre and Violaine Werquin for
their synthetic contributions whilst on placement at GSK.
References and notes
1. Methods according to Hamblin, J. N.; Angell, T.; Ballentine, S., et al Bioorg. Med.
Chem. Lett. 2008, 18, 4237. except compound 10d was soaked into an apo-
PDE4B crystal at 1 mM for ꢀ2 days, while compound 16 was soaked at 2 mM
for ꢀ1 day. Data were collected for compound 16 at station 17ID-B of the
Advanced Photon Source (Argonne, USA) using an ADSC Q210 CCD detector.
During refinement, the occupancy of 16 was set at 0.7 to account for
incomplete soaking of the compound into the crystal probably due to poor
compound solubility. The final R-factors achieved for the complexes 10d and
16 following structure refinement were 19.6% (Rfree = 26.0%) and 18.7%
(Rfree = 24.0%), respectively, and the coordinates deposited in the PDB as
entries 3O56 and 3O57.
2. Torphy, T. J.; Undem, B. J. Thorax 1991, 46, 512.
3. Torphy, T. J.; Undem, B. J.; Cieslinski, L. B.; Luttmann, M. A.; Reeves, M. L.; Hay,
D. W. P. J. Pharmacol. Exp. Ther. 1993, 265, 1213.
4. McKenna, J. M.; Muller, G. W. Cyclic Nucleotide Phosphodiesterases in Health and
Disease; CRC Press: LLC, Boca Raton, Florida ,USA, 2007; Dyke, Hazel J. Expert
Opin. Ther. Pat. 2007, 17, 1183; Smith, V. B.; Spina, D. Curr. Opin. Invest. Drugs
2005, 6, 1136.
5. Horowski, R.; Sastre-Y-Hernandez, M. Curr. Ther. Res. 1998, 38, 23.
6. Rabe, F.; O’Donnell, D.; Muir, F., et al Eur. Respir. J. 2004, 24. Abstr. 267.
7. The ability of compounds to inhibit catalytic activity of PDE4B (human
recombinant) was determined by scintillation proximity assay (SPA) in 96-well
format as described in WO2004024728.
Table 2
SAR of oxazoles
O
NH
R
N
8. The ability of compounds to inhibit LPS induced TNF-
a production in PBMCs
N
N
was determined by electrochemiluminescence using the Meso Scale Discovery
(MSD) technology as described in WO 2007036733.
9. Ersmark, K.; Nervall, M.; Hamelink, E., et al J. Med. Chem. 2005, 48, 6090.
10. Sahbari, J.; Russell, J. WO0032565.
11. The ability of compounds to inhibit catalytic activity at PDE4B (human
recombinant) was determined by IMAP Fluorescence Polarisation (FP) assay
(IMAP Explorer kit, available from Molecular Devices Corporation, Sunnydale,
CA, USA; Molecular Devices code: R8062) in 384-well format as described in
WO 2004056823.
Compd
R
PDE4B pIC50
8.5b
PBMC pIC50
7.7
O
3
N
*
H
12. Wang, P.; Ohelth, K. M.; Wu, P.; Billah, M. M.; Egan, R. W. Mol. Pharmacol. 1999,
56, 170.
N
13. The 4-methylamino-3-oxadiazole-pyridine model systems show
a clear
O
16
10.1
9.6
8.0
N
preference for the nitrogen coordination to the amino NH over the oxygen
**
coordination for the 1,2,4- (2.8 kcal/mol at B3LYP/6-31+G ) and the 1,3,4-
O
*
*
oxadiazoles (6.2 kcal/mol).
14. Buchanan, G. L. Chem. Soc. Rev. 1988, 17, 91.
N
8.6a
O
18
a
Value is a single experiment.
Data from an alternative assay format.7
b