Design and discovery of 6-[(3 s,4 s)-4-methyl-1-(pyrimidin-2-ylmethyl) pyrrolidin-3-yl]-1-(tetrahydro-2 h -pyran-4-yl)-1,5-dihydro-4 h -pyrazolo[3,4- d ]pyrimidin-4-one (PF-04447943), a selective brain penetrant pde9a inhibitor for the treatment of cognitive disorders

Article abstract of DOI:10.1021/jm3007799

6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl] -1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943) is a novel PDE9A inhibitor identified using parallel synthetic chemistry and structure-based drug design (SBDD) and has advanced into clinical trials. Selectivity for PDE9A over other PDE family members was achieved by targeting key residue differences between the PDE9A and PDE1C catalytic site. The physicochemical properties of the series were optimized to provide excellent in vitro and in vivo pharmacokinetics properties in multiple species including humans. It has been reported to elevate central cGMP levels in the brain and CSF of rodents. In addition, it exhibits procognitive activity in several rodent models and synaptic stabilization in an amyloid precursor protein (APP) transgenic mouse model. Recent disclosures from clinical trials confirm that it is well tolerated in humans and elevates cGMP in cerebral spinal fluid of healthy volunteers, confirming that it is a quality pharmacological tool for testing clinical hypotheses in disease states associated with impairment of cGMP signaling or cognition.

Full text of DOI:10.1021/jm3007799

Article
pubs.acs.org/jmc
Design and Discovery of 6‑[(3S,4S)‑4-Methyl-1-(pyrimidin-2-
ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro‑2H‑pyran-4-yl)-1,5-
dihydro‑4H‑pyrazolo[3,4‑d]pyrimidin-4-one (PF-04447943), a
Selective Brain Penetrant PDE9A Inhibitor for the Treatment of
Cognitive Disorders
Patrick R. Verhoest,* Kari R. Fonseca, Xinjun Hou, Caroline Proulx-LaFrance, Michael Corman,
Christopher J. Helal, Michelle M. Claffey, Jamison B. Tuttle, Karen J. Coffman, Shenpinq Liu,
Frederick Nelson, Robin J. Kleiman, Frank S. Menniti, Christopher J. Schmidt, Michelle Vanase-Frawley,
and Spiros Liras
Neuroscience Medicinal Chemistry, Pfizer World Wide Research and Development, 700 Main Street, Cambridge, Massachusetts
02139, United States
S
* Supporting Information
ABSTRACT: 6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-
(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
(PF-04447943) is a novel PDE9A inhibitor identified using parallel synthetic
chemistry and structure-based drug design (SBDD) and has advanced into
clinical trials. Selectivity for PDE9A over other PDE family members was
achieved by targeting key residue differences between the PDE9A and PDE1C
catalytic site. The physicochemical properties of the series were optimized to
provide excellent in vitro and in vivo pharmacokinetics properties in multiple
species including humans. It has been reported to elevate central cGMP levels in
the brain and CSF of rodents. In addition, it exhibits procognitive activity in
several rodent models and synaptic stabilization in an amyloid precursor protein
(APP) transgenic mouse model. Recent disclosures from clinical trials confirm
that it is well tolerated in humans and elevates cGMP in cerebral spinal fluid of
healthy volunteers, confirming that it is a quality pharmacological tool for testing
clinical hypotheses in disease states associated with impairment of cGMP signaling or cognition.
Although PDEs have proven to be druggable targets,
achieving high selectivity among the gene family can be
challenging and is desirable for adequate safety profiles, since
unwanted PDE inhibition has been reported to be associated
with side effects.6,7 PDE catalytic domains all share a
hydrophobic clamp that binds the “purine”-like core of the
cyclic nucleotides and form key hydrogen bonds to the
common glutamine. For the dual substrate PDEs, this residue
can rotate to form donor−acceptor hydrogen bonds to both
cGMP and cAMP.8
The widespread distribution of PDEs in the central nervous
system (CNS) and critical role of GPCR signaling in
neurotransmission prompted the development of a PDE family
initiative to identify potential therapeutic utility for centrally
penetrant PDE inhibitors.9 We have previously reported the
discovery of multiple series of PDE10A inhibitors10 including a
INTRODUCTION
■
Phosphodiesterase (PDE) enzymes catalyze the hydrolysis of
cGMP and cAMP, which are ubiquitously utilized second
messengers for intracellular signaling cascades invoked by a
wide variety of biological stimuli and are of particular functional
importance in the central nervous system (CNS).1 There are 21
PDE genes subclassified into 11 different families (PDE1−11).
Each family member is characterized by a unique cellular and
subcellular distribution, distinct regulatory domains, enzyme
kinetics, and substrate affinity for cGMP, cAMP, or both.2
cGMP-specific PDEs include PDEs 5, 6, and 9, while cAMP-
specific PDEs include 4, 7, and 8 and the PDEs 1, 2, 3, 10, and
11 are dual-substrate PDEs. The ability to potentiate specific
cyclic nucleotide cascades by selectively targeting specific PDEs
has led to the development of multiple drugs from this class of
enzyme inhibitors. To date, inhibitors for PDEs 3, 4, and 5 have
been approved for conditions that include congestive heart
failure, chronic obstructive pulmonary disease, erectile
dysfunction, and pulmonary hypertension.3−5
Special Issue: Alzheimer's Disease
Received: June 4, 2012
Published: July 10, 2012
© 2012 American Chemical Society
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selective PDE10A inhibitor (PF-02545920) that has been
administered to patients and completed a phase 2 trial in
schizophrenia.11 We have used the experience gained executing
these programs to explore the development of selective brain
penetrant PDE9A inhibitors.
It has been reported that cGMP elevation can cause an
increase in synaptic transmission and long-term potentiation.12
In addition it has been shown that cGMP can reverse Aβ
induced deficits in long-term potentiation in hippocampal
slices.13 PDE9A is a cGMP specific phosphodiesterase with the
highest affinity of all PDEs for cGMP with Km = 170 nM.14 The
exquisite potency for cGMP hydrolysis combined with the
widespread distribution15 of PDE9A makes it an excellent
target for globally elevating cGMP to test this hypothesis in the
treatment of cognitive disruption in diseases such as
Alzheimer’s disease.16
Figure 2. PDE9A inhibitor 1 (PDB code 3JSI) bound and structural
differences with PDE1C (pink).
RESULTS AND DISCUSSION
■
We have previously described the utilization of parallel
medicinal chemistry to enable the potential synthesis of
thousands of pyrazolopyrimidines exemplified by HTS hit 1.
Utilizing prospective physicochemical property based design
coupled with the targeting of key residue differences within the
PDEs and in particular the residue differences between PDE1C
and PDE9A, we developed a brain penetrant in vivo tool 2 (PF-
04181366) that can elevate cGMP in brain tissue (Figure 1).17
ring from a variety of commercial and custom hydrazines. This
effort identified a 4-tetrahydropyran derivative 3, which
significantly improved the human liver microsomal clearance
from 200 to 14 mL/min/kg without impacting the P-
glycoprotein (P-gp) efflux ratio (Table 1). Additionally this
came with an improvement in lipophilic binding efficiency
(LipE)19 from 6.75 to 8.5. These characteristics aligned the in
vitro ADME properties and binding efficiencies with what is
reported for CNS marketed drugs.20 By increase of the polarity,
a 14-fold improvement in selectivity over PDE1c was
established and validated our design strategy. On the basis of
the cocrystal structures, the improvement in selectivity
appeared to be driven by the pyran oxygen interacting through
a water hydrogen bonding network to tyrosine residue 424 in
PDE9A, which in PDE1C is a phenylalanine. The loss in
potency for PDE1C can be rationalized as being due to an
increase in the desolvation penalty to accommodate the more
polar tetrahydopyran into the more lipophilic environment in
PDE1C formed by the Tyr to Phe mutations (Figure 2).
Incorporating the tetrahydropyran group with the benzyl-
pyrrolidine motif in compound 2 was paramount to identifying
compounds with further improvements in PDE1C selectivity.
Condensing 4-hydrazinetetrahydropyran with the 2-
(ethoxymethylene)malononitrile with base provided the
cyanopyrazole (Scheme 1). Oxidation of the cyano to the
carboxamide was achieved with aqueous hydrogen peroxide.
Pyrrolidine esters were then coupled with the pyrazoloaminoa-
mide to provide the benzyl analogues. The benzylpyrrolidine is
an enabled monomer that can rapidly be deprotected and
functionalized via reductive amination or alkylation to afford
the fully functionalized products.
Figure 1. Development of in vivo active lead 2.
Although compound 2 had high potency against PDE9A (1.8
nM) and selectivity over PDE1C (50-fold), it possessed high
human liver microsomal clearance (149 mL/min/kg) resulting
in a projected large dose and a short half-life.18 High selectivity
had been achieved with 2 over all the PDEs except PDE1C.
PDE1C is expressed in cardiac tissue, so further selectivity was
desired to advance a molecule into clinical trials. Therefore, our
goal was to move beyond the development of a centrally
penetrant tool to identify a more selective PDE9A inhibitor
with improvements in pharmacokinetics and safety that could
be taken into clinical trials. Our strategy to improve the
pharmacokinetics of our lead molecule 2 again utilized parallel
chemistry to target rapid replacement of the lipophilic
cyclopentyl group with more polar functionality. On the basis
of the X-ray crystal structure of 1, which has been previously
cocrystallized in the PDE9A catalytic domain (Figure 2), we
hypothesized that an increase in polarity would also improve
our selectivity over PDE1C, since PDE9A has a more polar
inhibitor binding environment with the presence of tyrosine-
424 versus a corresponding phenyl alanine-441 in PDE1C.
In addition, this strategy to design molecules with reduced
lipophilicity should improve the P450-mediated clearance of
the targeted compounds. The replacement of the cyclopentyl
group in compound 1 was explored by building the pyrazole
As we had hypothesized, the combination of the pyran and
the pyrolidine moiety afforded a highly potent 4.9 nM and
selective PDE9 inhibitor with improved pharmacokinetic
properties. Significant improvements from compound 2
included increased selectivity (>200× over PDE1C) and a 5-
fold reduction in clearance measured by human liver micro-
somes. It was determined that most of the activity resided in
the enantiomer exemplified by compound 8, which was 30-fold
more potent than the less active enantiomer 9.
Structure-based design identified an opportunity to improve
potency by completely filling the pocket that the methyl on
pyrrolidine compound 8 accessed. Modeling of an ethyl group
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a
Table 1
a
PDE9A and PDE1C: potency reported in nM as average of n = 4. HLM human liver microsomal clearance is in mL/min/kg. MDR is the efflux ratio
BA/AB. LipE= −log Ki − ClogP.
a
Scheme 1
a
(a) Step 1: 2-(ethoxymethylene)malononitrile (1 equiv), triethyl-
amine (4 equiv), ethanol (0.3 M), reflux 2 h. Step 2: 35% aq H2O2, aq
ammonia, ethanol, 48 h. Step 3: pyrolidine ester (2 equiv), THF (0.1
M), 1.0 M t-BuOK (in THF, 2 equiv), reflux 16 h. Step 4: PdOH2
Figure 3. The methyl group on the pyrrolidine does not completely fill
(10%), conc HCl (1 equiv), methanol, 40 psi of H2 18 h. Step 5:
the lipophilic pocket of PDE9A (circled area) (PDB code 3JSW).
reductive amination, aldehyde, 1,2-dichloroethane, sodium triacetox-
yborohydride, 50 °C, 18 h; or alkylation, 2-(chloromethyl)-
pyrimidine−HCl, cesium carbonate, iron triflate, DMF, 60 °C, 24 h.
plasma protein binding and a moderate volume of distribution
could be dosed once or twice a day, it was necessary to have a
analogue into the crystal structure was projected to optimize
the space filling of the lipophilic pocket in PDE9A (see Figure
3).
Addition of an ethyl group in compound 10 did enhance
potency (3-fold) but did not show an improvement in LipE or
selectivity and displayed a higher human liver microsomal
clearance (Table 2), rendering this avenue less appealing.
Removal of the benzyl moiety in 11 afforded a compound that
had low human liver microsomal clearance but with a
significant loss in potency.21
While compound 8 displayed many desirable attributes to
advance as a candidate molecule into clinical trials, the human
liver microsomal clearance was still moderate. Compound 8 has
a significant plasma protein binding free fraction of 40% due to
the polarity of the molecule ClogP = 0.97. While plasma
protein binding does not impact the dose, high free fractions
can lead to a decreased half-life projection in humans, since a
large amount of free drug is available to metabolize.22 To
ensure that a clinical compound from this series with low
compound with very low human liver microsomal clearance to
achieve an acceptable half-life in humans. Lowering the
clearance of the molecule would also have a positive impact
on the dose projection in humans. The enabled pyrrolidine
monomer rapidly allowed for SAR generation to identify either
substituents on the phenyl ring or incorporation of heterocycles
to drive down P450-mediated clearance.
Fluorination of the phenyl ring did not reduce the clearance
of the molecules, and only the para-fluoro substituent had
equivalent clearance to the unsubstituted phenyl ring. This
suggested that utilizing a blocking strategy with a fluorine to
deactivate the phenyl ring to reduce P450-mediated oxidation
was not likely to improve the dose or half-life projection. The
increased clearance in these fluorinated analogues is probably
driven through the increase in lipophilicity. Alternatively,
lowering lipophilicity by incorporation of a heteroatom into
the phenyl ring proved to be fruitful on multiple fronts. As
expected, human liver microsomal clearance tracked with the
lipophilicity of the compounds. Incorporation of one
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a
Table 2
a
PDE9A and PDE1C: potency reported in nM as average of n = 4. HLM human liver microsomal clearance is in mL/min/kg. MDR is the efflux ratio
BA/AB. LipE= −log Ki − ClogP. NA = unable to measure flux in MDR potentially because of poor permeability.
heteroatom into the ring decreased the microsomal clearance
by half for compounds 16 and 17. Subsequently, pyrimidines
and pyrazines were incorporated to determine if the clearance
could be lowered further with addition of another heteroatom
and concomitant lowering of the lipophilicity. It was
determined that the pyrimidine compounds 19 and 20 could
effectively render the compounds completely stable to P450-
mediated oxidation in the in vitro human liver microsomal
system.
While these heteroaryl compounds were highly polar with
negative ClogP values, compounds within this series still
maintained brain penetration. Interesting trends were seen with
the P-gp MDR efflux ratios, which are predictive of brain
penetration in humans. CNS compounds that exhibit no central
penetration impairment typically have an efflux ratio of <2.5.
The 2-pyridyl substitution 16 provided significantly less P-gp
efflux liability than the 3-substituted pyridine 17 (1.44 vs 4.49)
(Table 3). To further explore this trend, the 2,6-pyrimidine was
compared to the 3,5-pyrimidine, and this effect was even more
pronounced. The 2,6-pyrimidine 6-[(3S,4S)-4-methyl-1-(pyr-
imidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-
yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-
04447943)30 20 with a ClogP of −1.5 displayed an MDR
ratio of 1.23, while the 3,5-pyrimidine showed an efflux ratio of
15.8. This ratio proved highly predictive in that compound 20
displayed good brain penetration in the rat and compound 19
showed a very low brain to plasma ratio (<0.05). The
differential brain penetration and P-gp efflux liability cannot
be explained by traditional physicochemical properties, since
the differences in molecular weight, basic pKa, lipophilicity, and
polar surface area are negligible. Future publications will
explore the cause and generality of these findings.
Alignment of the desired in vitro ADME properties was
achieved with compound 20, and just as importantly potency
for PDE9A inhibition was maintained as lipophilicity was
decreased. This is demonstrated with the heterocyclic
compounds exhibiting improved LipE over the phenyl
compounds. The LipE was further improved via incorporation
of the pyrimidines, culminating in compound 20 with a LipE of
9.58. LipE for 20 is exquisitely high when compared to typical
enzyme inhibitors and marketed CNS drugs.
The X-ray structure of 20 cocrystallized in the catalytic
domain of PDE9A (Figure 4) shows that unique features of
interaction with PDE9A. While the traditional interactions with
Gln-453 are maintained, the pyrimidine ring is stacking with the
Phe-441. It appears that this stacking is not driven by
lipophilicity in that the heterocyclic analogues are essentially
equipotent to the phenyl ring. An edge on interaction with Phe-
456 is also observed. The water molecule mediating the ligand
Tyr-424 interaction is also observed and could potentially
donate a hydrogen bond to the nitrogen of the pyrimidine ring
of 20 at a distance of 2.9 Å. Compound 20 possessed all of the
desired in vitro attributes that were targeted to advance into
candidate selection including potency (8 nM), selectivity (>10
μM versus all the PDEs except PDE1C at >1 μM), low human
liver microsomal clearance (<8), and no P-gp liability.
Evaluation of 20 in the CEREP panel screen showed less
than 50% activity against a 75+ panel of GPCRs, enzymes, and
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a
Table 3
a
PDE9A and PDE1C: potency reported in nM as average of n = 4. HLM human liver microsomal clearance is in mL/min/kg. MDR is the efflux ratio
BA/AB. LipE= −log Ki − ClogP.
ion channels when tested at 10 μM. This selectivity was
achieved by optimizing the physicochemical properties of the
molecule and by interacting with two unique residues in
PDE9A vs PDE1C. As was designed, polar interactions with
tyrosine-424 in PDE9A (vs phenylalanine in PDE1C) by the
pyrrolidine and incorporation of a more polar pyran provided
significant selectivity. Additionally selectivity was gained via a π-
stacking interaction between the 2,6-pyrimidine and phenyl-
alanine 441 in PDE9A, which is an isoleucine in PDE1C.
Compound 20 was centrally penetrant in rodents as
evidenced by brain to plasma ratios ranging from 0.59 to
0.88 in rats and mice, respectively. There was a slight
disequilibrium in free drug concentrations across the central
compartment, as evidenced by free brain/free plasma ratios
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the opportunity for a translatable biomarker to assess target
engagement. Electrophysiological evaluation of compound 20
under select conditions has revealed an impact on hippocampal
synaptic plasticity, as represented by long-term potentiation. In
vivo studies demonstrate that auditory gating deficits produced
by amphetamine can be reversed in a dose-dependent fashion
with 20, suggesting the potential for utility in a variety of
human disease states that exhibit a disrupted P50-gating
response including schizophrenia, Huntington’s disease, and
Alzheimer’s disease.24 Procognitive activity in a variety of
rodent models has been demonstrated by multiple groups.
Compound 20 was efficacious in both a spatial recognition
memory model and a novel object recognition model, which
both rely on time-dependent disruption of episodic memory.25
In addition, compound 20 was able to reverse scopolamine-
induced deficits in both the Morris water maze and novel object
recognition, suggesting potential therapeutic utility in patients
with slight impairments of the cholinergic system, such as mild
cognitive impairment or early Alzheimer’s disease. Evidence
that 20 can reverse deficits produced by the NMDA receptor
antagonist ketamine, at doses expected to produce moderate
inhibition of the PDE9A enzyme, in a spatial memory task
using an eight-arm radial maze suggests the potential for
remediation of cognitive deficits across a variety of disease
states exhibiting impaired glutamate neurotransmission.26
Finally, preliminary evidence of synaptic stabilization in the
Tg2576 mouse model of amyloid precursor protein (APP)
overexpression is suggested by data showing that chronic
exposure to 20 prevented the loss in hippocampal dendritic
spine density that normally occurs in this transgenic model
between 4 and 5 months of age.27 Efficacy in this potential
synaptoprotective model occurred with chronic exposures
equivalent to efficacy seen in the cognition models and
correlated with a CSF cGMP elevation of ∼125−150%
Figure 4. X-ray crystral structure of PDE9A inhibitor 20 (PDB code
4E90) bound in PDE9A making key interaction with the phenyl-
alanine and tyrosine to provide selectivity over PDE1C.
ranging from 0.32 to 0.52 (rats and mice, respectively) and by a
cerebrospinal fluid (CSF)/free plasma equal to 0.19 (rat).
These findings indicate some impairment in the compound’s
ability to cross the rodent blood−brain barrier. The
disequilibrium in free drug concentrations was further evaluated
to determine species specificity and potential human translation
and was predicted to be a rodent-specific finding mediated by
efflux via P-gp (cross-species in vitro transporter evaluation).
Additional experiments examining CSF and plasma concen-
tration−time profiles in the dog resulted in no evidence of
disequilibrium (AUCcsf/AUCp,u equal to 0.93) in this higher
species.
The pharmacokinetic properties of compound 20 were
determined in both the rat and dog. The in vivo clearance of
compound 20 was moderate in the rat and low in the dog, in
agreement with in vitro clearance estimates generated using
liver microsomes (Table 4). The volume of distribution was
CONCLUSION
■
Through parallel chemistry and structure-based design, clinical
candidate 20 was identified to answer biological questions for a
centrally penetrant PDE9A inhibitor in humans. In particular,
PDE9A inhibition may improve cognition in multiple disease
states. Compound 20 shows excellent selectivity within the
PDE family and in broad ligand profiling. The physicochemical
properties of the molecule show that a highly polar molecule
(ClogP < 0) can still have CNS penetration if other
physicochemical properties are balanced such as limiting the
number of hydrogen bond donors, lowering the basic pKa, and
minimizing the molecular weight.28 The broad efficacy in
multiple preclinical cognition models including the potential for
synaptic stabilization in the Tg2576 mouse model has
supported the advancement of 20 into clinical trials. The
promising preclinical pharmacokinetic properties has translated
to phase 1 clinical trials. In humans, 20 has excellent
tolerability, exposure, and a half-life of 19−31 h.29 The
preclinical CSF elevation of cGMP has translated to humans,
and it has been reported that a single dose of 40 mg elevated
CSF cGMP by ∼300%.30 The translation of the central
preclinical cGMP response to humans is proof that 20 is a
viable clinical compound to test in multiple clinical populations
that display cognitive deficits.
Table 4. Summary of Preclinical Pharmacokinetic
a
Parameters of Compound 20
rat
dog
human
b
microsomal CLb (mL/min/kg)
<22.1
32.1
<17.4
11.7
<5.3
NA
NA
in vivo CLb (mL/min/kg)
ivivc
1.5×
1.5×
a
CLb determined from rat and dog following iv administration at 1 and
0.3 mg/kg, respectively. NA = not applicable. ivivc = in vitro to in vivo
b
correlation. Calculated using the well-stirred model with the inclusion
of all binding factors.
determined to be moderate across species (∼2 L/kg), and the
bioavailability ranged from 73% to 91% in rats and dogs,
respectively. In humans, compound 20 was projected to have a
low systemic clearance through the use of preclinical/in vitro
clearance prediction methodology.23 Additional information on
the preclinical and clinical pharmacokinetics and exposure−
response relationships will be reported in a future publication.
With desirable potency, selectivity, and pharmacokinetic
properties, compound 20 has been the subject of extensive
biological profiling using models of CNS function and
dysfunction. It has been shown to elevate cGMP in multiple
brain regions and in cerebral spinal fluid (CSF), which provided
EXPERIMENTAL SECTION
All reagents and solvents were used as purchased from commercial
sources. Reactions were carried out under a blanket of nitrogen. Silica
■
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gel chromatography was done using the appropriate size Biotage
prepacked silica filled cartridges. Mass spectral data were collected on a
Micromass ADM atmospheric pressure chemical ionization instrument
(LRMS APCI). NMR spectra were generated on a Varian 400 MHz
instrument. Chemical shifts were recorded in ppm relative to
tetramethylsilane (TMS) with multiplicities given as s (singlet), bs
(broad singlet), d (doublet), t (triplet), dt (doublet of triplets), m
(multiplet). Compound purity is determined by combustion analysis
(Quantitative Technologies, Inc.) or high pressure liquid chromatog-
raphy (HPLC). The purity of title compounds used in pharmacology
testing was verified by HPLC−MS using the following method: 12
min gradient on a HP1100C pump of increasing concentrations of
acetonitrile in water (5% → 95%) containing 0.1% formic acid with a
flow rate of 1 mL/min and UV detection at λ 220 and 254 nm on a
Gemini C18 150 mm × 4.6 mm, 5 μm column (Phenomenex,
Torrance, CA). All final compounds either met combustion analysis
within 0.4% or were >95% pure by HPLC by one of the methods
above.
11.6, 4.6 Hz, 2H), 3.28 (t, J = 1.7 Hz, 2H), 2.12−2.02 (m, 2H), 1.80−
1.76 (m, 2H). MS: M+H m/z = 193.1.
5-Amino-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole-4-car-
boxamide (6). A stirred solution of 5-amino-1-(tetrahydro-2H-pyran-
4-yl)-1H-pyrazole-4-carbonitrile 5 (228 mmol) in ethanol (300 mL)
was treated with 35% aqueous H2O2 (100 mL) followed by aqueous
ammonia (300 mL). The reaction mixture was stirred for 48 h at
ambient temperature and then quenched with saturated sodium
thiosulfate (800 mL) and concentrated under reduced pressure to
remove most of the ethanol. The resulting solid was removed by
filtration and washed with water (2 × 200 mL) and ether (2 × 150
mL). The solid was dried in vacuo to constant weight (31 g, 65% yield
1
for two steps). 400 MHz H NMR (CD3OD) δ 7.67 (s, 1H), 4.27−
4.21 (m, 1H), 4.03 (dd, J = 11.6, 4.6 Hz, 2H), 3.28 (t, J = 1.7 Hz, 2H),
2.14−2.04 (m, 2H), 1.81−1.78 (m, 2H). MS: M+H m/z = 211.2.
6-[(3S,4S)-1-Benzyl-4-methylpyrrolidin-3-yl]-1-(tetrahydro-
2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-
one (8). To a mixture of 5-amino-1-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazole-4-carboxamide (5.0 g, 23.78 mmol) and (3,4-trans)-methyl 1-
benzyl-4-methylpyrrolidine-3-carboxylate (11.7 g, 47.57 mmol) in
tetrahydrofuran (0.1 M) were added molecular sieves (pellets). To the
stirred mixture was added a 1.0 M solution of t-BuOK in THF (48.0
mL, 47.57 mmol), and the resulting mixture was heated at reflux under
an atmosphere of nitrogen with vigorous stirring overnight. The
reaction mixture was cooled to ambient temperature, and solids were
removed by filtration. The solids were washed with EtOAc (2×), and
the combined filtrates were concentrated under reduced pressure. The
remainder was partitioned between CH2Cl2 and H2O, and the aqueous
and organic layers were separated. The aqueous phase was extracted
with CH2Cl2 (1×), and the combined organic extracts were dried over
Na2SO4, filtered, and concentrated under reduced pressure. The crude
material was purified by chromatography (silica gel, 1% Et3N in
EtOAc) to produce (6.6 g, 68% yield) as a white solid. The
enantiomers were separated utilizing chiral chromatography with
Chiralcel OD-H, mobile phase 70/30 heptane/EtOH, tR = 11.465
(more active enantiomer 3), to provide the two enantiomers in >98%
6-Benzyl-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole[3,4-d]-
pyrimidin-4(5H)-one (3). To a solution of 5-amino-1-(tetrahydro-
2H-pyran-4-yl)-1H-pyrazole-4-carboxamide 6 (100 mg, 0.476 mmol)
in 4.8 mL of absolute ethanol was added ethyl 2-phenylacetate (352
mg, 2.14 mmol, 4.5 equiv) and then NaH (99 mg, 2.48 mmol, 5.2
equiv). The resulting heterogeneous mixture was heated in a
microwave at 150 °C for 30 min. After cooling to ambient
temperature, the resulting mixture was concentrated. The crude
material was purified with a 0−10% NH4OH in methanol/ethyl
acetate gradient to afford the desired material (130 mg, 88% yield) as a
1
white solid. 400 MHz H NMR (CDCl3) δ 11.32 (s, 1H), 8.10 (s,
1H), 7.46−7.29 (m, 5H), 4.92 (m, 1H), 4.19 (dd, J = 11.5, 3.8 Hz,
2H), 4.11 (s, 2H), 3.67 (t, J = 11.4 Hz, 2H), 2.49−2.38 (m, 2H),
2.00−1.96 (m, 2H). MS: M+H m/z = 311.3.
1-(Tetrahydro-2H-pyran-4-yl)hydrazine (4). To a solution of
tetrahydropyran-4-one (71.6 g, 715 mmol) in methanol (2 L) was
added tert-butylcarbazate (100 g, 758 mmol) at ambient temperature.
The mixture was stirred at ambient temperature for 20 h. The reaction
mixture was concentrated under reduced pressure to dryness to afford
a white solid (154 g). To a suspension of the white solid (154 g, 715
mmol) in water (1 L) was added acetic acid (500 mL, 8.73 mol), and
the mixture was stirred for 30 min to get a clear solution. To this
solution, solid NaCNBH3 (44.5 g, 708 mmol) was added portionwise.
The mixture was stirred at ambient temperature for 2 h. The mixture
was then transferred to a 12 L flask, cooled to 0 °C, and quenched
with 1 N NaOH (8.73 L, 8.73 mol). The mixture was extracted with
CH2Cl2 (3 × 3 L) and dried over Na2SO4. The organic layer was
filtered and concentrated to afford a white solid (164 g, contains ∼15%
of N-acetyl-N′-Boc-hydrazine derivative). Chromatography [silica,
ethyl acetate/MeOH (95:5)] gave 94 g of 90% pure Boc-hydrazine.
A solution of Boc-hydrazine (50 g, 231 mmol) in methanol (500 mL)
was added to a solution of HCl in dioxane (462 mL, 1.85 mol, 4.0 M).
The mixture was stirred at ambient temperature overnight.
Concentration of the reaction mixture under reduced pressure
afforded the title compound as a white solid (43 g, 98%). 400 MHz
1H NMR (DMSO) δ 3.85−3.82 (m, 2H), 3.27−3.21 (m, 2H), 3.13−
1
ee. 400 MHz H NMR (CDCl3) δ 8.02 (s, 1H), 7.39−7.25 (m, 6H),
4.83−4.75 (m, 1H), 4.14−4.09 (m, 2H), 3.82 (m, 1H), 3.62−3.54 (m,
3H), 3.39−3.37 (m, 1H), 3.00 (m, 1H), 2.83 (m, 1H), 2.66−2.27 (m,
4H), 2.10−1.83 (m, 3H), 1.20 (d, J = 6.6 Hz, 3H). MS: M+H m/z =
394.2.
6-[(3R,4R)-1-Benzyl-4-methylpyrrolidin-3-yl]-1-(tetrahydro-
2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-
1
one (9). 400 MHz H NMR (CDCl3) δ 8.02 (s, 1H), 7.39−7.25 (m,
6H), 4.82−4.76 (m, 1H), 4.14−4.09 (m, 2H), 3.82−3.79 (m, 1H),
3.62−3.54 (m, 3H), 3.37 (d, J = 8.7 Hz, 1H), 3.00 (d, J = 9.9 Hz, 1H),
2.79 (dd, J = 6.3, 2.5 Hz, 1H), 2.52−2.48 (m, 1H), 2.42−2.30 (m,
3H), 1.94−1.82 (m, 3H), 1.20 (d, J = 6.6 Hz, 3H). MS: M+H m/z =
394.2.
6-[(3,4-trans)-1-Benzyl-4-ethylpyrrolidin-3-yl]-1-(tetrahydro-
2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-
one (10). Following the procedure for the preparation of 6-[(3S,4S)-
1-benzyl-4-methylpyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-15-di-
hydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (8) but substituting trans-
methyl 1-benzyl-4-ethylpyrolidine-3-carboxylate provided the title
compound. Preparative conditions: Chiralcel OJ-H, mobile phase
80/20 CO2/MeOH, tR = 3.27. Analytical: AD column, mobile phase
3.05 (m, 1H), 1.88−1.84 (m, 2H), 1.48−1.38 (m, 2H). MS: M + H
m/z = 117.2.
1
85/15 heptane/EtOH, tR = 12.896. 400 MHz H NMR (CDCl3) δ
5-Amino-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole-4-car-
bonitrile (5). To a mixture of 1-(tetrahydro-2H-pyran-4-yl)hydrazine
dihydrogen chloride 4 (18 g, 96 mmol) in 200 mL of ethanol was
added triethylamine (30 g, 40 mL, 288 mmol) at 0 °C (ice bath). The
resulting mixture was stirred for 1 h. Then a solution of 2-
(ethoxymethylene)malononitrile (12 g, 96 mmol) in 100 mL of
ethanol was added slowly to keep the reaction temperature below 5
°C. This mixture was stirred at ambient temperature overnight and
then heated to reflux for 2 h. After removal of the solvent under
vacuum, the residue was washed with 300 mL of water. The solid was
collected, washed with additional 200 mL of water and 200 mL of 1:1
of hexane and ether, and dried to give 17 g of yellow solid. 400 MHz
1H NMR (CD3OD) δ 7.71 (s, 1H), 4.29−4.21 (m, 1H), 4.02 (dd, J =
8.02 (s, 1H), 7.40−7.26 (m, 5H), 4.78 (m, 1H), 4.12−4.09 (m, 2H),
3.82−3.804 (m, 1H), 3.65−3.54 (m, 3H), 3.35 (t, J = 8.3 Hz, 1H),
2.98 (d, J = 9.9 Hz, 1H), 2.89−2.86 (m, 1H), 2.48−2.32 (m, 3H), 2.20
(m, 1H), 1.95−1.87 (m, 2H), 1.63−1.58 (m, 2H), 1.50−1.49 (m, 1H),
0.93 (t, J = 7.1 Hz, 3H). MS: M + H m/z = 408.1.
6-[(3S,4S)-4-Methylpyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-
4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (11). 6-[(3S,4S)-1-
Benzyl-4-methylpyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-15-di-
hydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (5.6 g) was dissolved in 100
mL of methanol, and the mixture was added to a Parr bottle. Palladium
hydroxide (3.76 g) was added along with 3.56 mL of concentrated
hydrochloric acid. The reaction mixture was placed on a hydrogenator
under 40 psi of H2 for 18 h. The reaction mixture was filtered through
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Celite and concentrated to provide 4.47 g of the title compound as the
hydrogen chloride salt. 400 MHz 1H NMR (CD3OD) δ 8.03 (s, 1H),
4.49 (m, 1H), 4.09−4.06 (m, 2H), 3.74−3.57 (m, 4H), 3.24 (m, 1H),
3.05 (m, 1H), 2.89 (m, 1H), 2.77 (m, 1H), 2.30 (m, 2H), 1.90 (m,
2H), 1.22 (d, J = 6.6 Hz, 3H). MS: M+H m/z = 304.2.
8.38 (d, J = 2.1 Hz, 1H), 7.99 (s, 1H), 7.77 (d, J = 2.07 Hz, 1H), 7.75
(d, J = 2.07 Hz, 1H), 4.94−4.83 (m, 1H), 4.09−4.05 (m, 2H), 3.78−
3.57 (m, 4H), 3.31−3.28 (m, 1H), 3.11−3.06 (m, 1H), 3.01−2.91 (m,
2H), 2.72−2.65 (m, 1H), 2.50 (s, 3H), 2.33−2.23 (m, 3H), 1.90−1.86
(m, 2H), 1.14 (d, J = 7.1 Hz, 3H). MS: M + H m/z = 409.2.
6-{(3S,4S)-4-Methyl-1-[(5-methylpyrazin-2-yl)methyl]-
pyrrolidin-3-yl}-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (18). Following the procedure for
the preparation of 6-{(3S,4S)-4-methyl-1-[(2-methylpyrimidin-5-yl)-
methyl]pyrrolidin-3-yl}-1-(tetrahydro-2H-pyran-4-yl)-15-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one 19 but substituting 5-methylpyrazine-
2-carbaldehyde provided the title compound. 400 MHz 1H NMR
(CDCl3) δ 8.53 (s, 1H), 8.47 (s, 1H), 8.03 (s, 1H), 4.83−4.79 (m,
1H), 4.12−4.03 (m, 2H), 3.78−3.75 (m, 1H), 3.61−3.55 (m, 2H),
3.46−3.40 (m, 1H), 3.12−3.09 (m, 1H), 2.87 (m, 1H), 2.64 (m, 1H),
2.53 (m, 2H), 2.47−2.28 (m, 4H), 2.16 (m, 2H), 1.91−1.84 (m, 2H),
1.23−1.20 (m, 3H). MS: M + H m/z = 410.3.
6-[(3S,4S)-1-(2-Fluorobenzyl)-4-methylpyrrolidin-3-yl]-1-
(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (12). Following the procedure for the preparation
of 6-{(3S,4S)-4-methyl-1-[(2-methylpyrimidin-5-yl)methyl]pyrrolidin-
3-yl}-1-(tetrahydro-2H-pyran-4-yl)-15-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (19) but substituting 2-fluorobenzaldehyde provided
1
the title compound. 400 MHz H NMR (CD3OD) δ 7.98 (s, 1H),
7.47−7.42 (m, 1H), 7.33−7.26 (m, 2H), 7.18−7.07 (m, 1H), 4.08−
4.04 (m, 2H), 3.85 (s, 2H), 3.69−3.56 (m, 3H), 3.20−2.93 (m, 4H),
2.69−2.62 (m, 1H), 2.40−2.19 (m, 3H), 1.88−1.85 (m, 2H), 1.14 (d, J
= 6.6 Hz, 3H). MS: M + H m/z = 412.1.
6-[(3S,4S)-1-(3-Fluorobenzyl)-4-methylpyrrolidin-3-yl]-1-
(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (13). Following the procedure for the preparation
of 6-{(3S,4S)-4-methyl-1-[(2-methylpyrimidin-5-yl)methyl]pyrrolidin-
3-yl}-1-(tetrahydro-2H-pyran-4-yl)-15-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (19) but substituting 3-fluorobenzaldehyde provided
6-{(3S,4S)-4-Methyl-1-[(2-methylpyrimidin-5-yl)methyl]-
pyrrolidin-3-yl}-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (19). To a solution of 6-[(3S,4S)-
4-methylpyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo-
[3,4-d]pyrimidin-4(5H)-one hydrogen chloride (493 mg) in 1,2-
dichloroethane (10 mL) were added acetic acid (174 mg), 2-
methylpyrimidine-5-carbaldehyde (236 mg), and sodium triacetox-
yborohydride (635 mg). The reaction mixture was heated at 50 °C
overnight. The reaction mixture was concentrated onto silica gel and
purified by CombiFlash chromatography to provide the title
1
the title compound. 400 MHz H NMR (CDCl3) δ 8.02 (s, 1H),
7.35−7.30 (m, 1H), 7.21−7.20 (m, 1H), 7.05 (d, J = 9.5 Hz, 1H),
6.98−6.93 (m, 1H), 4.83−4.77 (m, 1H), 4.14−4.08 (m, 2H), 3.80−
3.77 (m, 1H), 3.64−3.54 (m, 3H), 3.36 (t, J = 8.8 Hz, 1H), 3.01 (d, J =
9.9 Hz, 1H), 2.84−2.83 (m, 1H), 2.57 (m, 1H), 2.44−2.27 (m, 3H),
1.95−1.83 (m, 3H), 1.20 (d, J = 7.1 Hz, 3H). MS: M + H m/z = 412.4.
6-[(3S,4S)-1-(4-Fluorobenzyl)-4-methylpyrrolidin-3-yl]-1-
(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (14). Following the procedure for the preparation
of 6-{(3S,4S)-4-methyl-1-[(2-methylpyrimidin-5-yl)methyl]pyrrolidin-
3-yl}-1-(tetrahydro-2H-pyran-4-yl)-15-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one 19 but substituting 4-fluorobenzaldehyde provided
1
compound (146 mg). 400 MHz H NMR (CDCl3) δ 8.63 (s, 2H),
8.01 (s, 1H), 4.82−4.76 (m, 1H), 4.12−4.08 (m, 2H), 3.68 (d, J = 5.0
Hz, 3H), 3.64−3.54 (m, 2H), 3.28 (t, J = 8.3 Hz, 1H), 3.04 (d, J = 9.9
Hz, 1H), 2.89−2.86 (m, 1H), 2.71 (s, 3H), 2.66−2.62 (m, 1H), 2.49−
2.27 (m, 3H), 1.97 (t, J = 7.9 Hz, 1H), 1.91−1.83 (m, 2H), 1.19 (d, J =
7.05 Hz, 3H). MS: M + H m/z = 410.2.
6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-
yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-one (20). To a solution of 6-[(3S,4S)-4-methylpyrro-
lidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one hydrogen chloride (7.75 g) in dimethylformamide (115
mL) were added iron triflate (900 mg), 2-(chloromethyl)pyrimidine
hydrogen chloride (4.5 g), and cesium carbonate (22.2 g), and the
reaction mixture was heated at 60 °C for 24 h. The reaction mixture
was concentrated onto silica gel and purified by flash chromatography,
eluting with 0−15% methanol/ethyl acetate/1% saturated ammonium
1
the title compound. 400 MHz H NMR (CDCl3) δ 8.02 (s, 1H),
7.36−7.33 (m, 2H), 7.05−7.01 (m, 2H), 4.82−4.77 (m, 1H), 4.14−
4.09 (m, 2H), 3.79−3.76 (m, 1H), 3.62−3.54 (m, 3H), 3.34 (t, J = 8.3
Hz, 1H), 2.99 (d, J = 9.9 Hz, 1H), 2.85−2.83 (m, 1H), 2.60−2.55 (m,
1H), 2.45−2.30 (m, 3H), 1.99−1.82 (m, 3H), 1.20 (d, J = 7.1 Hz,
3H). MS: M + H m/z = 412.1.
6-[(3S,4S)-1-(4-Methoxybenzyl)-4-methylpyrrolidin-3-yl]-1-
(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (15). Following the procedure for the preparation
of 6-{(3S,4S)-4-methyl-1-[(2-methylpyrimidin-5-yl)methyl]pyrrolidin-
3-yl}-1-(tetrahydro-2H-pyran-4-yl)-15-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one 19 but substituting 4-methoxybenzaldehyde provided
the title compound. 400 MHz 1H NMR (CDCl3) δ 8.01 (s, 1H), 7.29
(d, J = 8.3 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 4.82−4.08 (m, 1H),
4.13−4.08 (m, 2H), 3.84−3.75 (m, 4H), 3.61−3.51 (m, 3H), 3.35 (t, J
= 8.7 Hz, 1H), 2.97 (d, J = 9.9 Hz, 1H), 2.82−2.80 (m, 1H), 2.53−
2.49 (m, 1H), 2.42−2.30 (m, 3H), 1.95−1.82 (m, 3H), 1.18 (d, J = 7.1
Hz, 3H). MS: M + H m/z = 424.5.
1
hydroxide to provide the title compound (6 g). 400 MHz H NMR
(CDCl3) δ 12.30 (s, 1H), 8.63 (d, J = 5.0, Hz, 2H), 8.03 (s, 1H), 7.20
(d, J = 5.0, Hz, 1H), 4.84−4.79 (m, 1H), 4.30−4.27 (m, 1H), 4.14−
4.08 (m, 2H), 3.87−3.82 (m, 1H), 3.63−3.55 (m, 2H), 3.47 (t, J = 7.9
Hz, 1H), 3.29 (d, J = 9.9 Hz, 1H), 2.88−2.86 (m, 1H), 2.60−2.56 (m,
1H), 2.46−2.24 (m, 4H), 1.93−1.84 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H).
MS: M + H m/z = 396.2. Anal. Calcd for C20H25N7O2: C, 60.74%; H,
6.37%; N, 24.79%. Found: C, 60.56%; H, 6.43%; N, 24.61%.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures for the enzyme assays and pharma-
cokinetic experiments. This material is available free of charge
via the Internet at http://pubs.acs.org.
6-{(3S,4S)-4-Methyl-1-[(6-methylpyridin-3-yl)methyl]-
pyrrolidin-3-yl}-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (16). Following the procedure for
the preparation of 6-{(3S,4S)-4-methyl-1-[(2-methylpyrimidin-5-yl)-
methyl]pyrrolidin-3-yl}-1-(tetrahydro-2H-pyran-4-yl)-15-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one 19 but substituting 6-methylnicotinal-
dehyde provided the title compound. 400 MHz 1H NMR (CD3OD) δ
8.38 (d, J = 2.1 Hz, 1H), 7.99 (s, 1H), 7.77 (d, J = 2.07 Hz, 1H), 7.75
(d, J = 2.07 Hz, 1H), 4.94−4.83 (m, 1H), 4.09−4.05 (m, 2H), 3.78−
3.57 (m, 4H), 3.31−3.28 (m, 1H), 3.11−3.06 (m, 1H), 3.01−2.91 (m,
2H), 2.72−2.65 (m, 1H), 2.50 (s, 3H), 2.33−2.23 (m, 3H), 1.90−1.86
(m, 2H), 1.14 (d, J = 7.1 Hz, 3H). MS: M + H m/z = 409.2.
6-{(3S,4S)-4-Methyl-1-[(6-methylpyridin-3-yl)methyl]-
pyrrolidin-3-yl}-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (17). Following the procedure for
the preparation of 6-{(3S,4S)-4-methyl-1-[(2-methylpyrimidin-5-yl)-
methyl]pyrrolidin-3-yl}-1-(tetrahydro-2H-pyran-4-yl)-15-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one 19 but substituting 6-methylnicotinal-
dehyde provided the title compound. 400 MHz 1H NMR (CD3OD) δ
■
S
Accession Codes
Coordinates of the PDE9A crystal structures have been
deposited in the Protein Data Base for compound 16 (PDB
code 4E90).
AUTHOR INFORMATION
Corresponding Author
*Phone: 617-395-0708. Fax: 860-686-0013. E-mail: patrick.r.
verhoest@pfizer.com.
Notes
■
The authors declare no competing financial interest.
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(17) Verhoest, P. R.; Proulx-Lafrance, C.; Corman, M.; Chenard, L.;
Helal, C. J.; Hou, X.; Kleiman, R.; Liu, S.; Marr, E.; Menniti, F. S.;
Schmidt, C. J.; Vanase-Frawley, M.; Schmidt, A. W.; Williams, R. D.;
Nelson, F. R.; Fonseca, K. R.; Liras, S. Identification of a Brain
Penetrant PDE9A Inhibitor Utilizing Prospective Design and
Chemical Enablement as a Rapid Lead Optimization Strategy. J.
Med. Chem. 2009, 52, 7946−7949.
ABBREVIATIONS USED
■
PDE, phosphodiesterase; cGMP, cyclic guanosine mono-
phosphate; CNS, central nervous system; SBDD, structure-
based drug design; P-gp, P-glycoprotein; LipE, lipophilic ligand
efficiency; LE, ligand efficiency; CSF, cerebral spinal fluid; LTP,
long-term potentiation
(18) Whalen, K.; Gobey, J.; Janiszewski, J. A. Centralized Approach
to Tandem Mass Spectrometry Method Development for High-
Throughput ADME Screening. Rapid Commun. Mass Spectrom. 2006,
20, 1497−1503.
(19) Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A. The
Maximal Affinity of Ligands. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
9997−10002.
(20) Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M.
D.; Verhoest, P. R.; Villalobos, A.; Will, Y. Defining Desirable Central
Nervous System Drug Space through the Alignment of Molecular
Properties, in Vitro ADME, and Safety Attributes. ACS Chem. Neurosci.
2010, 1, 420−434.
(21) Feng, B; Mills, J. B.; Davidson, R. E.; Mireles, R. J.; Janiszewski,
J. S.; Troutman, M. D.; de Morais, S. M. In Vitro P-Glycoprotein
Assays To Predict the in Vivo Interactions of P-Glycoprotein with
Drugs in the Central Nervous System. Drug Metab. Dispos. 2008, 36,
268−275.
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