Structure-based design of a potent, selective, and brain penetrating PDE2 inhibitor with demonstrated target engagement

Article abstract of DOI:10.1021/ml500262u

Structure-guided design led to the identification of the novel, potent, and selective phosphodiesterase 2 (PDE2) inhibitor 12. Compound 12 demonstrated a >210-fold selectivity versus PDE10 and PDE11 and was inactive against all other PDE family members up

Full text of DOI:10.1021/ml500262u

Letter
pubs.acs.org/acsmedchemlett
Structure-Based Design of a Potent, Selective, and Brain Penetrating
PDE2 Inhibitor with Demonstrated Target Engagement
Peter Buijnsters,*,† Meri De Angelis,‡ Xavier Langlois,† Frederik J. R. Rombouts,† Wendy Sanderson,§
Gary Tresadern,§ Alison Ritchie,∥ Andres A. Trabanco,‡ Greet VanHoof,§ Yves Van Roosbroeck,†
́
and Jose-Ignacio Andres‡
́
́
†Neuroscience Medicinal Chemistry, Janssen Research & Development, a Division of Janssen Pharmaceutica NV, Turnhoutseweg 30,
2340 Beerse, Belgium
‡Neuroscience Medicinal Chemistry, Janssen Research & Development, Janssen-Cilag S.A., C/Jarama 75, 45007 Toledo, Spain
§Discovery Sciences, Janssen Research & Development, a Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse,
Belgium
∥BioFocus, Chesterford Research Park, Saffron Walden, Essex CB10 1XL, U.K.
S
* Supporting Information
ABSTRACT: Structure-guided design led to the identification of the novel,
potent, and selective phosphodiesterase 2 (PDE2) inhibitor 12. Compound
12 demonstrated a >210-fold selectivity versus PDE10 and PDE11 and was
inactive against all other PDE family members up to 10 μM. In vivo
evaluation of 12 provided evidence that it is able to engage the target and to
increase cGMP levels in relevant brain regions. Hence, 12 is a valuable tool
compound for the better understanding of the role of PDE2 in cognitive
impairment and other central nervous system related disorders.
KEYWORDS: PDE2 inhibitor, target engagement, phosphodiesterase 2, [1,2,4]triazolo[4,3-a]quinoxalines
hosphodiesterases (PDEs) play a critical role in the
Pdegradation of the cellular levels of the secondary
messengers cAMP (3′,5′-cyclic adenosine monophosphate)
and cGMP (3′,5′-cyclic guanosine monophosphate).1 To date
11 different PDEs are known and are characterized by the cyclic
nucleotide substrate that they hydrolyze: PDE4, 7, and 8
hydrolyze cAMP, and PDE5, 6, and 9 hydrolyze cGMP, whereas
PDE1, 2, 10, and 11 hydrolyze both substrates. Both cAMP and
cGMP play a key role in intracellular signaling and in processes of
neuroplasticity such as long-term potentiation (LTP),2,3 which
could result in improved cognitive function and memory.4 Thus,
PDE inhibition is of interest in the treatment of cognitive
dysfunction.5
kg oral dose (B/P = 0.05, [P] = 72.9 ng/mL, and [B] = 3.6 ng/g),
meaning that in vivo data with this compound should be treated
with caution.11 We recently reported the identification and early
optimization of [1,2,4]triazolo[4,3-a]quinoxalines as dual
PDE2/PDE10 inhibitors, culminating in the identification of
compound 2, a potent PDE2/PDE10 inhibitor (IC50 2.8 nM for
PDE2 and 35 nM for PDE10) (Figure 1).12
PDE2 expression is highest in the brain, where it is mainly
localized in the cortex, hippocampus, and striatum.6 This
suggests that PDE2 may regulate intraneuronal cGMP and
cAMP levels in brain areas involved in emotion, perception,
concentration, learning, and memory. The main evidence was
generated with 2-[(3,4-dimethoxyphenyl)methyl]-7-[1-[(1R)-1-
hydroxy ethyl]-4-phenyl-butyl]-5-methyl-3H-imidazo[5,1-f ]-
[1,2,4] triazin-4-one, Bay 60-7550 (1),7 a highly selective
PDE2 inhibitor, but with suboptimal pharmacokinetic (PK)
properties. Several studies have postulated that 1 improved
memory acquisition and consolidation in object recognition
tasks in rats.8−11 However, a recent study demonstrated poor
brain uptake of 1 in rats 30 min after administration of a 10 mg/
Figure 1. Structure and PDE2/PDE10 inhibitory activity of Bay 60-
7550 1 and [1,2,4]triazolo[4,3-a]quinoxalines 2 and 3. All data was
generated at Janssen.
Received: June 23, 2014
Accepted: July 22, 2014
Published: July 22, 2014
© 2014 American Chemical Society
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the conserved glutamine (Gln859) into an alternative con-
formation compared to most PDE structures.
Consequently, space exists for a water molecule to form a
hydrogen bond bridge between the quinoxaline and the
conserved glutamine. In addition, a methyl is in the typical
position occupied by hydrogen bonding groups of substrate
cAMP or cGMP. The triazolo group of the ligand forms
hydrogen bonds to two different PDE-conserved water
molecules, which in turn H-bond to enzyme amino acids
including Tyr655 and Gln812. The N-benzylamide is located at
the site entrance and hugs the roof of the pocket interacting with
lipophilic amino acids Leu770 and Leu774. The planarity of the
amide with the tricyclic scaffold is broken (carbonyl oxygen is 36°
out of plane) to avoid a clash between the amide NH and
benzylic carbon atom with Met847. Another ligand solvent H-
bond interaction between the carbonyl oxygen to a highly
conserved water molecule likely compensates for the internal
ligand strain. This suboptimal conformation was therefore a
candidate for optimization.The phenyl substituent on the triazole
also interacts with Leu770. It was noticed that the water molecule
interacting with the amide carbonyl could be targeted from this
aromatic ring. Changing the phenyl to a 3-pyridyl would permit
the sp2 nitrogen to form a hydrogen bond to this water molecule.
In summary, structural analysis of 3 provided rationale for
structural modifications at both substituent positions on the
tricycle.
Docking of 1 into the apo-PDE2 structure (1Z1L) available at
the time provided a structural understanding of the high
selectivity (PDE10/PDE2 = 1000). Subsequently the exper-
imental structure of Zhu et al.15 confirmed that the 3-
phenylpropyl substituent of the asymmetric center accesses an
open pocket above the active site. This uncommon interaction
for PDEs is the source of the high selectivity for 1. In other PDE
structures, such as PDE10 2O8H,16 this channel is blocked by the
closer proximity of Leu625 and Leu665 (Leu770 and Leu809 in
PDE2).
To achieve PDE2 selectivity, a small number of molecules
were designed. First, replacement of the benzylamide in 3 with
N-morpholinylmethyl was previously shown to be an optimal
group at this position.12 Second, an ortho-halogen on the aryl
substituent of the triazole favors an orthogonal orientation with
respect to the tricycle and reduces rotational freedom providing
energetic gains for binding (Figure 3). From this twisted phenyl/
Figure 2. X-ray crystallographic binding mode of 3 (cyan) with PDE2A
viewed from the entrance to the active site (A) and top down (B); pdb
accession code 4D09. Important amino acids are highlighted in green,
and catalytic Zn2+ and Mg2+ ions are shown, along with active site water
molecules (red spheres).
In this letter we report the crystal structure of 3, as well as the
structure-guided optimization of 2 and 3 toward a suitable tool
compound for further validation of PDE2 as a target for
treatment of cognitive disorders. The goal was to identify a probe
molecule that should (1) be 100-fold selective for PDE2 versus
all other PDE family members; (2) have reasonable brain
exposure; and (3) have good physicochemical properties.
At the outset of our work only two PDE2 crystal structures
were available in the public domain.13,14 These structures showed
PDE2 folds similar to other PDEs with shared binding site
features: a conserved glutamine enables binding to the
nucleobase, the sulfate extends across the site and interacts
with the catalytic metals. It was recognized that structure-based
optimization would benefit greatly from a crystallographic
structure of our tricyclic hit series. The structure of 3 bound to
the PDE2A catalytic domain (amino acids 579−921) was
Figure 3. Design principles for novel compounds from compound 3.
successfully determined and refined to 2.5 Å resolution (Rwork
=
aryl group the selectivity pocket is accessible by a suitable
substituent on the same ring. Third, replacing phenyl with a 3-
pyridyl directs the aromatic nitrogen to interact with the
conserved water molecule, helping the aromatic ring adopt the
preferred twisted orientation without an ortho substituent. In
21.8% and Rfree = 29.8%). Binding of 3 sits in the traditional
hydrophobic clamp between Phe830 and Phe862. The binding
mode is unique as the ligand interacts with multiple water
molecules within the active site. Interestingly, the ligand forces
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turn, a lipophilic moiety in the 5 position of the pyridyl accesses
the lipophilic pocket and enhances selectivity versus PDE10.
In vitro hPDE2 and rPDE10 inhibition for target com-
pounds12,17 along with the PDE10/PDE2 selectivity ratio are
shown in Table 1. In general the o-chlorophenyl substituted
Table 1. In Vitro hPDE2 and rPDE10 Inhibitory Activity of
Compounds 2 and 4−13 and Their PDE10/PDE2 ratio
b
IC50 (nM)
compd
R
hPDE2
rPDE10
PDE10/PDE2
a
2
N/A
Me
2.8
35
13
4
10.4
6.1
106
10
5
Et
266
44
6
n-Pr
i-Pr
n-Bu
Me
6.5
322
50
7
4.8
1862
1622
271
388
211
22
8
7.7
9
12.1
30.5
404
10.1
123
10
11
12
13
n-Pr
i-Pr
n-Bu
6131
>10000
4823
7762
201
>25
482
63
MeOCH2CH2
a
b
Not available. IC50s were calculated from at least three independent
experiments. See Supporting Information for details.
compounds 4−8 had similar PDE2 potency as examples 2 and 3,
and their selectivity versus PDE10 improved as the lipophilic
character of the aliphatic chain increases (8 n-Bu > 6 n-Pr > 5 Et >
4 Me). With regard to PDE10/PDE2 selectivity, the linear n-
butyl chain (8) seemed slightly worse compared with the
branched isopropyl moiety (7). The PDE2 affinity for pyridyl
derivatives such as 9, 10, and 12 was similar to their
corresponding o-chloro analogues 4, 6, and 8, and selectivity
improved again with increasing chain length in the alkoxy
substituent. Thus, compound 12 containing an n-butyloxy
substituent possesses remarkable 480-fold selectivity for PDE2
versus PDE10. Introduction of polar groups in the aliphatic chain
or branching was detrimental for potency and selectivity as can
be seen for compounds 13 and 11.
Generally, there was a trend for the pyridyl derivatives being
less potent than the o-chloro substituted molecules. This may
arise from the rotational freedom of the pyridyl derivatives versus
the o-chlorophenyl substituted tricycles. One exception was
compound 12, which maintained good PDE2 activity of 10.1 nM
and 480-fold selectivity versus PDE10. The structure of
compound 12 in complex with PDE2 was determined and
refined to 1.9 Å and confirmed our design hypothesis (Figure 4;
Figure 4. X-ray crystallographic binding mode of 12 (cyan) with PDE2A
viewed from the entrance to the active site (A) and top down (B); pdb
accession code 4D08. Annotation and coloring is the same as Figure2
butyloxy-group to access the lipophilic pocket. Compared to the
structure of 3, 12 displaced a water molecule from this pocket,
which is formed by hydrophobic residues such as Leu770,
Leu809, and Ile866, explaining the preference for lipophilic alkyl
chains. The conformational locking of the pyridyl rotation via the
water mediated H-bond may only be successful with optimal 5-
position substitution as shown by the greater variability in the
PDE2 activity of the pyridyl analogues compared to the o-
chlorophenyl examples. The morpholine group in 12 goes to the
bottom of the active site, and its flexibility is represented in subtly
different conformations in the different crystallographic subunits.
In one of the four subunits the morpholine oxygen is able to form
a hydrogen bond with Asn704. The triazole forms the same
hydrogen bond interactions to two water molecules as seen in 3.
Conversely, in the X-ray structure of PDE2 with molecule 3
(Figure 2), the selectivity pocket is closed. This highlights the
value of using both the X-ray structure of 3 to confirm the
[1,2,4]triazolo[4,3-a]quinoxaline scaffold binding mode along
with the docking of 1 into apo-PDE2 to reveal the selectivity
pocket. Along with the recent work of Zhu et al.15 we provide
here additional evidence showing selective PDE inhibitors
accessing this unusual ligand-induced hydrophobic pocket.
R
work = 20.5% and Rfree = 24.7%).
Molecule 12 sits in the usual hydrophobic clamp between
Phe830 and Phe862 and the conserved glutamine and adopts a
conventional conformation interacting closely to the scaffold
forming a hydrogen bond to the quinoxaline nitrogen at a 2.2 Å
distance. The hydrogen bonds between the nitrogen atom of the
pyridyl moiety and the water in the bottom of the active site helps
the pyridyl adopt the rotational conformation permitting the 5-
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Compound 12 was tested for inhibition against other PDE
enzymes and in vitro ADME assays to evaluate its potential as a
candidate for in vivo studies. Compound 12 demonstrated >210-
fold selectivity versus PDE10 and PDE11, and it was inactive
against other PDE family members (Table 2).
Table 5. Brain Exposure Profile of 12 after s.c.
Administration
a
a
in vivo plasma kinetics in rat 2.5 mg/kg i.v. mean ( SD)
t1/2 (h)
0.31( 0.02)
7.8 ( 1.3)
2.7 ( 0.2)
305 ( 54)
Cl (L/h/kg)
V
dss (L/kg)
Table 2. PDE Profile of Compound 12
AUC0‑inf (ng·h/mL)
in vivo plasma kinetics in rat 10 mg/kg s.c. mean ( SD)
a
a
a
subtype
IC50 (nM)
subtype
IC50 (nM)
t1/2 (h)
max (ng/mL)
max (h)
AUC0‑inf (ng·h/mL)
1 ( 0.1)
hPDE1A
hPDE2A
hPDE3B
hPDE4D
hPDE5A
>10000
10
hPDE6AB
hPDE7A
hPDE9A
rPDE10A
hPDE11A
>10000
>10000
>10000
4823
C
658 ( 34)
0.67 ( 0.29)
1671 ( 90)
t
>10000
>10000
>10000
a
2138
Male Sprague−Dawley rat fed (n = 3), 10 mg/kg, 1 mg/mL in 20%
HP-β-CD, pH = 7.0.
a
IC50s were calculated from experiments each with eight concen-
trations.
concentration in brain, the unbound brain fraction (Fub) and
the unbound fraction to plasma proteins (Fup) were determined,
which amounted to 0.14 and 0.31, respectively. This then results
in a free concentration of 12 in the brain of 115 ng/g (67 nM) at
1 h postdose.
Molecule 12 had high metabolic turnover (Table 3, first two
entries), which prevents oral administration. The compound was
Table 3. In Vitro ADME and Physicochemical Properties of
Next, an ex vivo occupancy assay was used to evaluate PDE2
target engagement in rat brain slices using tritiated 12 as an
autoradioimaging agent. Coadministration of 12 and MP1017
(2.5 mg/kg, s.c.), a potent and selective PDE10 inhibitor, which
increases the signal-to-noise ratio, demonstrated that 12 occupies
PDE2 with an ED50 of 4.3 mg/kg (Figure 5), which corresponds
Compound 12
in vitro ADME and physicochemical properties
a
hLM
0
0
a
rLM
b
c
d
formulatability (mg/mL)
4, 15, 16
e
CYP450
>10 μM
f
f
AMESII
inactive, <125 μg/mL
a
Percent of compound remaining after 15 min incubation with rat or
b
c
human liver microsomes. Water pH 3.6. Ten percent β-HP-CD at
d
e
pH 3.6. Twenty percent HP-β-CD at pH 7.0. Inhibition of
cytochrome P450 (CYP) enzyme activity in the presence of test
compound at 1 μM; 1A2, 2C9, and 2D6, CYP inhibition measured
using the recombinant CYP inhibition screening assay, 10 μM test
compound; 2C19 and 3A4, CYP inhibition measured using the human
f
liver microsomal CYP inhibition assay, IC50 determination. AMESII:
in vitro colorimetric mutagenicity assay using an adaptation of the
AMESII Mutagenicity Assay kit from Xenometric.
Figure 5. In vivo PDE2 receptor occupancy against dosage of 12. Male
Wistar rats (n = 3 per dose). Occupancy was calculated as the inhibition
of specific [3H]-12 binding in drug-treated animals relative to vehicle-
treated animals.
inactive in a bacterial mutagenicity assay (AMESII). The poor in
vitro microsomal stability of 12 translated to rapid clearance from
rat plasma after i.v. injection (t1/2 = 0.31 h, Cl >100% of rat
hepatic blood flow, Table 5).
Nevertheless, a concentration of 658 34 ng/mL in plasma
was reached after s.c. administration of a 10 mg/kg dose of 12
(Table 5). Compound 12 displayed brain penetration with a B/P
ratio of approximately 1 over several hours (Table 4).
Furthermore, even after 4 h, promising brain levels could be
measured. In order to have an estimation of the free
to an EC50 of 21 nM. The increase of the signal-to-noise ratio can
be explained by the fact that PDE10 inhibition increases
intracellular cGMP, which is known to activate PDE2 through
its GAF domain. To confirm target engagement and central
functional effect, the levels of cyclic nucleotides versus baseline
concentrations were determined in rat striatum and hippo-
campus, regions involved in learning and memory. One hour
after dosing of 12 at 10 mg/kg s.c., cGMP levels increased in rat
striatum with 251% (range 186%−302%) compared to vehicle-
dosed animals over 5 independent experiments (n = 4−5 each).
The cAMP levels did not consistently increase (mean change
109%, range 82%−139%). The same profile was observed in
hippocampus with a significant increase in cGMP levels (298%)
and unchanged cAMP (91%) levels (one experiment, n = 5).
These data are in agreement with prior evidence that PDE2
inhibition in brain mainly influences cGMP levels rather than
cAMP.10,18
a
Table 4. Pharmacokinetic Parameters for Compound 12
time (h)
plasma (ng/mL) SD
brain (ng/g) SD
B/P
0.5
1
663 34
730 49
680 50
259 138
78 63
932 105
824 40
777 124
269 117
119 40
1.4
1.1
1.1
1.1
0.93
2
4
7
b
b
c
24
B.Q.L.
B.Q.L.
n.d.
a
Male Sprague−Dawley rat fed (n = 3) 10 mg/kg s.c., 1 mg/mL in
b
c
20% HP-β-CD, pH = 7.0. B.Q.L. = below quantification level. n.d. =
not determined.
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In summary, structure-based design optimized a series of
[1,2,4]triazolo[4,3-a]quinoxalines for their activity against PDE2
and selectivity versus PDE10. Crystallographic structures along
with docking of known selective molecules provided rationale for
synthesis. The most selective example, 12, displayed 10.1 nM
inhibition against PDE2, >210-fold selectivity versus PDE10 and
PDE11, and was inactive against other PDEs. X-ray crystallog-
raphy confirmed our design hypotheses that 12 accesses an
unusual ligand-induced lipophilic pocket. Molecule 12 displayed
an acceptable in vitro ADME and physicochemical profile and
good brain exposure. More importantly, 12 showed target
engagement by occupying PDE2 and increasing cyclic nucleotide
levels in relevant rat brain regions. Molecule 12 thus represents a
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ASSOCIATED CONTENT
* Supporting Information
■
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phosphodiesterase inhibitors improve performance on the ED/ID
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2 or type 10 reverses object memory deficits induced by scopolamine or
MK-801. J. Behav. Brain Res. 2013, 236, 16−22.
Docking protocols for Bay 60-7550 (1), protocols of in vitro
hPDE2 inhibitory activity of final compounds 4−13, in vitro PDE
profile of 12, in vivo PDE2 occupancy method, in vivo cAMP and
cGMP measurements in striatum and hippocampus, brain tissue
binding, plasma tissue binding, synthesis procedures, and
characterization. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(P.B.) E-mail: pbuijnst@its.jnj.com. Tel: +32(0)14607439.
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Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Lieve Heylen kindly provided support with the retrieval of all in
vitro and in vivo data. We also thank Ilse Lenaerts for performing
the occupancy experiments. We thank the ADME-Toxicology
department for performing all the ADME experiments.
ABBREVIATIONS
■
AUC0‑inf, area under the curve until infinite time; Cl, clearance;
CYP450, cytochrome P450; ED50, dose giving 50% effect; HP-β-
CD, (2-hydroxypropyl)-beta-cyclodextrin; hLM, human liver
microsomes; IC50, concentration giving 50% inhibition; i.v.,
intravenous; pdb, Protein Databank; PK, pharmacokinetic; p.o.,
per os; s.c., subcutaneous; SD, standard deviation; rLM, rat liver
microsomes; t1/2, half-life; tmax, time at maximum concentration;
Vdss, steady-state volume of distribution
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