Pyrazolopyridines as a novel structural class of potent and selective PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.05.052

Optimisation of a high-throughput screening hit resulted in the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective inhibitors of Phosphodiesterase 4 (PDE4). Herein, we describe early SAR studies around this novel template highlighting preferred substituents and rationalization of SAR through X-ray crystal structures of analogues bound to the PDE4 active site. Pyrazolopyridine 20a was found to be a potent and selective PDE4 inhibitor which also inhibits LPS induced TNF-α production from isolated human peripheral blood mononuclear cells and has an encouraging rat PK profile suitable for oral dosing.

Full text of DOI:10.1016/j.bmcl.2008.05.052

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4237–4241
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazolopyridines as a novel structural class of potent and selective
PDE4 inhibitors
*
J. Nicole Hamblin , Tony D. R. Angell, Stuart P. Ballantine, Caroline M. Cook, Anthony W. J. Cooper,
John Dawson, Christopher J. Delves, Paul S. Jones, Mika Lindvall , Fiona S. Lucas, Charlotte J. Mitchell,
Margarete Y. Neu, Lisa E. Ranshaw, Yemisi E. Solanke, Don O. Somers, Joanne O. Wiseman
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimisation of a high-throughput screening hit resulted in the discovery of 4-(substituted amino)-1-
alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective inhibitors of Phosphodiesterase
4 (PDE4). Herein, we describe early SAR studies around this novel template highlighting preferred sub-
stituents and rationalization of SAR through X-ray crystal structures of analogues bound to the PDE4
active site. Pyrazolopyridine 20a was found to be a potent and selective PDE4 inhibitor which also inhib-
its LPS induced TNF-a production from isolated human peripheral blood mononuclear cells and has an
encouraging rat PK profile suitable for oral dosing.
Received 8 April 2008
Revised 13 May 2008
Accepted 14 May 2008
Available online 17 May 2008
Keywords:
PDE4 inhibitors
SAR
Ó 2008 Elsevier Ltd. All rights reserved.
Crystallography
Pyrazolopyridine
Chronic obstructive pulmonary disease (COPD)1 is a leading
cause of morbidity and mortality worldwide, forecast to become
the fifth leading cause of death by 2020. Cigarette smoking is the
principal risk factor and the disease is characterised by progressive,
largely irreversible airflow obstruction, significant structural
damage and by the presence of one or more of the following
pathologies: chronic bronchitis, emphysema and bronchiolitis.
Phosphodiesterase 4 (PDE4) plays a key role in the regulation of
intracellular concentrations of the second messenger cyclic AMP
(cAMP), effecting its selective hydrolysis to 50-AMP. PDE4 is the
predominant phosphodiesterase enzyme in immune and inflam-
matory cells (e.g. neutrophils, monocytes, macrophages)2 and is
also a major contributor to cAMP metabolism in airway smooth
muscle and airway epithelial cells.3 In COPD, cAMP is believed to
suppress the activation of immunocompetent cells, mediate bron-
chodilation, modulate neuronal activity and inhibit smooth muscle
proliferation; hence elevated levels of cAMP achieved through
PDE4 inhibition may be beneficial for COPD patients.4
Rolipram 1 cause pronounced nausea and emesis at effective
anti-inflammatory doses in the clinic.7 Roflumilast (DaxasTM) 2 is
representative of a second generation of PDE4 inhibitors which
show moderate efficacy in COPD at tolerated doses,8 however,
the maximum dose that can be given (and hence efficacy) is still
limited by the aforementioned transient adverse effects.
Our objective was thus to develop an oral PDE4 inhibitor with
improved therapeutic index.
At the time we began our work, most of the advanced PDE4
inhibitors possessed a common catechol ether structural motif
(Fig. 1). Our initial strategy was to determine whether this was
contributing to the observed emetic effects and hence we sought
a lead molecule devoid of this structural feature. Compound 3
(Fig. 2) was discovered via high throughput screening, along with
several structurally related pyrazolopyridine analogues which all
showed good PDE4 potency and promising selectivity profiles ver-
sus PDE3 and PDE5.9 There are four sub-types of the PDE4 enzyme:
A, B, C and D, but the PDE4B sub-type is believed to play a central
PDE4 inhibitors are known to have a broad range of anti-inflam-
matory and anti-bronchoconstrictor effects in animal models5 and
as such have been well studied by numerous groups.6 In spite of
this however, bringing a drug of this class to market has been ham-
pered by undesirable side effects. Early PDE4 inhibitors such as
Cl
O
O
N
OMe
O
O
N
H
Cl
* Corresponding author. Fax: +44 (0)1438 768301.
E-mail address: nicole.j.hamblin@gsk.com (J.N. Hamblin).
Present address: Novartis Institute for Biomedical Research, 4560 Horton Street,
Emeryville, CA 74607, USA.
1
O
2
N
F
F
H
Figure 1. Structures of R-Rolipram 1 and Roflumilast 2.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.052

4238
J. N. Hamblin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4237–4241
Table 1
Pyrazolopyridine 4-position SAR
NH
N
PDE4B pIC50 6.8
PDE3 pIC50 4.3
PDE5 pIC50 4.7
R4
N
CO2Et
CO2Et
N
N
N
3
N
Figure 2. Initial pyrazolopyridine HTS hit.
a
R4
PDE4BpIC50
role in inflammation,10 being the predominant subtype in mono-
cytes and neutrophils and hence we used this isoform for routine
screening.
The series was considered highly tractable and sufficiently
structurally distinct from known PDE4 inhibitors to be an interest-
ing start point. Initial plans were to explore the SAR around the
three existing substituents using parallel synthesis techniques.
Synthesis of pyrazolopyridines is well described in the litera-
ture and we chose to use the precedented high temperature reac-
N
H
8a
8b
8c
8d
8e
8f
6.8
N
H
7.2
6.2
8.0
7.8
7.8
6.7
6.0
6.5
N
N
H
tion between N-1-ethyl-5-aminopyrazole
4
and diethyl
ethoxymethylenemalonate 5 to form the ring (Scheme 1).11,12
4-Hydroxypyrazolopyridine intermediate 6 was treated directly
with phosphoryl chloride to give key 4-chloro-derivative 7, which
underwent thermal displacement with a diverse range of amines to
give 8a–j, allowing us to probe SAR at the 4-position.
N
H
O
N
H
The data showed that a significant increase in potency is
observed where the 4-substituent is a branched or cycloalkyl ami-
no-group, with 6-membered saturated rings more potent than
5-membered rings (Table 1). Particularly interesting was tetrahy-
dropyran analogue 8e with the data suggesting that polar function-
ality can be tolerated and that this position may therefore offer a
handle for future modification of physicochemical properties,
should this be required during lead optimisation.
N
H
8g
8h
8i
OMe
F
N
H
N
H
Tertiary amines such as pyrrolidine in 8c were less preferred, as
were N-methylated analogues such as 8j suggesting the impor-
tance of the NH group. These observations were later explained
by crystal structures of related pyrazolopyridine analogues bound
to PDE4B (Fig. 3), which showed the presence of an internal hydro-
gen bond between the 4-NH group and the 5-carbonyl oxygen, to
maintain planarity with the template. Unbranched alkyl amines
gave poor selectivity over PDE5, in contrast to branched analogues
which routinely gave >100-fold selectivity (data not shown).
We also chose to investigate the nature of the linking group at
the 4-position (Scheme 1). Displacement of chlorine in intermedi-
ate 7 with azide and reduction under Staudinger conditions13
afforded 4-amino intermediate 10, which was derivatised under
standard conditions to provide amide and sulphonamide deriva-
tives. Unfortunately these changes afforded a general loss of
potency (Table 1), again supported by the crystal structure which
showed that the conformational restriction required to allow the
6.6b
N
O
8j
Me
11a
12a
NHCOPh
NHSO2Ph
5.6
<4.5
a
Values are mean of P2 experiments in all tables unless otherwise stated.
Value is a single experiment.
b
aforementioned intramolecular hydrogen bond was unfavourable
with these alternative linkers.
Chemistry to introduce variation at the 1-position followed the
published method of Bare et al.11 (Scheme 2). Preparation of the
versatile 1-H pyrazolopyridine intermediate 13 from compound 7
first required transformation of the 4-chloro substituent into the
R4
R3
Cl
N
OH
EtO2C CO2Et
CO2Et
c
a
CO2Et
CO2Et
b
N
+
NH2
N
N
N
N
EtO
N
N
N
N
N
N
5
4
NHCOR4
CO2Et
7
6
8a-j
N
d
N
N
g
11
NH2
N
N3
CO2Et
CO2Et
e, f
N
N
NHSO2R4
CO2Et
N
N
N
h
N
9
10
N
N
12
Scheme 1. Reagents and conditions: (a) 150 °C, neat, 16 h; (b) POCl3, 120 °C, 20 h (58% over two steps); (c) R4R3NH, EtOH, Et3N, 80 °C, 16 h; (d) NaN3, DMF, 16 h (84%); (e)
PPh3, THF, 2.5 h; (f) H2O 2 h (60% over two steps); (g) R4COCl, CHCl3, Et3N, 16 h; (h) R4SO2Cl, CHCl3, pyridine, reflux, 72 h.

J. N. Hamblin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4237–4241
4239
Table 2
Pyrazolopyridine 1-position SAR
O
N
N
CO2Et
N
N
R1
R1
PDE4B pIC50
17a
17b
17c
17d
17e
17f
17g
17h
18
H
Me
Et
Pr
i-Pr
CH2Ph
(CH2)2CO2Et
(CH2)2OH
Ph
5.7
6.6
8.0
7.0
6.7b
5.4
5.3
6.8
5.5
Figure 3. Overlay of the crystal structure of compound 20a (orange) and R-Rolip-
ram 1 (cyan) bound to PDE4B 152-503. Key amino acids are shown in grey and
metal ions within the active site are shown in magenta.
b
Value is a single experiment.
able inhibitors, we also prepared an array of 5-amides. For this
work we elected to use our preferred tetrahydropyran amino sub-
stituent at the 4-position. Ester hydrolysis of 8e was carried out
using sodium hydroxide in aqueous ethanol and was followed by
amide formation via the acid chloride under standard conditions.11
The acid chloride was not isolated but was treated directly with a
range of amines in a parallel fashion (Scheme 3).
The data showed the 5-position to be tolerant of a wide range of
amide substituents (Table 3), again consistent with the crystal
structure wherein this position is directed towards the opening
of the active site (Fig. 3). In general, lipophilic secondary amides
were well tolerated, with benzyl amides such as 20a and saturated
analogues such as 20j preferred. Primary amide 20e was accept-
able but tertiary amides such as 20b and cyclic amines such as
20f afforded reduced potency.
Several amides of interest were selected for progression to fur-
ther assays (Table 3). The data show that good cellular potency
(inhibition of TNF-a in PBMCs14) can be achieved across this ser-
ies. >100-fold selectivity versus PDE3 and PDE5 is also
demonstrated.
Examination of oral rat PK profiles for selected pairs of ana-
logues showed, not surprisingly, that the 5-amide derivatives rou-
tinely gave improved bioavailability as compared with the 5-ester
analogues. Compound 20a was shown to have particularly encour-
aging rat oral PK [rat t1/2 1.6 h, F 35%].
OEt
N
OEt
N
OEt
N
CO2Et
CO2Et
CO2Et
b
a
c
7
N
N
N
N
N
N
H
14
16
d
15
O
N
O
N
N
N
CO2Et
CO2Et
e or f or g
N
N
N
N
R1
H
13
17a-h
Scheme 2. Reagents and conditions: (a) Na, EtOH, reflux, 2 h, 90%; (b) NBS, CCl4,
reflux, 3 h; (c) Na2CO3, THF, 18 h, 51% over two steps; (d) 4-aminotetrahydropyran,
neat, 90 °C, 6 h, 91%; (e) p-BEMP resin, R1Br, DMF, 16 h; (f) NaH, DMF, R1Br, 16 h; (g)
K2CO3, DMF, R1Br, 55 °C, 45 h.
less reactive 4-ethoxy group in 14, in order to avoid undesirable
hydrolysis during subsequent steps. Bromination of the 1-ethyl
group and in situ elimination then afforded vinyl derivative 15
which was used directly in the next step. N-dealkylation under ba-
sic conditions to give 16 was followed by incorporation of the pre-
ferred 4-amino tetrahydropyran moiety, to afford 13 which could
be used to probe the 1-position SAR via alkylation.
A variety of different bases were used to deprotonate the pyraz-
olopyridine template in 13, including sodium hydride, potassium
carbonate and the polymer bound base BEMP (2-tert butylimino-
2-diethylamino-1,3-dimethyl-perhydro-1,3,2-diazaphosphorine).
The latter showed the greatest utility for our needs, allowing facile
work up of multiple parallel reactions. The resultant anion was
then treated with a range of alkyl halides.
As referred to above, the crystal structure of compound 20a
bound to PDE4B 152–50315 (Fig. 3) shows the tetrahydropyran
moiety directed towards the metal ions in the active site. An intra-
molecular hydrogen bond, between the 4-amino NH group and the
carbonyl of the amide, maintains co-planarity with the template
and orients the 5-benzylamine group towards the opening of the
active site. The template itself sits in a ‘hydrophobic clamp’ formed
by Ile410, Phe414 and Phe446. The pyridine-like nitrogen forms a
hydrogen bond with Gln443 and the N-ethyl substituent fills the
same small lipophilic pocket as occupied by the methoxy group
of Rolipram.
Compound 18 where R1 = phenyl was prepared according to
Scheme 1 starting with N-1-phenyl-5-aminopyrazole.
Unfortunately, this work failed to uncover a superior substitu-
ent to ethyl with respect to potency, the trend for simple alkyls
and aryls following Et > Me, Pr, i-Pr > H, CH2Ph, Ph (Table 2). Incor-
poration of more highly functionalised groups, such as the
hydroxyethyl derivative 17h, was generally detrimental for
potency.
X-ray crystallography of related molecules bound to PDE4B (Fig.
3) later highlighted that the 1-ethyl substituent optimally fills a
small pocket, the size constraint of which supports the observed
SAR.
O
O
O
NH
N
NH
O
NH
N
CO2Et
NR1R2
CO2H b, c
a
N
N
N
N
N
N
N
19
8e
20a-j
Since we were concerned about the potential for metabolic
instability of the ester functionality and we desired orally bioavail-
Scheme 3. Reagents and conditions: (a) NaOH, EtOH/H2O (95:5),16 h, 97%; (b)
SOCl2, CHCl3, 1 h; (c) R1R2NH, 0 °C, 0.5 h.

4240
J. N. Hamblin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4237–4241
Table 3
Pyrazolopyridine 5-position SAR
O
N
N
O
NR5R6
N
N
NR5R6
PDE4B pIC50
8.5
PDE3 pIC50
<4.5
PDE5 pIC50
5.7
PBMC TNF-a pIC50
20a
7.7
N
20b
7.3
7.7
7.9
7.6
6.8
7.3
6.9
7.0
8.1
—
—
—
N
20c
<4.5
<4.5
<4.5
—
4.8
<4.7
4.8
—
7.1
7.4
7.1
—
N
20d
20e
20f
20g
20h
20i
N
F
NH2
N
N
H
<4.5
—
<4.7
—
6.8
—
O
N
N
—
—
—
HN
HN
20j
<4.5
5.5
7.2
USA, 2007; p 667; Dyke, Hazel J. Exp. Opin. Ther. Patents 2007, 17, 1183; Smith,
V. B.; Spina, D. Curr. Opin. Invest. Drugs 2005, 6, 1136.
7. Horowski, R.; Sastre-Y-Hernandez, M. Curr. Ther. Res. 1998, 38, 23.
8. Rabe, F.; O’Donnell, D.; Muir, F., et al Eur. Respir. J. 2004, 24(Suppl. 48). Abst.
267.
9. The ability of compounds to inhibit catalytic activity at PDE4B (human
recombinant), PDE3 (from bovine aorta) and PDE5 (human recombinant) was
determined by scintillation proximity assay (SPA) in 96-well format as
described in WO2004024728.
10. Wang, P.; Ohleth, K. M.; Wu, P.; Billah, M. M.; Egan, R. W. Mol. Pharmacol. 1999,
56, 170.
11. Bare, T. M.; McLaren, C. D.; Campbell, J. B.; Firor, J. W.; Resch, J. F.; Walters, C. P.;
Salama, A. I.; Meiners, B. A.; Patel, J. B. J. Med. Chem. 1989, 32, 2561.
12. Yu, G.; Mason, H. J.; Ximao, W.; Wang, J.; Chong, S.; Dorough, G.; Henwood, A.;
Pongrac, R.; Seliger, L.; He, B.; Adam, L.; Krupinski, J.; Macor, J. J. Med. Chem.
2001, 44, 1025.
In summary, a new series of 4-(substituted amino)-1-alkyl-pyr-
azolo[3,4-b]pyridine-5-carboxamides has been identified as potent
and selective inhibitors of PDE4. The early SAR has been rational-
ized by crystallography and representatives from the series show
good cellular potency, selectivity versus PDE3 and PDE5 and rat
PK suitable for oral dosing. In particular, 20a has emerged as a
compound of significant interest.
Progress with in vivo studies towards the ultimate goal of pro-
viding a potent PDE4 inhibitor with a good therapeutic index from
this series will be reported in due course.
Acknowledgements
13. Vaultier, M.; Knouzi, N.; Carrié, R. Tetrahedron Lett. 1983, 24, 763.
14. PBMCs were prepared from heparinised human blood by centrifugation on
hystopaque at 1000g for 30 min. The cells were collected from the interface,
washed by centrifugation (1300g, 10 min) and resuspended in assay buffer
The authors thank Giancarlo Trani for synthetic contributions
whilst on placement at GSK.
(RPMI1640 containing 10% foetal calf serum, 1% L-glutamine and 1% penicillin/
streptomycin) at 5 ꢀ 105 cells/ml. One hundred microlitres of cells were added
to microtitre wells containing 0.5 or 1.0 ll of an appropriately diluted
compound solution. After the assay plates had been incubated for 1 h at
37 °C, 5% CO2, 25 ll LPS (1 ng/ml final) was added and the samples incubated at
37 °C, 5% CO2 for a further 20 h. The supernatant was then assayed for TNF-a
using a commercially available ELISA (Pharmingen).
References and notes
1. Krishna, G.; Sankaranarayanan, V.; Chitkara, R. K. Exp. Opin. Invest. Drugs 2004,
13, 255. and references therein.
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. Wang, D.; Cui, X. Int. J. COPD 2006, 1, 373.
5. Bundschuh, D. S.; Eltze, M.; Barsig, J.; Wollin, L.; Hatzelmann, A.; Beume, R. J.
Pharmacol. Exp. Ther. 2001, 297, 280.
6. For recent reviews see McKenna, J. M.; Muller, G. W. In Cyclic Nucleotide
Phosphodiesterases in Health and Disease, CRC Press LLC: Boca Raton, Florida,
15. PDE4B2B protein (152–503) was derived from PDE4B2B (152–528) by the
action of endogenous protease activity during extraction and purification
procedures from insect cells. Purification was performed essentially as
described in Rocque et al. (Protein Expr. Purif. 1997, 9, 191) but with some
modifications. During cell breakage and extraction of Sf9 insect cells
expressing human PDE4B2B (152–528) protease inhibitors were not included
to promote cleavage. After clarification by centrifugation and filtration PDE4B2

J. N. Hamblin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4237–4241
4241
protein was purified on Q-Sepharose FF and Cibacron Blue Sepharose FF as
previously described. The eluate was applied to a 10 ml Q-Sepharose HP
column and eluted with a NaCL gradient to separate cleaved from uncleaved
structure was obtained from a crystal soaked with 1 mM inhibitor (compound
20a) for approximately 3 days in well solution containing 18% PEG 3000. X-ray
diffraction data were collected at 100 K at wavelength 0.933 Å using beamline
ID14.4 with an ADSC Quantum 4 CCD detector at the European Synchrotron
Radiation Facility. The data were processed with HKL data processing package
(Otwinowski. Z., Minor, W. Methods Enzymol. 1997, 276: Macromolecular
Crystallography, part A, p. 307) and the structure solved using molecular
replacement starting from the native pde4b coordinates (molecule A of PDB
entry 1f0j) as the search model in Amore (Navaza J. Acta Cryst. 1994, A50, 157)
followed by refinement by REFMAC (Murshudov, et al. J. Acta Cryst. 1997, D53,
240). Three surface cysteines in the crystal structure have been modelled with
an associated Arsenic atom as they appear to be chemically modified (at least
partially) possibly due to cacodylate in the crystallisation buffer (Maignan et al.
JMB 1998, 282, 359). The final R-factor achieved for the complex was 19.9% (R-
free = 24.1%) and the coordinates deposited in the PDB as entry 3D3P.
protein. The cleaved protein was further purified by size exclusion on
a
Superdex 200 column (26 ꢀ 60) and finally dialysed into 10 mM Hepes, pH 7.0,
20 mM NaCL, 0.1 mM EDTA for crystallography. The site of cleavage was
confirmed by LC–MS/MS on the C-terminal peptide from a trypsin digest using
an LCQ Deca XP Plus ion trap mass spectrophotometer. Purified protein was
concentrated to 16–20 mg/ml in 10 mM Hepes, pH 7.0, 20 mM NaCl, 0.1 mM
EDTA. Diffraction quality crystals were obtained using a modified condition to
that described by Xu, et al. (Science 2000, 288, 1822). The crystals were grown
at 4 °C by vapour diffusion using an equal volume of protein to well solution
(14–18% PEG 3000, 10% glycerol, 50 mM sodium cacodylate, pH 6.5, 100 mM
sodium acetate, 1 M sodium chloride, 1% DMF and 5 mM DTT). Crystals
typically grew up to 0.5 mm in length within 1–4 weeks. The complex