A new chemical tool for exploring the physiological function of the PDE2 isozyme

Article abstract of DOI:10.1016/j.bmcl.2005.10.005

Oxindole (2) is a potent and selective PDE2 inhibitor with a favorable ADME, physiochemical and pharmacokinetic profile to allow for use as a chemical tool in elucidating the physiological role of PDE2.

Full text of DOI:10.1016/j.bmcl.2005.10.005

Bioorganic & Medicinal Chemistry Letters 16 (2006) 307–310
A new chemical tool for exploring the physiological
function of the PDE2 isozyme
Robert J. Chambers,a,* Kristin Abrams,a Norman Y. Garceau,a Ajith V. Kamath,a
Christopher M. Manley,c Susan C. Lilley,a Douglas A. Otte,a Dennis O. Scott,b
Alissa L. Sheils,a David A. Tess,b A. Samuel Vellekoop,c Yan Zhanga and Kelvin T. Lama
aResearch Technology Center, Pfizer, Inc., 620 Memorial Drive, Cambridge, MA 02139, USA
bGroton Laboratories, Pfizer, Inc., Eastern Point Road, Groton, CT 06340, USA
cAlbany Molecular Research, Inc., 21 Corporate Circle, Albany, NY 12212, USA
Received 2 September 2005; revised 30 September 2005; accepted 3 October 2005
Available online 3 November 2005
Abstract—Oxindole (2) is a potent and selective PDE2 inhibitor with a favorable ADME, physiochemical and pharmacokinetic
profile to allow for use as a chemical tool in elucidating the physiological role of PDE2.
ꢀ 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 II (PDE2) possesses a low affinity cata-
lytic domain and an allosteric domain specific for
cGMP. The low affinity catalytic site can hydrolyze both
cAMP and cGMP with a lower apparent KM for cGMP
over cAMP. However, when cGMP binds to the alloste-
ric site, the catalytic site undergoes a conformational
change showing high affinity for cAMP. PDE2 is ex-
pressed throughout the body and therefore has a broad
array of functions and potential therapeutic utility.1 It
has been shown that EHNA (erythro-9-(2-hydroxy-3-
nonyl)adenine) (1) (Fig. 1), a potent adenosine deami-
nase inhibitor (Ki = 10À9 M),2 selectively inhibits
PDE2.3 However, the use of EHNA (1) as a chemical
tool in determining the pharmacological role of PDE2
is limited due to low PDE2 potency and high potency
in inhibiting adenosine deaminase. A recent advance-
ment in the development of PDE2 inhibitors is marked
by the discovery that analogs of phosphatidylinositol
3-kinase (PI3K) inhibitor LY294002 inhibit PDE2 with
potency approaching that of EHNA (1).4 Another re-
cent advancement in the area is the report that Bay
60-7550, a potent and selective PDE2 inhibitor structur-
N
NH2
N
N
O
N
S
N
N
N
NH
O
O
CH3
OH
H3C
CH3
2
EHNA 1
Figure 1. PDE2 inhibitors EHNA (1) and oxindole (2).
ally derived from EHNA (1), elevates intracellular
cGMP in cultured neurons and is efficacious in animal
models of memory and cognition.5 These recent findings
prompt us to report that oxindole (2) is a potent and
selective PDE2 inhibitor with a favorable pharmacolog-
ical profile to allow the use as a chemical tool in further
defining the role of PDE2 across a broad range of
disease states.
In a comparative evaluation of potency and selectivity
across the phosphodiesterase isozyme family, oxindole
(2) was found to be a potent inhibitor of PDE2 being
over an order of magnitude more potent than EHNA
(1) and both compounds lacked significant inhibition
of other isozymes indicating a high degree of selectivity
toward PDE2 (Table 1).6,7 Also, 2 shows PDE2 potency
higher than those reported for LY294002 analogs and
PDE1 selectivity higher than that reported for Bay 60-
7550.4,5
Keywords: Phosphodiesterase; PDE2.
*
Corresponding author. Tel.: +1 860 441 5695; fax: +1 860 441 1758;
e-mail: Robert.J.Chambers@pfizer.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.005

308
R. J. Chambers et al. / Bioorg. Med. Chem. Lett. 16 (2006) 307–310
Table 1. Comparative PDE2 potency and selectivity of EHNA (1) and
oxindole (2)
Table 2. Broad ligand binding profile of oxindole (2)
Target
% binding at 10 lMa
PDE isozyme
EHNA (1)
Oxindole (2)
Adenosine A1
Adenosine A2a
Adenosine A3
Adrenergic a1
Adrenergic a2
Adrenergic b1
Adrenergic b2
Norepinephine Uptake
Angiotensin-I
16
24
35
0
PDE1 IC50, nM (n)
PDE2 IC50, nM (n)a
PDE3 IC50, nM (n)
PDE4A IC50, nM (n)
PDE4B IC50, nM (n)
PDE4C IC50, nM (n)
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)
PDE2 selectivity
>16,000 (1)
635 134 (2)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>25X
>16,000 (1)
40 18 (9)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
>16,000 (1)
11,600 (1)
290X
16
15
12
12
11
4
Angiotensin-II
Benzodiazepine
Bradykinin B1
Bradykinin B2
Dopamine D1
Dopamine D2
Dopamine D3
Dopamine D4
Dopamine Uptake
GABA
11
0
5
5
12
0
a Values are means of a number (n) experiments with standard
deviation.
0
11
3
GABA Uptake
AMPA
Kainate
8
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 54 receptors and ion channels
where no significant level of binding relevant to its
PDE2 potency was found (Table 2). In addition, oxin-
dole (2) showed no inhibition of 5-lipoxygenase (5-LO)
or cyclooxygenase (COX-1), two enzymes which other
oxindole derivatives inhibit and derive anti-inflammato-
ry activity (Table 3).8,9 The chemical structure of 2 dis-
plays proximal hydrogen bond donor and acceptor pairs
which is associated with kinase inhibition which in turn
would effect cell signaling.10 Against a panel of thirty ki-
nases, 2 showed no significant inhibition relative to the
level of PDE2 inhibition (Table 3).11
20
0
NMDA
7
Histamine H1
Histamine H2
Histamine H3
MCR4
7
0
0
12
9
Muscarinic M1
Muscarinic M2
Muscarinic M3
Muscarinic M4
Choline Uptake
Neurokinin K1
Nicotinic (neuronal)
Nicotinic (muscle)
Opiate d
0
0
1
0
0
7
0
3
Opiate j
0
Opiate l
PAF
3
In comparing the solubility and ADME properties of
EHNA (1) and oxindole (2), both compounds showed
an appreciable level of solubility and cell permeability
however, oxindole (2) shows over a twofold increase in
metabolic stability over EHNA (1) as measured by
half-life in rat liver microsomes (Table 4). 12 In parental
Madin–Darby canine kidney (MDCK) and transfectant
multidrug resistant (MDR) overexpressing human
p-glycoprotein (P-gp) cell lines, oxindole (2) showed a
moderate rate of absorption with no apparent efflux.13
21
23
15
0
Serotonin 5-HT1A
Serotonin 5-HT2A
Serotonin 5-HT2C
Serotonin 5-HT3
Serotonin 5-HT4
Serotonin 5-HT7
Serotonin Uptake
Glucocorticord
Thyroid hormone
Vasopressin V1
Vasopressin V2
Ca2+ channel D (dihydro pyridine)
Ca2+ channel D (dilitiazem)
Ca2+ channel L (verapamil)
Ca2+ channel N
0
0
0
0
0
0
5
13
0
Having found a favorable solubility and ADME profile
for oxindole (2), the in vivo pharmacokinetic profile was
determined in rats, which showed a moderate rate of
clearance and a low volume of distribution leading to
a moderate half-life and level of oral bioavailability
(Table 5).14 Furthermore, unbound plasma concentra-
tions of oxindole (2) were achieved which exceeded its
IC50 value for over 2 h.
0
0
0
a Values are from a single experiment with duplicate determinations.
lecular Friedel–Crafts alkylation occurs regioselectively
to yield 5.16 Acylation of 5 with chloroacetyl chloride
followed by Fries rearrangement of ester 6 affords ke-
tone 7.17 Reduction of 7 and subsequent acid-catalyzed
dehydration of alcohol 8 gives furan 9. Acylation of 9
with diethylcarbonate followed by aminolysis of ester
10 with 2-aminothiadiazole affords 2 as a crystalline
solid.
The synthesis of oxindole 2 proceeds via an eight-step
linear sequence in a 33% overall yield starting from Me-
tol 3, an inexpensive and readily available starting mate-
rial (Scheme 1). Acylation of 3 with chloroacetyl
chloride occurs chemoselectively at the aniline nitrogen
to give amide 4.15 Cyclization of 4 by way of a intramo-

R. J. Chambers et al. / Bioorg. Med. Chem. Lett. 16 (2006) 307–310
309
Table 3. Off target inhibition profile of oxindole (2)
HO
Cl
O
HO
HO
b
a
O
Target
% inhibition at 10 lMa
N
N
NH
CH3
CH3
CH3
5-Lipoxygenase
Cyclooxygenase
MKK1
MAPK2
JNK1a1
SAPK2a
SAPK2b
SAPK3
SAPK4
RSK1
35
0
4
3
5
c
0
O
O
O
e
d
Cl
O
O
12
0
O
O
N
N
N
CH3
CH3
HO
CH3
O
8
7
6
0
N
5
f
N
7
O
N
O
S
O
O
13
1
NH
O
O
O
O
g
h
O
MAPKAP-K2
MSK1
PRAK
8
N
N
6
CH3
CH3
CH3
2
9
10
28
16
13
0
PKA
PKCa
Scheme 1. Reagents and conditions: (a) ClCOCH2Cl, TEA, DMAP,
DMF, 0 ꢁC, 83%; (b) AlCl3, 180 ꢁC, 89%; (c) ClCOCH2Cl, Py CH2Cl2,
0 ꢁC; 94% (d) AlCl3, 180 ꢁC, 76%; (e) NaBH4, MeOH, 0 ꢁC, 92%; (f)
TFA, MeCN, 20 ꢁC, 88%; (g) (EtO)2CO, Na, EtOH, reflux, 97%; (h) 2-
PDK1
PKBa
SGK
14
0
˚
amino-1,3,4-thiadiazole, 4 A Sieves, PhH, reflux, 80%.
p70S6K
GSK3b
ROCK-II
AMPK
CHK1
23
16
20
8
poxygenase (5-LO) or cyclooxygenase (COX-1), two en-
zymes which other oxindole derivatives inhibit and
derive anti-inflammatory activity. In regard to mediat-
ing off target cell signaling pathways, oxindole (2)
showed no significant inhibition against a panel of 30 ki-
nases. Thus, oxindole (2) is positioned to be a useful
chemical tool in defining the role of PDE2 in a broad
range of disease states.
0
CK2
PHK
32
0
LCK
CSK
5
5
CDK2
CK1
2
10
53
7
DYRK1A
NEK6
NEK2
0
References and notes
a Values are from a single experiment with duplicate determinations.
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EHNA
(1)
Oxindole
(2)
Rat liver microsome t1/2 (min)
31
34
83
5
CACO-2 apical Papp (·10À6 cm/s)
CACO-2 basolateral Papp (·10À6 cm/s)
MDCK apical Papp (·10À6 cm/s)
MDR basolateral Papp (·10À6 cm/s)
Turbidimetric solubility (pH 7, lg/ml)
18
32
3.0
4.6
55
NT
NT
>65
6. For preparations of PDE1, PDE2, PDE3, PDE4A,
PDE4B, PDE4C, PDE4D, PDE5, PDE8A, PDE8B,
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NT, not tested.
Table 5. Rat pharmacokinetics of oxindole (2)
Clearance Cl (ml/min/kg)
Volume of distribution Vdss (L/kg)
Half life t1/2 (h)
8.8
0.1
1.6
0.8
Tmax (h)
Cmax (ng/ml)
Oral bioavailability % F
8303
41
Oxindole (2) is a potent and selective PDE2 inhibitor
that possesses a suitable solubility and in vitro ADME
profile leading to a moderate level of in vivo oral
bioavailability and half-life in rats. Against a panel of
54 receptors and ion channels, oxindole (2) shows no
significant interaction nor is effective in inhibiting 5-li-
7. EHNA (1) and oxindole (2) were evaluated for phos-
phodiesterase inhibition activity using a scintillation
proximity assay (SPA) in 96-well plates. The PDE SPA

310
R. J. Chambers et al. / Bioorg. Med. Chem. Lett. 16 (2006) 307–310
yttrium silicate beads (Amersham Biosciencesꢂ) bind
Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P.
Biochem. J. 2000, 351, 95.
preferentially to the linear nucleotide compared to the
cyclic nucleotide. In the case of PDE2, 3H-cGMP and
unlabeled cGMP are added to the reaction and when the
product, H-GMP, is in close proximity to the beads, the
scintillant within the bead is excited, which is detected
12. For rat liver microsome assay see: (a) Obach, R. S.; Baxter,
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venously, 1 mg/kg in EtOH:PEG 400:H2O (1:4:5) and
orally, 30 mg/kg in 0.5% methylcellulose and plasma
samples were drawn at 0.083, 0.25, 0.5, 1, 2, 4, 6, and 8 h
post dose for analysis. Two animals were used for each
dosing route and mean plasma concentrations were used in
subsequent calculations.
3
using
a Packard scintillation counter. The enzyme
concentration used is in the linear range and the KM of
the enzyme was determined (15 lM). The final substrate
concentration is <1/3 of KM (1 lM) so that IC50 values
would approximate the Ki values (a) Bardelle, C.; Smales,
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2004, 12, 2233.
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cyclooxygenase (COX-1) inhibition activities.
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Cohen, University of Dundee, Dundee, Scotland, UK