Design, synthesis and evaluation of [3H]PF-7191, a highly specific nociceptin opioid peptide (NOP) receptor radiotracer for in vivo receptor occupancy (RO) studies

Article abstract of DOI:10.1016/j.bmcl.2014.09.069

Herein we report the identification of (+)-N-(2-((1H-pyrazol-1-yl)methyl)-3-((1R,3r,5S)-6′-fluoro-8-azaspiro[bicyclo[3.2.1]octane-3,1′-isochroman]-8-yl)propyl)-N-[³H]-methylacetamide {[³H]PF-7191 [(+)-11]} as a promising radiotracer for the nociceptin opioid peptide (NOP) receptor. (+)-11 demonstrated high NOP binding affinity (K_i = 0.1 nM), excellent selectivity over other opioid receptors (>1000×) and good brain permeability in rats (C_b,u/C_p,u = 0.29). Subsequent characterization of [³H](+)-11 showed a high level of specific binding and a brain bio-distribution pattern consistent with known NOP receptor expression. Furthermore, the in vivo brain binding of [³H](+)-11 in rats was inhibited by a selective NOP receptor antagonist in a dose-responsive manner. This overall favorable profile indicated that [³H](+)-11 is a robust radiotracer for pre-clinical in vivo receptor occupancy (RO) measurements and a possible substrate for carbon-11 labeling for positron emission tomography (PET) imaging in higher species.

Full text of DOI:10.1016/j.bmcl.2014.09.069

Accepted Manuscript
3
Design, synthesis and evaluation of [ H]PF-7191, a highly specific nociceptin
opioid peptide (NOP) receptor radiotracer for in vivo receptor occupancy (RO)
studies
Lei Zhang, Elena Drummond, Michael A. Brodney, Julie Cianfrogna, Susan E.
Drozda, Sarah Grimwood, Michelle A. Vanase-Frawley, Anabella Villalobos
PII:
S0960-894X(14)01025-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.09.069
BMCL 22035
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 August 2014
19 September 2014
24 September 2014
Please cite this article as: Zhang, L., Drummond, E., Brodney, M.A., Cianfrogna, J., Drozda, S.E., Grimwood, S.,
3
Vanase-Frawley, M.A., Villalobos, A., Design, synthesis and evaluation of [ H]PF-7191, a highly specific
nociceptin opioid peptide (NOP) receptor radiotracer for in vivo receptor occupancy (RO) studies, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.09.069
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
3
Design, synthesis and evaluation of [ H]PF-
Leave this area blank for abstract info.
7
191, a highly specific nociceptin opioid
peptide (NOP) receptor radiotracer for in
vivo receptor occupancy (RO) studies
Lei Zhang*, Elena Drummond, Michael A. Brodney, Julie Cianfrogna, Susan E. Drozda, Sarah Grimwood,
Michelle A. Vanase-Frawley, Anabella Villalobos
Total Binding
Non-specific binding
[3
H]PF-7191

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
3
Design, synthesis and evaluation of [ H]PF-7191, a highly specific nociceptin
opioid peptide (NOP) receptor radiotracer for in vivo receptor occupancy (RO)
studies
a, *
b
a
d
c
Lei Zhang , Elena Drummond , Michael A. Brodney , Julie Cianfrogna , Susan E. Drozda , Sarah
b
e
a
Grimwood , Michelle A. Vanase-Frawley , Anabella Villalobos
a
Neuroscience medicinal chemistry, Pfizer Inc., Cambridge, MA 02139, USA
Neuroscience research unit, Pfizer Inc., Cambridge, MA 02139, USA
b
c
Neuroscience medicinal chemistry, Pfizer Inc., Groton, CT 06340, USA
d
Pharmacokinetics, dynamics and metabolism, Pfizer Inc., Groton, CT 06340, USA
e
Primary pharmacology group, Pfizer Inc., Groton, CT 06340, USA
*
Corresponding author: Tel: +1-617-295-0640; e-mail: lei.zhang3@pfizer.com
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Herein we report the identification of (+)-N-(2-((1H-pyrazol-1-yl)methyl)-3-((1R,3r,5S)-6'-
3
fluoro-8-azaspiro[bicyclo[3.2.1]octane-3,1'-isochroman]-8-yl)propyl)-N-[ H]-methylacetamide
3
{[ H]PF-7191 [(+)-11]} as a promising radiotracer for the nociceptin opioid peptide (NOP)
Accepted
i
receptor. (+)-11 demonstrated high NOP binding affinity (K = 0.1 nM), excellent selectivity
Available online
over other opioid receptors (>1000x) and good brain permeability in rats (Cb,u/Cp,u= 0.29).
Subsequent characterization of [ H](+)-11 showed a high level of specific binding and a brain
3
Keywords:
NOP receptor
ORL1
Radiotracer
Receptor occupancy
PET
bio-distribution pattern consistent with known NOP receptor expression. Furthermore, the in
3
vivo brain binding of [ H](+)-11 in rats was inhibited by a selective NOP receptor antagonist in a
3
dose-responsive manner. This overall favorable profile indicated that [ H](+)-11 is a robust
radiotracer for pre-clinical in vivo receptor occupancy (RO) measurements and a possible
substrate for carbon-11 labeling for positron emission tomography (PET) imaging in higher
species.
2
009 Elsevier Ltd. All rights reserved.
The nociceptin opioid peptide (NOP) receptor, formerly also
known as opioid receptor-like 1 (ORL-1), was first identified in
were interested in developing a selective NOP receptor
radiotracer, specifically a [ H] radiotracer that would allow
3
1
th
1
994. It was widely considered as the 4 member of the opioid
rodent in vivo receptor occupancy (RO) measurements, which
could then be used for effective lead characterization and
prioritization, and ultimately clinical candidate selection.
Development of a suitable radiotracer for in vivo NOP RO
studies has been challenging due to its relatively low expression
level (Bmax) and high lipophilicity of literature leads. Prior to
initiation of our effort, only one NOP antagonist PET radiotracer,
receptor family, sharing high sequence homology to µ, κ and δ
opioid receptors. A heptadecapeptide, termed as nociceptin or
2
orphanin FQ (N/OFQ), was identified as the endogenous agonist
for NOP receptors, which interestingly shows no appreciable
affinity for other opioid receptor subtypes despite of its structural
similarity to opioid peptide agonist dynorphin A. Like other
opioid receptors, NOP receptors are widely expressed in the
central nervous system (CNS) and peripheral organs. In the brain,
NOP receptors are enriched in the cortex, hippocampus,
hypothalamus and brain stem while expressed less in striatum
1
1
[ C]CPEB (1, Figure 1), was reported which failed to
5
demonstrate specific binding in vivo. Since our effort was
initiated, two additional positron emission tomography (PET)
1
1
tracers, [ C]-(S)-3-(2’-fluoro-4’,5-dihydrospiro[piperidine-4,7’-
thieno-[2,3-c]pyran-1-yl)-2(2-fluorobenzyl)-N-
3
and cerebellum.
6
18
7
methylpropanamide (2) and [ F]MRK-0911 (3) have been
reported, showing specific to non-specific ratios of 1.28 and ~2
respectively in non-human primates (NHP).
Pharmacologically, the NOP receptor is coupled to the
+
activation of phospholipase C (PLC) and K channels and the
inhibition of adenylyl cyclase, with a main effect of inhibiting the
release of key neurotransmitters, e.g. acetylcholine,
catecholamines, glutamate etc. in neuronal tissues. Therefore
there has been an interest in developing selective NOP
antagonists as potential treatment for depression, anxiety,
3
A NOP receptor [ H] radiotracer needs to meet a number of
specific requirements in order to be viable in vivo. Structurally, it
must be amenable to tritiation, typically requiring incorporation
of three tritiums to offer sufficient specific activity for in vivo
studies (~75 Ci/mmol). Pharmacokinetic (PK)-wise, it needs to
4
Parkinson’s disease (PD) and obesity. Within our group, we

be brain permeable and has low non-specific binding (NSB) in
brain tissues. Pharmacologically, it needs to be potent and
selective towards the NOP receptor. The requisite binding
the pendant pyrazole (9 vs. 7) also yielded similar selectivity
increase (1.4-fold to 106-fold).
Based on this information, we proposed a total of 6 analogs by
combining the potency and selectivity handles identified from the
SAR analysis. All analogs have an N-Me amide moiety as a
potential tritiation site and could be divided into two structural
classes based on the choice of their selectivity handles (Scheme
affinity for a radiotracer is dictated by B
with a ratio of
max
8
Bmax/K >10 typically required. The B of NOP receptor has
d
max
been estimated at 237 fmol/mg protein in rat brain (~12 nM in
9
the whole brain, assuming 50 mg protein/g wet tissue). The B
max
information on higher species, however, has been sparse. Only a
single report on macaque brain exists, showing a less defined
Bmax estimation (> 6.1 fmol/mg protein in receptor rich regions,
1
). Analogs 10-12 utilized a reversed amide moiety for
selectivity, with variations on the pendant R groups: a thiazole
(10), an unsubstituted pyrazole (11), and 4-Cl pyrazole (12) with
an intention to maximize selectivity by combining the two
selectivity handles shown in Figure 1B. Analogs 13-15 utilized
substituted heteroaryls (R groups) for selectivity, probing
variations in heteroaryls (pyrazole for 13, 14; imidazole for 15)
and substituents (4-methyl for 13, 15; 4-chloro for 14). Prior to
the synthesis of any analogs, we prioritized this group of targets
according to their physicochemical properties (CNS PET MPO)
and absorption, distribution, metabolism and excretion (ADME)
1
0
e.g. cortex, caudate and hippocampus). Therefore, potency in
the sub-nM range would likely be required to achieve in vivo
engagement of NOP receptors.
1
7
properties using in silico models. As shown in Scheme 1, all six
proposed analogs reside in the desirable physicochemical
property space (CNS PET MPO >3). Analogs 10 and 15 were
predicted to have high Pgp liability (cMDR BA/AB 5.32 and
8
.95 respectively), suggesting a high risk of poor brain
permeability. They were therefore not pursued despite their
otherwise favorable property profile. In comparison, analogs 11-
Figure 1. Known NOP receptor antagonist radiotracers
1
4 were predicted to have good passive permeability (cRRCK >
Our group has previously disclosed a set of design and
selection criteria for novel CNS PET tracer development.
1
1
5) and low Pgp liability (cMDR BA/AB < 2.5), suggesting good
brain permeability, among which 11 and 14 have the best overall
profile, meeting all PK criteria for radiotracers. Analogs 12 and
Considering the similarity in property requirements between a
3
PET tracer and a [ H] radiotracer, we were keen to explore
1
3, however, were predicted to have low Fu_b, which would
whether we could apply similar design principles for PET tracers
3
require close attention to their potential risk of high NSB.
in our in vivo [ H] NOP radiotracer development effort.
Specifically we targeted analogs with suitable physicochemical
properties [CNS PET multi-parameter optimization (MPO) score
>
3], good passive permeability [RR canine kidney (RRCK) > 5 x
-6 12
1
0 cm/sec] and low p-glycoprotein (P-gp) efflux [multidrug
13 14
resistance (MDR) BA/AB < 2.5] for brain permeability 5and
appropriate fraction unbound in brain (Fu_b > 0.05) to
1
minimize the risk of NSB. With this set of parameters in mind,
we started our effort by data-mining of our NOP antagonist
chemical matter for viable tracer leads. We were particularly
interested in the spirocyclic series previously developed by our
Nagoya colleagues for its extensive SAR and overall excellent
1
6
NOP receptor activities. In-depth SAR analysis revealed
intriguing trends on potency and selectivity. As shown in Figure
2
A, switching the piperidine ring of 4 to a tropane ring led to ~5-
fold increase in potency (5 vs. 4). Further improvement in
potency (14-fold) was achieved by placing a fluorine at the 4-
position of the distal phenyl ring (6 vs. 5), yielding compounds
with sub-nM potency. The overall profile of 6 was attractive as it
has many of the desirable attributes for a radiotracer lead,
including a viable tritiation site (amide N-methyl), potent NOP
binding affinity (K = 0.59 nM), high RRCK (> 10), low MDR (<
i
2
.5) and favorable predicted Fu_b (> 0.05). However, while it
showed no appreciable potency for κ and δ opioid receptors (K >
i
1
0 µM), it was potent at µ opioid receptor (K = 5.9 nM), which
i
we hypothesized would confound interpretation of in vivo RO.
This prompted us to further investigate SAR specifically related
to selectivity over µ opioid receptor. While the NOP potency was
mainly influenced by the spiropiperidine portion of the
molecules, we found that the µ opioid receptor selectivity could
be modulated by varying the right hand N-alkyl portion (Figure
2
B). For example, reversing the amide connectivity (8 vs. 7)
Figure 2. A) historical SAR on NOP potency; B) historical SAR on
selectivity over µ opioid receptor
significantly increased the NOP affinity while reducing µ opioid
binding, leading to a net increase in selectivity from 1.4-fold to
1
05-fold. Alternatively, adding a substituent at the 4-position of

The synthesis of analogs 11-14 was accomplished via a
convergent synthetic route depicted in Scheme 2. The
construction of the right-hand portion started from commercially
available phenyl bromide 16. Lithium-Br exchange, followed by
trapping with N-protected tropanone 17, afforded diol 18, which
upon treatment with mesyl chloride in the presence of pyridine
yielded cyclic ether 19. Removal of the ethyl carbamate
protecting group gave the key intermediate (1R,3r,5S)-6'-fluoro-
8-azaspiro[bicyclo[3.2.1]octane-3,1'-isochromane] (20).
Scheme 1. Design of potent and selective NOP receptor antagonists for radiotracer development
The synthesis of the right hand portion of the molecules began
with conversion of ethyl 2-(hydroxymethyl)acrylate (21) to the
corresponding bromide 22. N-alkylation with pyrazole 23 bearing
1
variations at the R position yielded intermediate 24. Coupling of
2
0 and 24 via 1,4-addition followed by hydrolysis of the ethyl
ester gave carboxylic acid 25. From this intermediate, analogs 13
and 14 were synthesized via amide formation with diethylamine,
while analogs 11 and 12 were synthesized in a 3-step sequence
involving amide formation with methylamine to give
intermediate 26, amide reduction to N-methyl amine, and
acylation. Finally chiral separation of analogs 11-14 yielded
individual enantiomers for further profiling.
The potency, selectivity and in vitro ADME properties of (±)-
1
1 to 14 are summarized in Table 1. Compound chirality
appeared to have significant impact on NOP receptor affinity.
The (+)-enantiomers of analogs 11-14 demonstrated significantly
higher NOP receptor binding affinity than the corresponding (-)-
enantiomers (~9-20x). Affinity discrepancies between (+) and (-)
enantiomers were also observed for µ receptor binding, but to a
lesser extent. Taken together, the (+)-11-14 achieved comparable
potency to the early lead 6, but with much improved µ receptor
selectivities ranging from 66x for (+)-14 to > 1000x for (+)-
1
1(PF-7191). In terms of in vitro ADME properties, (+)-11 and
(+)-14 were superior with favorable RRCK, MDR and fractions
unbound values. In comparison, (+)-12 and (+)-13 showed lower
than predicted passive permeability (RRCK = 3.4 and 4.2
respectively), and low fraction unbound in brain (0.02 and 0.01
respectively) consistent with the in silico predictions. Overall
(+)-11 emerged as the most promising tracer lead. It met all of
our design objectives including sub-nM NOP affinity (K = 0.1
i
nM), high µ receptor selectivity (1036x) and favorable ADME
-
6
profile for brain permeability (RRCK = 8.0 x10 cm/s; MDR1
BA/AB= 1.8) and low risk of NSB (Fu_b= 0.06).
Scheme 2. Synthesis of analogs 11-14. Reagents and conditions: (a)
o
n-BuLi, THF, -78 C; (b) MsCl, pyridine, Et N, CH Cl , rt; (c) KOH,
Prompted by the favorable profile of (+)-11, we further
examined its selectivity and brain permeability, the data of which
are summarized in Table 2. Similar to the early lead 6, (+)-11
3
2
2
o
ethylene glycol, reflux; (d) PBr , Et O, 0 C; (e) K CO , CH CN, 23,
3
2
2
3
3
rt; (f) KF, Al O , CH CN, reflux;(g) LiOH, MeOH, H O, rt; (h)
2
3
3
2
Me NH.HCl, Et N, DMF, HATU, rt; (i) MeNH .HCl, Et N, DMF,
2
3
2
3
o
o
HATU, rt; (j) BH , THF, 0 C; (k) AcCl, Et N, 0 C.
3
3

exhibited no appreciable activity in δ and κ opioid receptors.
Furthermore, it showed a clean profile in the Bioprint panel
consisting of 73 targets, demonstrating no significant activity at
the high passive permeability and low Pgp liability of (+)-11
indeed translated into good brain permeability, with a C /C
ratio of 0.29. This overall profile supported further progressing
b,u
p,u
1
8
1
0 µM. Its brain permeability was evaluated in a time-course
(+)-11 to tritiation and subsequent tracer characterization studies.
1
9
neuroPK study in rats (0.5 mg/kg, iv). We were glad to see that
Table 1. Potency, selectivity and in vitro ADME profiles of (±)-11-14.
µ Opioid
-
6
NOP receptor
RRCK (x10
MDR1
e
f
Compound
receptor K
i
Selectivity
rFu_b
rFu_p
a
c
d
binding K
i
(nM)
cm/sec)
BA/AB
b
(nM)
PF-7191, (+)-11
0.1
1.7
145.5
231.4
1036x
139x
8.0
1.80
1.63
0.06
0.08
ND
g
(-)-11
13.8
ND
(
(
(
+)-12
0.3
2.8
25.2
75.4
84x
27x
3.4
2.9
2.42
2.11
0.02
ND
0.03
ND
(-)-12
+)-13
0.3
24.7
82x
2x
4.2
5.0
2.01
2.59
0.01
ND
0.02
ND
(-)-13
50.4
119.6
+)-14
0.2
13.2
66x
4x
18.3
15.4
1.26
1.30
0.04
ND
0.06
ND
(
-)-14
44.3
191.1
a
K
i
(geometric mean, n≥ 3 measurements unless specified) determined using a fluorescence polarization assay with Cy
3
B labeled nociceptine in CHO cells
b
expressing human NOP receptors;
K
i
(geometric mean, n≥ 3 measurements unless specified) determined using a fluorescence polarization assay with Cy
3
B
c
labeled dermorphin in CHO cells expressing human µ opioid receptors; RRCK cells with low transporter activity were isolated from Mardin-Darby canine
kidney cells and were used to estimate passive permeability; Ratio from the MS-based quantification of Papp,A→B and Papp,B→A of the test compound (1 µM)
across contiguous monolayers from MDR1-transfected MDCK cells; Unbound fraction of compound in rat brain homogenate; Unbound fraction of compound
in rat plasma; ND, not determined
d
e
d
g
Table 2. Broad spectrum selectivity and NeuroPK of (+)-11
tritiation was accomplished by alkylation of the amide N-H with
tritiated methyl iodide, yielding [ H](+)-11in high radiochemical
3
Property
Result
purity (99%) and specific activity (80 Ci/mmol), which was used
in subsequent ex vivo and in vivo tracer characterization studies.
a
3
δ opioid receptor binding
-1%@ 10 uM
30% @ 10 uM
All < 30%@ 10 uM
141 ng.hr/mL
11 ng.hr/mL
54 ng.hr/mL
3.2 ng.hr/mL
0.29
The bio-distribution of [ H](+)-11 in rat brain was determined
b
through an ex vivo binding study, following a literature protocol
µ opioid receptor binding
3
20
using [ H] N/OFQ. Four brain regions (cortex, hippocampus,
striatum and cerebellum) were examined. The NSB was
determined using a high dose of a selective NOP receptor
antagonist (PF-04926965, 10 µM). As shown in Figure 3, the
c
Bioprint panel
d
C (AUC0–2)
p
3
e
distribution of [ H](+)-11 binding was consistent with known
Cp, u (AUC0–2
)
NOP receptor expression: high in cortex and hippocampus and
low in striatum and cerebellum. Robust specific binding was
observed in all four brain regions tested, with cortex showing the
highest specific binding with a specific/non-specific binding ratio
of ~18.
f
C (AUC0–2)
b
g
Cb, u (AUC0–2
)
h
Cb, u/Cp,u
a
3
Percentage inhibition of [ H]DADLE binding @ 10 µM (n=1) in CHO
b
3
2
cells expressing human δ opioid receptors; Percentage inhibition of [ H]U-
6
9593 binding @ 10 µM (n=1) in CHO cells expressing rat κ opioid
c
d
receptors; Broad-spectrum selectivity panel consisting of 73 targets; AUC0–
e
2
hr of total plasma compound concentration in rats (0.5 mg/kg, iv);
Calculated AUC0–2hr of free plasma compound concentration using fraction
e
unbound in rat plasma (rFu_p); AUC0–2hr of total brain compound
e
concentration in rats (0.5 mg/kg, iv); Calculated AUC0–2hr of free brain
h
compound concentration using fraction unbound in rat brain (rFu_b); AUC0–
2
hr-derived ratio of unbound brain compound concentration-to-unbound
plasma compound concentration in rats.
The tritiation of (+)-11 was carried out using a synthetic route
outlined in Scheme 3. Starting from intermediate 25, amide
formation with ammonia, reduction to the corresponding primary
amine 27 and acylation yielded the racemic precursor, which
upon chiral separation gave the labeling precursor (+)-28. The
3
Scheme 3. Synthesis of radiotracer [ H](+)-11. Reagents and
o
conditions: (a) NH Cl, DIPEA, DMF, BOP, rt; (b) BH , THF, 0 C;
4
3
(
c) AcCl, pyridine, CH Cl , rt; (d) chiral separation; (e) NaH, CT I,
2
2
3
o
DMF, 0 C to rt.

physicochemical and PK properties. From this effort, (+)-11 (PF-
7191) emerged as the promising lead, possessing many desirable
attributes for a radiotracer, e.g. favorable physicochemical
properties, high NOP receptor affinity and selectivity, high
passive permeability, low MDR asymmetry and low risk of NSB.
This favorable in vitro profile translated into good brain
permeability (C /C = 0.29) and high specific binding levels
3
Encouraged by the high specific binding window of [ H](+)-
1
1 in the ex vivo binding study, we next examined it in a rat in
vivo time-course binding study (Figure 4). Rat cortex was chosen
for analysis as it had showed the highest specific binding levels
ex vivo. As shown in Figure 4, [ H](+)-11 demonstrated rapid
brain uptake, reaching a steady state from 5 min (1354 ± 84
DPMs) to 30 min (1496 ± 72 DPMs) followed by a slow wash-
out phase (918 ± 55 DPMs at 60 min). Using a homogenate
filtering procedure, a high percentage (~80%) of the total binding
was determined to be receptor bound at steady state (5-30 min).
Based on this time-activity curve, the 10 min time point was
3
b,u
p,u
(
specific/non-specific = 18 in rat cortex, ex vivo). Importantly,
3
the binding of [ H](+)-11in rat cortex was shown to be inhibited
by a selective NOP receptor antagonist in a dose-response
manner, providing robust in vivo RO determinations. Overall,
3
[
H](+)-11 is a viable radiotracer to support preclinical in vivo
RO measurement in rodents. Its overall favorable profile and
structural amenability to C-11 labeling also warrant further PET
effort for imaging NOP receptors in higher species.
selected for a subsequent in vivo RO study. As shown in Figure
, the rat in vivo binding of [ H](+)-11 was inhibited by PF-
4926965 in a dose-responsive manner, yielding an ED50 of 0.9
3
5
0
mg/kg (Figure 5). Inhibition of binding reached a plateau
between 10 mg/kg and 17.8 mg/kg (data not shown), indicating
that a saturating dose was achieved. This overall favorable profile
%
inhibition
00
3
1
indicated that [ H](+)-11 is a robust radiotracer measuring pre-
clinical in vivo RO.
Total Binding
Non-specific binding
5
0
ED = 0.9 mg/kg
50
0
0
.1
0.32
1
3.2
10
PF-04926965(mg/kg)
3
Figure 5. Dose response inhibition of [ H](+)-11 in rat cortex (100
µCi/kg iv, 10 min) by PF-04926965 (0.1, 0.32, 1, 3.2 and 10 mg/kg
sc, 60 min, n= 3-4/group)
Acknowledgments
3
Figure 3. Bio-distribution of [ H](+)-11 in rat brain (1 nM, 30 min
The authors would like to thank WUXI Pharmatech and GVK
Bio for synthesis support. The authors also thank the ORL-1
project scientist at PGRD Nagoya laboratories for the historical
SAR information and the Pfizer ADME technology group for
generating the in vitro pharmacokinetic data.
incubation); non-specific binding was determined by a high dose of
PF-04926965 (10 µM), a selective NOP receptor antagonist.
.
Total
Bound
References and notes
1
2
.
.
Mollereau, C.; Parmentier, M.; Mailleux, P.; Butour, J. L.;
Moisand, C.; Chalon, P.; Caput, D.; Vassart, G.; Meunier, J. C.
FEBS Lett. 1994, 341, 33.
a) Meunier, J. C.; Mollereau, C.; Toll, L.; Suaudeau, C.; Moisand,
C.; Alvinerie, P.; Butour, J. L.; Guillemot, J. C.; Ferrara, P.;
Monsarrat, B.; Mazarguil, H.; Vassart, G.; Parmentier, M.;
Costentin, J. Nature 1995, 377, 532; b) Reinscheid, R. K.;
Nothacker, H. P.; Bourson, A.; Ardati, A.; Henningsen, R. A.;
Bunzow, J. R.; Grandy, D. K.; Langen, H.; Monsma, F. J., Jr.;
Civelli, O. Science 1995, 270, 792.
3
Time (min) after iv injection of [ H] (+)-11
3
4
.
.
Mollereau, C.; Mouledous, L. Peptides 2000, 21, 907.
Lambert, D. G. Nat. Rev. Drug. Discov. 2008, 7, 694.
3
Figure 4. In vivo time course of [ H](+)-11 in rat cortex (100 µCi/kg
5. Ogawa, M.; Hatano, K.; Kawasumi, Y.; Ishiwata, K.; Kawamura,
iv; 1, 5, 10, 20, 30 and 60 min, n= 3-4/group; bound radioactivity
was measured in filtered cortex homogenate washed with 2x 5 mL
ice-cold buffer (50 mM Tris, pH=7.4); total radioactivity levels were
determined using unfiltered cortex homogenate.
K.; Ozaki, S.; Ito, K. Nucl. Med. Biol. 2003, 30, 51.
6
7
.
.
Pike, V. W.; Rash, K. S.; Chen, Z.; Pedergal, C.; Statnick, M. A.;
Kimura, Y.; Hong, J.; Zoghbi, S. S.; Fujita, M.; Toledo, M. A.;
Diaz, N.; Gackenheimer, S. L.; Tauscher, J. T.; Barth, V. N.;
Innis, R. B. J. Med. Chem. 2011, 54, 2687.
Hostetler, E.; Sanabria-Bohorquez, S.; Eng, W. S.; Joshi, A.;
Patel, S.; Gibson, R.; O’Malley, S.; Krause, S. M.; Ryan, C.;
Riffel, K.; Bi, S.; Okamoto, O.; Kawamoto, H.; Ozaki, S.; Ohta,
H.; de Groot, T.; Bormans, G.; Depre, M.; de Hoon, J.; De
Lepeleire, I.; Reynders, T.; Cook, J.; Burns, H. D.; Egan, M.; Cho,
W.; van Laere, K; Hargreaves, R. J. Neuroimage 2013, 68, 1.
Patel, S.; Gibson, R. Nucl. Med. Biol. 2008, 35, 805.
In conclusion, guided by a set of design and selection criteria
for novel CNS radiotracer development, we carried out a
radiotracer-specific SAR effort in the spirocyclic NOP antagonist
series with a specific focus on improving NOP receptor affinity
and µ opioid receptor selectivity, while maintaining favorable
8
.

9
.
Bojnik, E.; Farkas, J.; Magyar, A.; Tömböly, C.; Güclü, U.;
Gündüz, O.; Borsodi, A.; Corbani, A.; Benyhe, S. Neurochem. Int.
Koike, H.; Matsumoto, Y.; Mihara, S.; Nakamura, H. WO
2006134486 A2, 2006.
2
009, 55, 458.
17. In silico ADME models were developed based on the wealth of
experimental data from high-throughput screening in vitro ADME
assays, which provide large training sets of data points for
structurally diverse compounds. For example, a calculated RRCK
(cRRCK) in silico model was developed based on the RRCK
1
1
0. Bridge, K. E.; Wainwright, A.; Reilly, K.; Oliver, K. R.
Neuroscience 2003, 118, 513.
1. Zhang, L.; Villalobos, A.; Beck, E. M.; Bocan, T.; Chappie, T. A.;
Chen, L.; Grimwood, S.; Heck, S. D.; Helal, C. J.; Hou, X.;
Humphrey, J. M.; Lu, J.; Skaddan, M. B.; McCarthy, T. J.;
Verhoest, P. R.; Wager, T. T.; Zasadny K. J. Med. Chem. 2013,
passive permeability data for
a
diverse set of
>100,000
compounds. The prediction performance of the cRRCK model is
continuously monitored prospectively, typically providing results
within 2-fold of experimental values for 80% of new compounds
meeting the prediction confidence cutoff. For details, please see:
Keefer, C. E.; Kauffman, G. W.; Gupta, R. J. Chem. Inf. Model.
2013, 53, 368.
5
6, 4568.
1
2. RRCK cells were generated in-house (Pfizer Inc., Groton, CT,
USA) as a subclone of Mardin-Darby canine kidney wild-type
(MDCK-WT) cells that displayed low expression of endogenous
P-gp (approximately 1-2% of MDCK-WT cells, based on mRNA
level). For detailed information, see: Callegari, E.; Malhotra, B.;
Bungay, P. J.; Webster, R.; Fenner, K. S.; Kempshall, S.; LaPerle,
J. L.; Michel, M. C.; Kay, G. G. Br. J. Clin. Pharmacol. 2011, 72,
18. For complete broad spectrum selectivity data of (+)-11 (PF-7191),
please see the supplementary material.
19. Shaffer, C. L. Annu. Rep. Med. Chem. 2010, 45, 55.
20. Hayashi, S.; Hirao, A.; Imai, A.; Nakamura, H.; Murata, Y.;
Ohashi, K.; Nakata, E. J. Med. Chem. 2009, 52, 610.
2
35.
1
1
1
1
3. Feng, B.; Mills, J. B.; Davidson, R. E.; Mireles, R. J.; Janiszewski,
J. S.; Troutman, M. D.; de Morais, S. M. Drug Metab. Dispos.
2
008, 36, 268.
Supplementary Material
4. Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M. D.;
Verhoest, P. R.; Villalobos, A.; Will, Y. ACS Chem. Neurosci.
2
010, 1, 420-434.
The broad spectrum selectivity data and preparation procedure
of (+)-11 (PF-7191) can be found in the online version.
5. Di, L.; Umland, J. P.; Chang, G.; Huang, Y.; Lin, Z.; Scott, D. O.;
Troutman, M. D.; Liston, T. E. Drug Metab. Dispos. 2011, 39,
1
270.
6. a) Hirota, M.; Mihara, S.; Nakamura, H.; Koike, H.; Matsumoto,
Y. WO 2005092858 A2, 2005; b) Hashizume, Y.; Hirota, M.;
Click here to remove instruction text...