A new chemical tool for exploring the role of the PDE4D isozyme in leukocyte function

Article abstract of DOI:10.1016/j.bmcl.2005.10.026

Nicotinamide (2) is a potent and selective inhibitor of the PDE4D isozyme and as a chemical tool selectively blocks eosinophil mediator release and chemotaxis thus linking the role of PDE4D to eosinophil function.

Full text of DOI:10.1016/j.bmcl.2005.10.026

Bioorganic & Medicinal Chemistry Letters 16 (2006) 718–721
A new chemical tool for exploring the role of the PDE4D isozyme
in leukocyte function
b,
b
a
a
*
Robert J. Chambers, Kristin Abrams, Tessa A. Castleberry, John B. Cheng,
a
b
a
a
Douglas A. Fisher, Ajith V. Kamath, Anthony Marfat, David O. Nettleton,
Joann D. Pillar, Eben D. Salter, Alissa L. Sheils, John T. Shirley, Claudia R. Turner,
a
a
b
a
a
a
b
John P. Umland and Kelvin T. Lam
a
Groton Laboratories, Pfizer, Inc., Eastern Point Road, Groton, CT 06340, USA
Research Technology Center, Pfizer, Inc., 620 Memorial Drive, Cambridge, MA 02139, USA
b
Received 29 August 2005; revised 5 October 2005; accepted 6 October 2005
Available online 2 November 2005
Abstract—Nicotinamide (2) is a potent and selective inhibitor of the PDE4D isozyme and as a chemical tool selectively blocks
eosinophil mediator release and chemotaxis thus linking the role of PDE4D to eosinophil function.
ꢀ
2005 Elsevier Ltd. All rights reserved.
The phosphodiesterase (PDE) enzyme family controls
intercellular levels of secondary messenger cAMP or
cGMP through regulation of their hydrolysis. Phospho-
diesterase Type IV (PDE4) regulates cAMP through its
O
O
CH3
H CO
3
N
H
S
O
N
O
0
hydrolysis to 5 -AMP which is blocked by Rolipram
1
N
H
(
1). The PDE4 gene encodes four distinct isoenzyme
subtypes designated PDE4A, PDE4B, PDE4C, and
PDE4D in which their catalytic sites are conserved but
differ in their regulatory domain. Insights into the func-
Rolipram (1)
2
F
2
Figure 1. Chemical structures of Rolipram (1) and nicotinamide (2).
tion of these subtypes have been obtained largely
through expression profiling and deficient mice, yet
much about the function of these subtypes is unknown
or not well understood. The PDE4D subtype is found
be ineffective as a chemical tool in defining the physio-
logical function of these isozymes (Table 1). In recent
years, a series of naphthyridine PDE4D inhibitors, such
as NVP-ABE171, have been described which show effi-
cacy in blocking leukocyte oxidative burst and mediator
release in vitro and antigen induced influx and activa-
2
in most human tissues and organs but is highly ex-
3
pressed in eosinophils, a key leukocyte believed to be
4
involved in the progression of respiratory diseases. In
addition, studies in PDE4D deficient mice indicate that
PDE4D plays a critical role in airway tone and hyper-re-
activity, suggesting a possible further link between
7
tion in vivo. We have identified nicotinamide derivative
as a potent and selective inhibitor of the PDE4D iso-
2
5
PDE4D and respiratory diseases. Archetypical PDE4
inhibitor Rolipram (1) has been successfully used as a
zyme and as such can be used as a chemical tool in fur-
ther exploring the role of PDE4D in leukocyte function.
chemical tool in linking PDE4 with an array of disease
6
states (Fig. 1). However, Rolipram (1) shows no signif-
icant selectivity in inhibition between the PDE4A,
PDE4B, and PDE4D isozymes, and therefore would
In evaluation of potency and selectivity across the PDE
family, both Rolipram (1) and (2) show selectivity in
inhibiting PDE4 over other isozymes (Table 1). Howev-
8
er, 2 showed increased potency and selectivity toward
PDE4D compared to Rolipram (1), which showed no
significant differentiation between the PDE4A, PDE4B,
Keywords: Phosphodiesterase; PDE4D; Eosinophil.
*
Corresponding author. Tel.: +1 860 441 5695; fax: +1 860 441
758; e-mail: Robert.J.Chambers@pfizer.com
9
1
and PDE4D isozymes.
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.026

R. J. Chambers et al. / Bioorg. Med. Chem. Lett. 16 (2006) 718–721
719
Table 1. Potency and selectivity of Rolipram (1) and nicotinamide (2)
inhibiting PDE4B, the predominant isozyme in mono-
cytes which has been shown to regulate TNF-a
production.
PDE isozyme
Rolipram (1)
Nicotinamide (2)
1
6
PDE2 IC50, nM (n)
PDE3 IC50, nM (n)
PDE4A IC50, nM (n)
>16,000 (1)
>16,000 (1)
11 (8)
>16,000 (1)
>16,000 (1)
107 (6)
a
Leukocyte chemotaxis is a key step in the inflammatory
response and PDE4 inhibitors are known to inhibit leu-
17
kocyte chemotaxis by elevating intracellular cAMP.
a
PDE4B IC50, nM (n)
PDE4C IC50, nM (n)
34 (8)
3410 (2)
33 (8)
185 (6)
1378 (2)
4 (6)
a
a
PDE4D IC50, nM (n)
PDE5 IC50, nM (n)
PDE8A IC50, nM (n)
PDE8B IC50, nM (n)
PDE9 IC50, nM (n)
PDE10 IC50, nM (n)
PDE11 IC50, nM (n)
PDE4D selectivity
With this in mind and considering that PDE4D may
be linked to eosinophil function, we wished to examine
the effect of 2 on in vitro human eosinophil chemotax-
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
<1X
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
27X
18
is. Against chemoattractant mediators leukotriene B4
(LTB ), C5a, platelet-activating factor (PAF), and eo-
4
taxin, 2 proved to be several orders of magnitude more
potent than Rolipram (1) in blocking eosinophil chemo-
taxis (Table 3). Having demonstrated that 2 was effective
in inhibiting eosinophil chemotaxis in vitro, we next
wished to examine the effect of 2 on leukocyte chemo-
taxis in vivo. In regard to in vivo evaluation of nonselec-
tive PDE4 inhibitors, there has been increasing evidence
suggesting that the occurrence of emesis in nonselective
PDE4 inhibitors like Rolipram (1) may be due to inhibi-
a
Values are means of a number (n) of experiments.
In considering that 2 might interact with other pharma-
cological targets that in turn could then compromise
interpretation of results from further in vitro and in vivo
functional profiling, 2 was assayed for interaction
against a collection of 52 receptors and ion channels
where no significant level of binding relative to its
1
9
tion of the PDE4D subtype. It was found that 2 pos-
sesses suitable physiochemical and ADME properties
for aerosol delivery directly into the airways, which
would allow for evaluation of effects on leukocyte che-
motaxis in vivo while minimizing potential emetic side
1
0
PDE4D potency was found.
Having established selectivity in 2 for PDE4D over
other potential pharmacological targets, we next wanted
to profile it for functional activity. Inhibition of PDE4
elevates intracellular levels of cAMP by inhibiting
2
0
effects due to systemic exposure. Rolipram (1) has
been shown to be efficacious in reducing the influx of
neutrophils and eosinophils in bronchoalveolar lavage
(BAL) fluid from cynomolgous monkeys exposed to
0
hydrolysis to 5 -AMP and PDE4D has been found to
2
2
1
,11
Ascaris suum. In this model, aerosol delivery of 2 sig-
nificantly reduced the infiltration of eosinophils (% inhi-
bition = 65 ± 14, p < 0.05) but had no significant effect
be expressed in U937 cells.
In these cells, 2 was found
to be 50-fold more potent in elevating intracellular
cAMP compared to Rolipram (1) (Table 2). The eleva-
tion of intracellular cAMP has been shown to block leu-
2
2
on neutrophil influx. The selectivity observed in reduc-
ing in vivo eosinophil over neutrophil influx is likely due
to 2 selectively inhibiting PDE4D, the predominant iso-
zyme in eosinophils, and consequently being less potent
than Rolipram (1) in inhibiting PDE4B, a predominant
1
2
kocyte mediator release. With this in mind and
considering that PDE4D may be linked to eosinophil
function, we wished to evaluate 2 against several cellular
functional endpoints. Degranulation and respiratory
burst of eosinophils result in the release of the intercel-
lular mediators eosinophil derived neurotoxin (EDN)
2
3
isozyme in neutrophils.
The synthesis of 2 is carried out using a linear three-step
sequence and a resolution by chiral HPLC (Scheme 1).
and cysteinyl leukotrienes (LTE ), which are associated
4
with airway inflammation and constriction responses,
3
1
respectively. In blocking the release of EDN and
LTE in human whole blood, 2 proved to be approxi-
4
1
4
mately 20-fold more potent than Rolipram (1). Tumor
necrosis factor (TNF-a) is another inflammatory media-
tor that is regulated by intercellular cAMP and is pro-
Table 3. Comparative effect of Rolipram (1) and nicotinamide (2) on
in vitro human eosinophil chemotaxis
Chemoattractant
Rolipram (1)
Nicotinamide (2)
1
5
duced primarily by monocytes. Interestingly, 2 had
little effect on blocking the release of TNF-a in human
whole blood relative to those on EDN and LTE4.
This effect is likely due to 2 selectively inhibiting
PDE4D, the predominant isozyme in eosinophils, and
consequently being less potent than Rolipram (1) in
a
LTB IC50, nM (n)
4
6500 (2)
17,000 (2)
3000 (2)
<1 (2)
1 (2)
1 (2)
3 (2)
a
C5a IC50, nM (n)
PAF IC50, nM (n)
a
a
Eotaxin IC50, nM (n)
13,000 (2)
a
Values are means of a number (n) of experiments.
Table 2. Comparative in vitro functional activity of Rolipram (1) and nicotinamide (2)
Functional assay
Rolipram (1)
Nicotinamide (2)
a
cAMP elevation U937 cells EC50, nM (n)
a
EDN human whole blood IC30, nM (n)
1100 (16)
283 (5)
41 (3)
22 (2)
16 (4)
2 (4)
a
4
LTE human whole blood IC50, nM (n)
a
TNF-a human whole blood IC50, nM (n)
625 (2)
925 (3)
a
Values are means of a number (n) of experiments.

720
R. J. Chambers et al. / Bioorg. Med. Chem. Lett. 16 (2006) 718–721
CO CH CH
CO
2
CH
2
CH
3
2. Muller, T.; Engels, P.; Fozard, J. R. Trends Pharm. Sci.
996, 17, 294.
2
2
3
a
1
(
71%)
3. Souness, J. E.; Maslen, C.; Webber, S.; Foster, M.;
Raeburn, D.; Palfreyman, M. N.; Ashton, M. J.; Karlsson,
J-A. Br. J. Pharmacol. 1995, 115, 39.
N
Cl
N
O
3
4
4
5
6
. Bousquet, J.; Chanez, P.; Lacoste, J. Y.; Barneon, G.;
Ghavanian, N.; Enander, I.; Venge, P.; Ahlstedt, S.;
Simony-Lafontaine, J.; Godard, P.; Michel, F.-B. N. Engl.
J. Med. 1990, 323, 1033.
. Hansen, G.; Jin, S.-L. C.; Umetsu, D. T.; Conti, M. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 6751; Mehats, C.; Jin, C.;
Wahlstrom, J.; Law, E.; Umetsu, D. T.; Conti, M. FASEB
J. 2003, 17, 1831.
F
b
(
83%)
O
O
CH3
CO H
2
N
CH
3
H
S
N
H N
2
N
O
. Zhu, J.; Mix, E.; Winblad, W. CNS Drug Rev. 2001, 7,
387; Dyke, H.; Montana, J. Expert Opin. Invest. Drug
2002, 11, 1.
2
6
S
5
c, d
(
25%)
7. Hersperger, R.; Bray-French, K.; Mazzoni, L.; Muller,
T. J. Med. Chem. 2000, 43, 675; Trifilieff, A.; Wyss,
D.; Walker, C.; Mazzoni, L.; Hersperger, R. J. Pharm.
Exp. Ther. 2002, 301, 241; Hersperger, R.; Dawson, J.;
Mueller, T. Bioorg. Med. Chem. Lett. 2002, 12,
F
F
Scheme 1. Reagents and conditions: (a) 4-fluorophenol, Cs
DMF, 80 ꢁC, 18 h; (b) 1 N NaOH, EtOH, reflux, 3 h; (c) (i)
ClCO CH CH(CH N-methylmorpholine, CH Cl
ꢀ10 ꢁC,
0 min; (ii) 6, ꢀ10 ꢁC to 20 ꢁC, 18 h; (d) Chiral HPLC, Chiralcel AS
column, EtOH/heptane (1:9) elutant.
2 3
CO ,
2
2
3
)
2
,
2
2
,
2
33.
2
8
. For preparations of PDE2, PDE3, PDE5, PDE8A,
PDE8B, PDE9, PDE10, and PDE11 isozymes, see: Dick-
inson, N.; Jang, E.; Haslam, R. Biochem. J. 1997, 323, 371;
Fisher, D.; Smith, J.; Pillar, J.; Denis, S.; Cheng, J.
Biochem. Biophys. Res. Commun. 1998, 246, 570; Hayashi,
M.; Matsushima, K.; Ohashi, H.; Tsunoda, H.; Murase,
S.; Kawarada, Y.; Tanaka, T. Biochem. Biophys. Res.
Commun. 1998, 250, 751; Fisher, D.; Smith, J.; Pillar, J.;
Denis, S.; Cheng, J. J. Biol. Chem. 1998, 273, 15559;
Fujishige, K.; Kotera, J.; Omori, K. Eur. J. Biochem. 1999,
Displacement of the activated 2-chloro substituent in 3
with 4-fluorophenol in the presence of cesium carbonate
in dimethylformamide at 80 ꢁC gives 4 which in turn is
saponified with aqueous sodium hydroxide in refluxing
ethanol to give carboxylic acid 5. Coupling of the
mixed isobutyl anhydride of 5 with 6 followed by reso-
lution and isolation of the dextrorotatory enantiomer
by chiral HPLC affords 2 in 99% enantiomeric purity
which is assigned the R-configuration based on litera-
2
4
266, 1118; Fawcett, L.; Baxendale, R.; Stacey, P.; McG-
routher, C.; Harrow, I.; Soderling, S.; Hetman, J.; Beavo,
J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3702; For
evaluation of compounds using a FlashPlate assay, the
2
5
ꢂ
ture analogy.
format used against these isozymes, see: Ilona, K.;
Stevens, M.; Behrens, D.; Oldenburg, K. J. Biomol.
Screen. 1999, 4, 27.
. For the preparation of PDE4A, PDE4B, PDE4C, and
PDE4D isozymes and method for evaluating compounds,
see: Cohan, V.; Showell, H.; Fisher, D.; Pazoles, C.;
Watson, J.; Turner, C.; Cheng, J. J. Pharm. Exp. Ther.
Nicotinamide (2) is a potent and selective inhibitor of
the PDE4D subtype over the PDE4A, PDE4B, and
PDE4C subtypes and shows no inhibition of other
PDE isozymes. In addition, when evaluated against a
collection of 52 receptors and ion channels, 2 shows
no significant binding. In cellular function, by selectively
inhibiting PDE4D, 2 effectively elevates intracellular
cAMP and blocks the release of eosinophil associated
9
1
996, 278, 1356.
1
0. Nicotinamide (2) was profiled by Cerep (France) and data
are reported as percent binding at 10 lM concentration as
a single experiment of duplicate determinations: adenosine
A (12), adenosine A (23), adenosine A (31), adrenergic
mediators EDN and LTE in human whole blood. How-
4
ever, 2 is ineffective in blocking the release of monocyte
associated mediator TNF-a from human whole blood
compared to Rolipram (1), presumably due to the pre-
dominant subtype in monocytes being PDE4B, which
has been shown to selectively regulate TNF-a. Nicotin-
amide (2) is effective in blocking chemokine stimulated
eosinophil chemotaxis in vitro and in vivo selectively
reduces the influx of eosinophils over neutrophils in
bronchoalveolar lavage (BAL) fluid from cynomolgous
monkeys exposed to Ascaris suum, presumably due to
PDE4D being the predominant PDE4 subtype in
eosinophils over neutrophils. These findings demon-
strate that PDE4D inhibitor 2 is an effective chemical
tool implicating PDE4D in playing a unique role in
eosinophil chemotaxis and mediator release.
1
2a
3
a
1
(0), adrenergic a
(6), norepinephine uptake (14), angiotensin-I (3), angio-
2
(0), adrenergic b
1
(12), adrenergic b
2
tensin-II (0), benzodiazepine (6), bradykinin B1 (0),
bradykinin B
dopamine D
2
(0), dopamine D
(0), dopamine D
1
(27), dopamine D
(9), dopamine Uptake
2
(0),
3
4
(
(
23), GABA (0), GABA uptake (22), AMPA (10), kainate
0), NMDA (0), histamine H1 (4), histamine H2 (0),
histamine H
(0), muscarinic M
M (12), choline uptake (60), neurokinin K (27), nicotinic
3
(0), melanocortin MCR4 (22), muscarinic
M
1
2
(1), muscarinic M (5), muscarinic
3
4
1
(neuronal, 3) nicotinic (muscle, 22) opiate d (18), opiate j
(21), opiate l (15), platelet activating factor (23), serotonin
5-HT1A (5), serotonin 5-HT
serotonin 5-HT (0), serotonin uptake (0), glucocorticord
3 4
(4), serotonin 5-HT (14),
7
(
V
(
0), thyroid hormone (8), vasopressin V
(9), calcium channel L (DHP, 25) calcium channel L
dilitiazem, 6) calcium channel L (verapamil, 0) calcium
1
(26), vasopressin
2
channel N (0).
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