Synthesis and evaluation of novel radioligands for positron emission tomography imaging of the orexin-2 receptor

Article abstract of DOI:10.1021/jm400772t

Orexin receptors (OXRs) in the brain have been implicated in diverse physiological and neuropsychiatric conditions. Here we describe the design, synthesis, and evaluation of OXR ligands related to (1R,2S)-2-(((2-methyl-4- methoxymethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoropyridin-2-yl) -2-(3-fluorophenyl)cyclopropanecarboxamide (9a) applicable to positron emission tomography (PET) imaging. Structural features were incorporated to increase binding affinity for OXRs, to enable carbon-11 radiolabeling, and to adjust lipophilicity considered optimal for brain penetration and low nonspecific binding. 9a displayed nanomolar affinity for OXRs, and autoradiography using rat brain sections showed that specific binding of [¹¹C]9a was distributed primarily to neocortical layer VI and hypothalamus, consistent with reported localizations of orexin-2 receptors (OX₂Rs). In vivo PET study of [¹¹C]9a demonstrated moderate uptake of radioactivity into rat and monkey brains under deficiency or blockade of P-glycoprotein, and distribution of PET signals in the brain was in agreement with autoradiographic data. Our approach and findings have provided significant information for development of OX₂R PET tracers.

Full text of DOI:10.1021/jm400772t

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Novel Radioligands for Positron
Emission Tomography Imaging of the Orexin‑2 Receptor
Norihito Oi,†,‡,§ Michiyuki Suzuki,†,‡ Taro Terauchi,† Masaki Tokunaga,‡ Yosuke Nakatani,†,‡,§
Noboru Yamamoto,† Toshimitsu Fukumura,‡ Ming-Rong Zhang,‡ Tetsuya Suhara,‡,§
and Makoto Higuchi*,‡,§
†Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan;
‡Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan;
§Graduate School of Medicine, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
S
* Supporting Information
ABSTRACT: Orexin receptors (OXRs) in the brain have
been implicated in diverse physiological and neuropsychiatric
conditions. Here we describe the design, synthesis, and
evaluation of OXR ligands related to (1R,2S)-2-(((2-methyl-
4-methoxymethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoropyri-
din-2-yl)-2-(3-fluorophenyl)cyclopropanecarboxamide (9a)
applicable to positron emission tomography (PET) imaging.
Structural features were incorporated to increase binding
affinity for OXRs, to enable carbon-11 radiolabeling, and to
adjust lipophilicity considered optimal for brain penetration
and low nonspecific binding. 9a displayed nanomolar affinity for OXRs, and autoradiography using rat brain sections showed that
specific binding of [11C]9a was distributed primarily to neocortical layer VI and hypothalamus, consistent with reported
localizations of orexin-2 receptors (OX2Rs). In vivo PET study of [11C]9a demonstrated moderate uptake of radioactivity into rat
and monkey brains under deficiency or blockade of P-glycoprotein, and distribution of PET signals in the brain was in agreement
with autoradiographic data. Our approach and findings have provided significant information for development of OX2R PET
tracers.
Due to the therapeutic potential of modulating these
receptors, this has been an active area of research, primary
for the treatment of insomnia.10−15 Development of almorexant
(1, a dual OX1−OX2R antagonist) from Actelion/GlaxoS-
mithKline was terminated at phase III clinical trial for the
treatment of insomnia. SB-649868 (2, a dual OX1−OX2R
antagonist) for sleep disorders had proceeded to phase II
clinical development prior to 1. Further advancement is
exemplified by suvorexant (3).16 a dual OX1−OX2R antagonist
from Merck, and application of this new drug for the treatment
of insomnia was filed for approval by United States Food and
Drug Administration in 2012.
A suitable radioligand would help to examine the relationship
between the therapeutic effect and receptor occupancy of this
novel class of medicinal agents. A few tritiated tracers, such as
[3H]1,17 [3H]N-ethyl-2-[(6-methoxypyridin-3-yl)-(toluene-2-
sulfonyl)amino]-N-pyridin-3-ylmethyl-1-acetamide
([3H]EMPA, [3H]4),18 and [3H]SB674042 ([3H]5),19 are
known to be usable for in vitro analysis of OXRs.
INTRODUCTION
■
Orexin or hypocretin receptors (OXRs) are G-protein-coupled
receptors (GPCRs) that mediate the central actions of the
endogenous neuropeptides orexin-A/hypocretin-1 (33 amino
acid residues long) and orexin-B/hypocretin-2 (28 amino acid
residues long) produced in the posterior and lateral
hypothalamus.1,2 Though synthesized by only a small
population of neurons in the posterior and lateral hypothal-
amus, orexins exert multiple functions particularly in areas
related to energy homeostasis, sleep/arousal, and brain reward
mechanisms via signaling derived from orexin-1 receptor
(OX1R) and orexin-2 receptor (OX2R) (also known as Hcrt1
and Hcrt2 receptors, respectively). Activation of orexin neurons
contributes to the promotion of maintenance of wakefulness,
and conversely, relative inactivity of orexin neurons induces the
onset of sleep. Preclinical and clinical studies have revealed that
the orexin pathway plays a critical role in motivation and
sleep−wake regulation.3,4 Abnormalities in orexin signaling
have been observed in sleep disorders such as human
narcolepsy−cataplexy syndrome,5 irregularities in central
vestibular motor control,6 a variety of feeding and gastro-
intestinal disorders,7,8 and addiction.9 However, functional
differences between signaling pathways derived from OX1R and
OX2R have not been completely elucidated yet.
Positron emission tomography (PET) is a useful tool for in
vivo quantification of diverse biological processes, but only a
Received: January 30, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Scheme 1. Chemical Structures of Orexin Receptor Antagonists and Radioligands
Scheme 2. Chemical Structures of PET Tracer Candidates and Their 11C-Labeling Versions
small number of PET tracers, including [11C]BBAC ([11C]
6a),20 [11C]BBPC ([11C]6b),20 and [11C]4,21 have been
evaluated for imaging of OXRs. However, in vivo PET studies
of [11C]6a and [11C]6b did not demonstrate the uptake of this
radioligand into the brain of rhesus monkeys.20 [11C]4 also
showed very low uptake into the brain of a baboon as measured
with PET.21 Hence, a novel PET tracer for OXRs would be
useful to understand the relationships between dose and
receptor occupancy, efficacy, and adverse events of therapeutic
agents, to better guide the appropriate dose setting in clinical
trials. Additionally, receptor occupancy could be used as an
objective outcome measure in a therapeutic assessment. This
would particularly serve evaluation of drugs for diseases in
which functional biomarkers such as polysomnography (in case
of insomnia) are unavailable.
It has been documented that mRNAs encoding OX1R and
OX2R are expressed in several distinct regions of the rat
brain.22,23 High structural and functional homology is also
reported for rat and human OX1R and OX2R, and orexins are
well conserved across mammalian species.24 It is of particular
interest to target either OX1R or OX2R by synergistically using
therapeutic and imaging agents. OX2R is present with a
relatively high density in the hippocampal CA3 region,
neocortical layer VI, tuberomammilliary nucleus, induseum
B
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Pharmacological Properties of PET Tracer Candidates
a
b
cLogP value was calculated by Daylight Software ver. 4.94 (Daylight Chemical Information Systems, Inc., Niguel, CA). Log D values were
c
quantified in n-octanol/phosphate buffer (pH 7.4) by the shake-flask method (n = 3; maximum range, 5%). See P-gp Transcellular Transport
Study in Experimental Section. Not determined.
d
griseum, medial thalamic areas, and nucleus accumbens, in
distinction from abundant localization of OX1R to the
prefrontal cortex, hippocampus, paraventricular thalamic
nucleus, ventromedial hypothalamic nucleus, dorsal raphe
nucleus, and locus coeruleus.23 This is consistent with reported
distribution of in vitro autoradiographic labeling with [3H]4,18
which is selective for OX2R, and supports the notion that
specific binding of a PET ligand to the neocortex (particularly
layer VI) could be an indicator of its reactivity with OX2R.
We have attempted to identify suitable PET tracer candidates
for OXRs among cyclopropane derivatives, one of our novel
classes of compounds with relatively highly binding affinity for
OX2R than for OX1R.25 We have selected several cyclopropane
analogs 7a−12a (Scheme 2) as PET tracer candidates for
OX2R imaging based on their binding affinity, lipophilicity, and
flux ratio (FR) for efflux transporter to maximize their ability to
penetrate the blood−brain barrier (BBB) and overall feasibility
of radiolabeling.
RESULTS AND DISCUSSION
■
Selection of PET Tracer Candidates. One hundred and
thirty compounds with potent binding affinity (inhibition
constant Ki < 10 nM) for OX2R were selected as initial PET
tracer candidates. The lipophilicity of this series of compounds
was relatively high with cLogP values ranging from 2.4 to 5.6
(cLogP values; 4.12 0.65, mean SD), and compounds with
cLogP > 4.7 were not taken into consideration. We also looked
at corrected FR calculated between LLC-PK1 cells and
multidrug resistance protein 1 (MDR1) expressed in LLC-
PK1 cells (LLC-MDR1) to narrow down our list of candidate
compounds likely to penetrate BBB, because corrected FR was
demonstrated to be well correlated to and thus highly
predictive of in vivo function of P-glycoprotein (P-gp) located
on cerebral endothelial cells.26 Therefore, compounds with FR
around 3.0 or below were selected. Consequently, 74
compounds met our criteria. Several compounds were then
chosen as for PET tracer candidates based on their affinity and
suitable characteristics as described below.
We report here a systematic strategy for discovering suitable
radioligands that can be used as PET tracers for imaging of
OX2R in the central nervous system (CNS). First, we calculated
the maximal number of binding sites (Bmax) for OX2R in the
brain and estimated the affinity of a compound required for
PET imaging on the basis of this Bmax value. A set of
compounds were then selected in consideration of these affinity
data along with FR values. We also conducted radiosynthesis
and in vivo evaluation of these compounds in rats and a
monkey and found that [11C]9a may have potential as a lead
for further development of PET tracer candidates.
While assessing these candidates, we evaluated the density of
OX2R in layer VI of the neocortex in the rat brain and
calculated Bmax for [3H]4 was 6.5 nM (see Experimental
Section). Because the binding potential for a ligand depends on
the ratio between Bmax and dissociation constant (Kd, in this
context equivalent to inhibition constant, Ki), we prioritized the
Ki value.27−30 High priority was given to the affinity for OX2R
over selectivity for OX2R versus OX1R, because these two
receptor subtypes were assumed to be distinguishable by their
distinct localizations in imaging assays. Six compounds, which
have Ki values equivalent to or smaller than Bmax, displayed
C
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3. Syntheses of Common Intermediate 18
a
Reagents and conditions: (a) NaHMDS, THF, 0 °C, 3 h, then KOH, EtOH, reflux, 8 h, then HCl, 0 °C−rt, 3 h; (b) NaBH4, MeOH−THF, 0 °C−
rt, 1 h; (c) Novozym 435, vinyl acetate, rt, 17 h; (d) DIAD, PPh3, 0 °C−rt, 15 h, then NaOH, EtOH−H2O, rt, 1 h; (e) (COCl)2, DMSO, TEA,
DCM, −60 °C−rt, 1 h, then 2-methyl-2-butene, NaClO2, NaH2PO4, acetone−H2O, rt, 14 h.
favorable properties by meeting most of our requirements as
listed in Table 1. We thereafter radiolabeled all six compounds
for subsequent autoradiographic characterization to validate the
usefulness of selecting these criteria when screening for an
imaging agent.
methoxymethyl (MOM) group was introduced prior to the
first step. To prepare precursors of 9a and 12a, a methanol
moiety on the pyrimidine ring was required. Therefore, hydroxy
precursors, 9c and 12c were converted to 9b and 12b,
respectively, in three steps via bromination of side chain by
using Br2/CHCl3; note that the reaction mainly gave 4-
bromination with a trace amount of 2,4-dibromide. Subsequent
substitution for the acetoxy group with sodium acetate followed
by hydrolysis produced 9b and 12b. Moreover, 10a and its
precursor 10b could be prepared by reacting the common
starting material 18b with 2-amino-5-iodopyridine and 2-
amino-5-cyanopyridine, respectively.
Chemistry.25 Preparation for all common key intermedi-
ates, carboxylic acids 18a−e, is summarized in Scheme 3. The
novel unlabeled compounds 7a−12a were synthesized
according to reaction sequences delineated in Scheme 4.
Cyclopropane ring formation was carried out by the reaction of
an appropriate phenyl acetonitrile 13 with (R)-(−)-epichlor-
ohydrin to give lactone 14.31 Reduction of lactone 14 with
sodium borohydride readily gave corresponding diol 15, which
could be regioselectively acetylated at the 2-methanol position
by Novozyme (lipase acrylic resin from Candida antarctica), to
give the desired monoacetylated compound 16. Mitsunobu
reaction of the corresponding alcohol with various kinds of
hydroxyl pyrimidines led to the production of 17 as an
intermediate. Hydrolysis of 17 with sodium hydroxide
(NaOH), followed by treatment with an appropriate oxidizer,
afforded corresponding carboxylic acid 18.
Radiosynthesis. Radiosynthesis of [11C]7a−12a was
performed using a handmade automated synthesis system
equipped with two units for [11C]methylation and [11C]-
cyanation. The O-[11C]methyl ligands [11C]7a, [11C]8a, [11C]
9a, [11C]11a, and [11C]12a were prepared by reacting
precursor 7b, 8b, 9b, 11b, and 12b with [11C]methyl iodide
([11C]CH3I), respectively, with specific activity of 37−185
GBq/μmol (Scheme 5). [11C]CH3I was prepared by reducing
cyclotron-produced [11C]CO2 with LiAlH4, followed by
iodination with 57% hydroiodic acid.32 For generation of
radiolabeled 7a, 8a, 9a, 11a, and 12a, [11C]CH3I was trapped in
a N,N-dimethylformamide (DMF) solution of precursors 7b,
8b, 9b, 11b, and 12b, respectively, with an appropriate amount
of base at −15 °C. For example, [11C]methylation of 9b with
[11C]CH3I under treatment with NaH proceeded efficiently for
3 min at 40 °C. Semipreparative HPLC purification for the
All of the selected PET tracer candidates 7a−12a were
prepared in an unlabeled form. Also, the corresponding
precursors 7b−12b for radiosynthesis were prepared, as
shown in Scheme 4. Condensation of 18 with various kinds
of pyridines to form an amide bond was carried out via the
amidation of carboxylic acid with aminopyridines or palladium
coupling of carboxamide and chloropyridines.
For candidates with a methoxy group, we prepared
corresponding hydroxy derivative as a radiolabeling precursor.
8b and 11b were prepared from target molecule 8a and 11a,
respectively, by cleavage of the methoxy group with pyridine
hydrochloride. To prepare 7a and its precursor 7b,
reaction mixtures gave [11C]9a with 26
4% (n = 5)
radiochemical yields (decay-corrected) based on [11C]CO2
(Supporting Information: Figure 4). [11C]12a was obtained
from 12b in the same manner. [11C]7a, [11C]8a, and [11C]11a
were prepared from their precursors, 7b, 8b, and 11b,
D
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. Syntheses of 7a−12a and Their Radiolabeling Precursors 7b−12b
a
Reagents and conditions: (a) WSC, HOBt, NH4Cl, DIPEA, DMF, rt, 7 days; (b) 2-chloro-5-fluoropyridine, K3PO4, Xant phos, Pd2(dba)3, dioxane,
80 °C, 1 day; (c) i: Br2, CHCl3, 0 °C−rt, 4 days; ii: AcONa, DMF, 50 °C, 2 h; iii: 1 M NaOH, MeOH, rt, 1.5 h, (d) pyridine−HCl salt, 120 °C, 2 h;
(e) i: (COCl)2, DMF, DCM, rt, 1 h; ii; DIPEA, 2-aminopyridines, THF, 60 °C, 1 h; (f) 2-amino-5-fluoropyridines, HATU, DIPEA, DMF, 0 °C−rt, 3
h; (g) 5 M HCl, THF, rt, 2 h; (h) CH3I, Cs2CO3, DMF, rt, 2 h.
respectively, according to the procedure described for the
synthesis of [11C]9a using K2CO3 instead of NaH. In the case
of [11C]10a, we performed [11C]cyanation of 10b to obtain
[11C]10a, because both 10a and 10b could be readily prepared
in one step from their common precursor 18b.
radioligands did not show radiolysis at room temperature (rt)
for 90 min after formulation, indicating radiochemical stability
over the period of at least one PET scan.
In Vitro Autoradiography Study. Figure 1 shows
representative in vitro autoradiographic images of coronal rat
brain sections with all labeled compounds examined. Among
these radioligands, only [11C]8a and [11C]9a exhibited
detectable specific binding to OX2R in layer VI (red
arrowhead) of the neocortex and hypothalamus, in line with
known distribution of OX2R, and [11C]9a showed the highest
contrast between total (Figure 1E) and nonspecific binding
(Figure 1F) of all tested compounds. Moreover, the local-
izations of [11C]8a and [11C]9a signals were very similar to that
of [3H]4 (Figure 1M, 1N).18 As for the current series of
The identities of [11C]7a−12a were confirmed by
coinjection with the corresponding unlabeled 7a−12a on
reverse phased−analytical HPLC. In the final product solutions,
their radiochemical purities were higher than 99%. Additionally,
specific activity of each product was calculated from the UV
absorption area at 254 nm based on standard curves from
known concentrations of unlabeled samples in common ratio.
The amount of carrier in the final product solution was
measured by the same analytical HPLC. Moreover, these
E
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 5. Radiosynthesis of [11C]7a−12a
a
Reagents and conditions: (a) LiAlH4, THF, −15 °C, 2 min; (b) hydroiodic acid, 180 °C, 2 min; (c) K2CO3, DMF, 50 °C, 3 min; (d) NaH, DMF,
40 °C, 3 min; (e) H2, Ni, 400 °C; (f) NH3, Pt, 960 °C; (g) Na2S2O5, CuSO4, H2O, 80 °C, 2 min; (h) DMF, 165 °C, 5 min.
Figure 1. Representative in vitro autoradiographic images of rat brains. Representative in vitro autoradiographic images of rat brains treated with
[11C]7a (7.6 nM, A, B), [11C]8a (2.0 nM, C, D), [11C]9a (2.5 nM, E, F), [11C]10a (59.0 nM, G, H), [11C]11a (1.6 nM, I, J), [11C]12a (2.6 nM, K,
L), [3H]4 (5.0 nM, M, N). All coronal slices were collected about −4 mm from bregma. (A) [11C]7a only; (B) [11C]7a with 7a (10 μM); (C) [11C]
8a only; (D) [11C]8a with 8a (10 μM); (E) [11C]9a only; (F) [11C]9a with 9a (10 μM); (G) [11C]10a only; (H) [11C]10a with 10a (10 μM); (I)
[11C]11a only; (J) [11C]11a with 11a (10 μM); (K) [11C]12a only; (L) [11C]12a with 12a (10 μM); (M) [3H]4 only; (N) [3H]4 with 4 (10 μM).
compounds, autoradiography results indicate that [11C]9a is
featured by high affinity and relatively low nonspecific binding
related as predicted by cLogP values. By contrast, [11C]7a,
[11C]10a, and [11C]11a did not produce signals reflecting the
distribution of OX2R. Despite the Ki values for [11C]11a
comparable to that of [11C]9a (Table 1), this radioligand
homogeneously bound to the brain slices presumably due to its
higher lipophilicity relative to [11C]9a. To clarify the
relationship between lipophilicity of the compounds and their
performance in autoradiographic imaging, we quantified log D
values of selected chemicals using n-octanol and phosphate
buffer (Table 1). The log D value of [11C]8a (2.93) was
comparable to [11C]9a (2.97), and both radioligands were
capable of producing autoradiographic signals consistent with
OX2R. Meanwhile, the log D value of [11C]11a (3.60) was
higher than those of [11C]8a and [11C]9a, presumably
accounting for homogeneous binding of this compound to
the brain slices. Hence, the log D value may serve as an
informative index in search for appropriate imaging agents.
Failure of staining OX2R with [11C]7a and [11C]10a with a
larger Ki than [11C]8a and [11C]9a indicates that Ki values far
below Bmax are required for high-contrast imaging of OX2R (see
Receptor Binding Assay for Bmax Evaluation in Experimental
Section).
Specificity of Autoradiographic Labeling with [11C]9a
for OX2R versus OX1R. To clarify specificity of [11C]9a
signals in the neocortex for OX2R versus OX1R and other
binding elements, in vitro autoradiographic assays were
performed for rat brain sections under conditions with 3
(OX1R and OX2R dual antagonist) and 4 (OX2R-selective
F
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. In vitro autoradiographic binding of [11C]9a to the rat neocortex in the absence (control) and presence of nonradioactive OXR ligands.
(A) Total binding of [11C]9a (3.6 nM) to the cortical layer VI at baseline and in coincubation with 4 (200 nM), 3 (20 μM), and unlabeled 9a (10
μM). (B) Inhibition of specific radioligand binding as percentage of inhibition by 10 μM unlabeled 9a. We conceived that a decrease of total [11C]9a
binding in the presence of 10 μM unlabeled 9a corresponds to the full abolishment of specific [11C]9a binding, and reduction of total binding in each
condition was normalized by this full abolishment to determine inhibition of specific radioligand binding.
antagonist). Specific binding of [11C]9a in the neocortical layer
VI was decreased by 85% in the presence of 20 μM 3 (Figure
2), suggesting that specific binding sites for [11C]9a are
primarily composed of OXRs. Addition of 200 nM 4 to the
reaction also diminished specific radioligand binding by 70%,
and we accordingly conceived that OX2R was a major specific
binding component for [11C]9a in the neocortex.
unlabeled 9a, because pharmacological effects of orally
administrated 9a at this dose were clearly demonstrated in
our spontaneous locomotion and sleep efficacy tests for wild-
type mice. As shown on the time−radioactivity curves (Figure
3E), pretreatment with unlabeled 9a markedly reduced the
radioactivity compared to control (Figure 3C). Radioligand
retention was significantly inhibited in all brain regions except
the cerebellum, and the distribution of radioactivity became
fairly uniform throughout the brain. This resulted in a decrease
in target-to-cerebellum ratios of radioactivity by the blockade
(Figure 3F), supporting the in vivo imaging of [11C]9a with its
specific and saturable binding sites in the brain.
Activity of 9a on Off-Target Binding Components.
Pharmacological profile of 9a was further investigated by GPCR
panel assays. 9a (10 μM) was inactive on representative 24 off-
target GPCRs, except its weak and modest antagonistic activity
on cannabinoid CB1 and melatonin MT1 receptors, respectively
(Table 2 in Supporting Information). In consideration of the
concentration of 9a reacted here, it is unlikely that a significant
portion of [11C]9a at much lower concentrations in autoradio-
graphic and PET experiments reacted with CB1. In addition,
Additional PET scans were conducted using [11C]8a in P-gp-
knockout rat and wild-type rat, respectively. Overall, [11C]8a
showed time−radioactivity curves similar to that of [11C]9a, but
there was however relatively lower retention compared with
that for [11C]9a (see Supporting Information, Figure 5).
Ex Vivo Metabolite Study in a P-gp-knockout Rat. An
ex vivo metabolite study was performed by using P-gp-
knockout rat (identical to one of the individuals used for
PET study), and we identified a radiometabolite which was
more hydrophilic than the parent tracer in plasma and brain.
Retention times (tRs) for this metabolite and parent compound
were 2.0 and 5.2 min, respectively. More than half of
radioactivity in the brain was derived from the parent tracer
at 30 min postinjection, while less unchanged [11C]9a remained
in plasma (Table 2). These data indicate that in vivo PET
signals in the brains of P-gp-knockout rats (Figure 3A,C) were
composed of [11C]9a and its metabolite.
In Vivo PET Study in Rhesus Monkey. The uptake of
radioactivity in all brain regions peaked at 30 s after intravenous
[11C]9a injection (Figure 4A,B), followed by a rapid decline of
radioactivity and low level retention in the brain (Figure 4B),
suggesting that radioactivity was rapidly cleared from the brain
by P-gp transporter in the initial phase of the scan. To examine
this notion, an additional PET scan with [11C]9a was
conducted under blockade of P-gp transporter. The monkey
used for the baseline PET assay underwent a [11C]9a-PET
measurement (Figure 4C,D), which was initiated at 0.25 h after
intravenous administration of tariquidar (XR9576, 8 mg/kg).34
Pretreatment with tariquidar markedly increased the uptake of
radioactivity into the brain compared to the baseline data.
33
distribution of MT1 is distinct from that of OXRs, and
therefore autoradiographic binding of [11C]9a to MT1 in the
neocortex is presumed to be negligible.
These data are consistent with autoradiographic observations
indicating that specific binding of [11C]9a is attributable to its
interaction with OX2R, although the possibility that 9a binds to
GPCRs and non-GPCR components unexamined here could
not be ruled out. On the basis of these in vitro results, we
further evaluated [11C]8a and [11C]9a using in vivo PET
imaging.
In Vivo PET Studies in Rats. PET scans were conducted
using [11C]9a, because it produced the clearest contrast in
autoradiography imaging. Meanwhile, in vitro assay indicated
that 9a exhibited FR larger than that of the other selected
compounds (Table 1), and therefore P-gp-knockout rats were
used for the PET scan in addition to wild-type rats. Notable
retention of radioactivity in OX2R-rich brain regions in contrast
with its low uptake in the cerebellum, which lacks the receptors,
was observed in P-gp-knockout rats (n = 3) (Figure 3A,C).
However, differences in the radioligand retention in the
neocortex, hippocampus, and thalamus could not be demon-
strated clearly. Additionally, there was only minimal uptake of
radioactivity into the brains of wild-type rats (n = 3) (Figure
3B,D). To further examine the specificity and displaceability of
[11C]9a binding in P-gp-knockout rats, a PET scan was
conducted 1 h after oral administration of 10 mg/kg of
G
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Rat PET study of [11C]9a. (A, B) Representative PET images of P-gp-knockout (A) and wild-type (B) rat brains generated by averaging
dynamic data at 0−90 min after intravenous injection of [11C]9a. PET images are superimposed on a magnetic resonance imaging (MRI) template.
(C, D) Time−radioactivity curves for [11C]9a in the neocortex (CTX), hippocampus (HIP), thalamus (THA), and cerebellum (CER) of P-gp-
knockout (C) and wild-type (D) rat brains (triplicated experiments). (E) Time−radioactivity curves for [11C]9 in the medial prefrontal cortex
(MPFC), HIP, CTX, and CER of P-gp-knockout rat brains after blockade with 9a (10 mg/kg po, 1 h before radioligand injection). (F) Target-to-
cerebellum ratio of AUC in the P-gp-knockout rat brain in control (closed bars) and blockade (open bars) experiments. The radioactivity is
expressed as percentage of injected radioligand dose per unit volume of tissue (% ID/mL).
Similar to in vivo PET data in P-gp-knockout rats (Figure 3C),
radioactivity retention was the highest in the neocortex,
implying that PET signals in the brain of the tariquidar-treated
monkey may reflect distribution of OX2R. As indicated by
autoradiography and PET results, [11C]9a had affinity for
OX2R sufficient for in vivo PET imaging, but its poor BBB
permeability and rapid clearance from the brain did not allow
us to sensitively detect OXRs except for the brains of animals
H
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. Fraction of Unchanged [11C]9a as Percentage of
Total Radioactivity in P-gp-knockout Rat Plasma and Brain
deficient in P-gp or treated with a P-gp inhibitor, highlighting
the significance of FR values for efflux transporters.
fraction of unmetabolized compound
in P-gp-knockout rat tissues (%)
(n = 1)
CONCLUSIONS
■
We developed a novel series of cyclopropane derivatives that
potentially set the path for the development of future potent
PET imaging tracers for OXRs. Based on affinity (Ki ≤ Bmax
6.5 nM), lipophilicity (2.4 < cLogP < 4.7), and efflux properties
of the P-gp transporter (FR ≤ 3), we selected and radiolabeled
time after injection (min)
30
brain
54
plasma
12
=
Figure 4. Rhesus monkey PET study of [11C]9a. (A, C) Orthogonal PET images of rhesus monkey brain generated by averaging dynamic scan data
at 0−90 min after intravenous injection of [11C]9a. Orthogonal images at baseline (A) and under blockade of P-gp by pretreatment with tariquidar
(8 mg/kg) at 0.25 h before radioligand injection (C) are superimposed on individual MRI data. (B, D) Time−radioactivity curves for [11C]9a in the
CTX, HIP, and CER of a rhesus monkey at baseline (B) and under P-gp blockade (D). Radioactivity is expressed as percentage of standardized
uptake value (% SUV).
I
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2. Chemistry. The purity of all compounds, radiolabeling
precursors 7b−12b and unlabeled 7a−12a, was determined by an
analytical HPLC method and was found to be greater than 95% for all
compounds (see Supporting Information, Table 1).
several compounds as PET tracer candidates. Among them,
[11C]9a showed specific binding for OXRs indicated by both in
vitro autoradiography of rat brain slices and in vivo PET
imaging of P-gp-knockout rats, along with the blockade of
radioligand binding by unlabeled compounds and insignificant
reactivity with GPCRs other than OXRs. The Bmax value for the
receptors obtained from in vitro binding assays enabled us to
estimate correctly the affinity of a ligand required for producing
a sufficient imaging contrast, and also the lipophilicity
(measured as log D value) of the compounds had an impact
on the performance of the radioligands in the autoradiographic
imaging well. According to the autoradiographic data of [11C]
8a and [11C]9a (Figure 1C, 1E), a compound with a Bmax/Ki
ratio of 5.0 or higher seems to be required for a PET tracer
candidate to detect specific binding to OX2R.28−30 By
characterizing the binding profile of 9a, it has potential to
visualize neocortical OX2R with adequate specificity (Figure 2).
For in vivo visualization of OX2R in the brain, the permeability
into the CNS, which could be estimated by FR and
lipophilicity, was equally important (Figures 3 and 4).
Moreover, robust metabolic stability is required for further
improvement (Table 2). We could not identify a PET tracer
that can be used to clearly visualize OX2R in wild-type rat and
nonhuman primate. However, we succeeded in clarifying the
preferred profile for an OX2R PET tracer. Hence, our approach
and findings have provided substantial insights into the
development of next-generation OX2R PET tracers.
(1R,2S)-2-(((2,4-Dimethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoro-
4 - h y d r o x y p y r i d i n - 2 - y l ) - 2 - ( 3 - fl u o r o p h e n y l ) -
cyclopropanecarboxamide (8b). A mixture of 8a (36 mg, 0.08 mmol)
and pyridine hydrochloride was stirred at 120 °C for 2 h. The reaction
mixture was allowed to cool to rt, and brine was added to the reaction
mixture. The mixture was extracted with ethyl acetate (AcOEt), dried
over magnesium sulfate (MgSO4), and evaporated in vacuo. The
residue was purified by silica gel column chromatography using n-
heptane/AcOEt (19/1 to 0/1, v/v) to give 8b (34.3 mg, 100% yield)
as a colorless powder; mp: 132−134 °C. 1H NMR (400 MHz,
CD3OD, δ): 1.59−1.68 (m, 1H), 1.84−1.96 (m, 1H), 2.23 (s, 3H),
2.37−2.45 (m, 1H), 2.49 (s, 3H), 4.38−4.47 (m, 1H), 4.64−4.72 (m,
1H), 6.46−6.67 (brs, 1H), 6.95−7.10 (m, 1H), 7.26−7.45 (m, 3H),
7.69 (d, J = 5.1 Hz, 1H), 8.11 (s, 1H). HRMS (FAB) calcd for
C22H20F2N4O3, 427.1582; found, 427.1544.
(1R,2S)-2-(((2-Methyl-4-hydroxymethylpyrimidin-5-yl)oxy)-
methyl)-N-(5-fluoropyridin-2-yl)-2-phenylcyclopropanecarboxa-
mide (12b). A solution of bromine (40 μL, 0.76 mmol) in dry CHCl3
(300 uL) was added dropwise to the solution of (1R,2S)-2-(((2,4-
dimethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoropyridin-2-yl)-2-
phenyl)cyclopropanecarboxamide (12c, 150 mg, 0.38 mmol) in dry
CHCl3 (3 mL) at 0 °C and the reaction mixture stirred at rt for 4 days.
The reaction mixture was quenched with 1 M sodium thiosulfate and
extracted with AcOEt. The organic layer was dried over MgSO4 and
evaporated in vacuo. The residue was purified by silica gel column
chromatography using n-heptane/AcOEt (7/3, v/v) to give (1R,2S)-2-
(((2-methyl-4-bromomethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoro-
pyridin-2-yl)-2-phenylcyclopropanecarboxamide (75 mg, 41.6% yield)
1
as a colorless powder. H NMR (300 MHz, CDCl3, δ): 1.64 (dd, J =
EXPERIMENTAL SECTION
■
5.2, 8.1 Hz, 1H), 1.92 (t, J = 5.2 Hz, 1H), 2.15 (dd, J = 5.2, 8.1 Hz,
1H), 2.58 (s, 3H), 4.24 (s, 2H), 4.56 (s, 2H), 7.30−7.43 (m, 4H),
7.48−7.54 (m, 2H), 8.05−8.12 (m, 1H), 8.12 (d, J = 3.2 Hz, 1H), 8.13
(s, 1H), 8.33 (brs, 1H). HRMS (FAB) calcd for C22H20BrFN4O2,
471.0832; found, 471.0849.
1. Materials and Methods. Melting points were measured using a
micro melting point apparatus (MP-500P; Yanaco, Tokyo, Japan) and
1
are uncorrected. H NMR spectra were recorded on a JEOL-AL-300
spectrometer (operating at 300 MHz, JEOL, Tokyo, Japan) or Varian
Mercury 400 spectrometer (operating at 400 MHz, Varian, Palo Alto,
CA) or Bruker Avance 600 spectrometer (operating at 600 MHz,
Bruker BioSpin, Ettlingen, Germany), with tetramethylsilane as an
internal standard. All chemical shifts (δ) were reported in parts per
million (ppm) downfield relative to the chemical shift of
tetramethylsilane. Signals are quoted as s (singlet), d (doublet), dt
(double triplet), t (triplet), q (quartet), or m (multiplet). Fast atom
bombardment mass spectra (FAB−MS) and high-resolution mass
spectra (HRMS) were obtained on a JEOL-AL-300 spectrometer
(JEOL) and recorded on the spectrometer. Electrospray ionization
mass spectra (ESI-MS) were recorded on ThermoFisherScientific
LTQ-Orbitrap XL spectrometer (ThermoFisherScientific Inc., Wal-
tham, MA). Column chromatography was performed using Fuji Silysia
BW-300 (300 mesh). Reverse phase HPLC was performed using a
JASCO HPLC module (JASCO, Tokyo, Japan) equipped with YMC
Pack Pro C18 columns (S-5 μm, 10 mm ID × 250 mm, YMC, Kyoto,
Japan) utilizing a 0.1% triethylamine (TEA) in water/methanol or
0.1% TEA in water/acetonitrile (MeCN) for semiseparative
purification. Chemical purity analysis was carried out by using YMC
Pack Pro C18 columns (S-5 μm, 4.6 mm ID × 150 mm, YMC) with
UV detector at 254 nm. Under radio-HPLC purification and analysis,
an effluent of radio activity was monitored by using a NaI (Tl)
scintillation detector system. All chemical reagents and solvents were
purchased from commercial sources (Sigma-Aldrich, St. Louis, MO;
Wako Pure Chemical Industries, Osaka, Japan; Tokyo Chemical
Industries, Tokyo, Japan) and used as supplied. [3H]4 was synthesized
in PerkinElmer Life & Analytical Sciences (Waltham, MA). Tariquidar
was purchased from Sequoia Research Products (Pangbourne, UK).
Carbon-11 (11C) dioxide was produced by the 14N(p,α)11C nuclear
reactions using CYPRIS HM-18 cyclotron (Sumitomo Heavy Industry,
Tokyo, Japan). If not otherwise stated, radioactivity was measured with
an IGC-3R Curiemeter (Aloka, Tokyo, Japan).
Sodium acetate (37 mg, 0.45 mmol) was added to the solution of
the above-mentioned bromide (70 mg, 0.15 mmol) in DMF (2 mL),
and the reaction mixture was heated at 50 °C for 2.5 h. The reaction
mixture was partitioned between AcOEt and brine and extracted with
AcOEt. The organic layer was dried over MgSO4 and evaporated in
vacuo. The residue was dissolved in MeOH (10 mL), and 1 M NaOH
(10 mL) was added to the reaction mixture at rt and stirred for 1.5 h.
The reaction mixture was partitioned between AcOEt and brine and
extracted with AcOEt. The organic layer was dried over MgSO4 and
evaporated in vacuo. The residue was purified by silica gel column
chromatography using n-heptane/AcOEt (4/6, v/v) to give 12b (49
1
mg, 81.0% yield) as a colorless amorphous substance. H NMR (300
MHz, CDCl3, δ): 1.62 (dd, J = 5.3, 7.9 Hz, 1H), 1.89 (t, J = 5.3 Hz,
1H), 2.13 (dd, J = 5.3, 7.9 Hz, 1H), 2.60 (s, 3H), 3.89 (brs, 1H), 4.43
(d, J = 17.6 Hz, 1H), 4.44 (d, J = 9.5 Hz, 1H), 4.52 (d, J = 9.5 Hz,
1H), 4.53 (d, J = 17.6 Hz, 1H), 7.27−7.47 (m, 6H), 8.02 (s, 1H), 8.07
(dd, J = 3.9, 9.1 Hz, 1H), 8.13 (d, J = 2.9 Hz, 1H), 8.35 (s, 1H).
HRMS (FAB) calcd for C22H21FN4O3, 409.1676; found, 409.1629.
(1R,2S)-2-(((2-Methyl-4-hydroxymethylpyrimidin-5-yl)oxy)-
methyl)-N-(5-fluoropyridin-2-yl)-2-(3-fluorophenyl)-
cyclopropanecarboxamide (9b). Compound 9b was prepared
according to the procedure described for the synthesis of 12b using
(1R,2S)-2-(((2-methyl-4-bromomethylpyrimidin-5-yl)oxy)methyl)-N-
(5-fluoropyridin-2-yl)-2-(3-fluorophenyl)cyclopropanecarboxamide
(100 mg, 0.21 mmol), sodium acetate (60 mg, 0.62 mmol) in DMF
(10 mL), and 1 M NaOH (2 mL) in EtOH (10 mL). The product was
purified by silica gel column chromatography using n-heptane/AcOEt
(3/7, v/v) to give 9b (53 mg, 61% yield) as a colorless powder; mp:
73−75 °C. 1H NMR (600 MHz, CD3OD, δ): 1.56 (t, J = 6.0 Hz, 1H),
1.85 (t, J = 6.0 Hz, 1H), 2.49 (t, J = 6.0 Hz, 1H), 2.53 (s, 3H), 4.41 (d,
J = 12.0 Hz, 1H), 4.49 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 12.0 Hz, 1H),
4.66 (d, J = 12.0 Hz, 1H), 6.98−7.04 (m, 1H), 7.32−7.36 (m, 1H),
J
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7.36−7.39 (m, 2H), 7.44−7.50 (m, 1H), 7.90−7.95 (m, 1H), 8.13 (s,
1H), 8.17 (brs, 1H). ESI-MS: m/z 449 (M + Na). HRMS (FAB) calcd
for C22H20F2N4O3, 427.1582; found, 427.1554.
3. Radiochemistry. All labeled compounds were formulated with
sterile saline (3 mL) containing Tween 80 (100 μL) and ascorbic acid
(25 mg) after HPLC purification. HPLC analytical and purification
conditions for all labeled compounds [11C]7a−12a are shown in the
Supporting Information.
[11C]9a, [11C]12a was obtained from 12b (2.40 mg, 5.0 μmol). The
reaction mixture was purified by HPLC (YMC Pack Pro C18) using the
mobile phase of MeCN/H2O/TEA (4.5/5.5/0.01, v/v/v) at a flow
rate of 5.0 mL/min to give 1.24 GBq of [11C]12a. The tR of [11C]12a
was 11.3 min for purification and 8.2 min for analysis on HPLC. The
synthesis time from EOB, 30.1 min; radiochemical yield decay-
corrected, 22.1% based on [11C]CO2; radiochemical purity, >99%;
specific activity at EOS, 101 GBq/μmol.
(1R,2S)-2-(((2,4-Dimethylpyrimidin-5-yl)oxy)methyl)-N-(5-[11C]-
cyanopyridin-2-yl)-2-(3-fluorophenyl)cyclopropanecarboxamide
([11C]10a). [11C]HCN Preparation. [11C]HCN was synthesized by a
handmade device in a two-step sequence of reaction. After [11C]CO2
in N2 gas from the cyclotron was trapped at −196 °C, it was heated to
50 °C, moved under N2 stream (flow rate of 10 mL/min), and mixed
with H2 gas at a flow rate of 10 mL/min. The mixed gas was passed
through a Ni wire tube at 400 °C in the methanizer to give a mixture
of [11C]CH4 in the carrier gas. Then it was mixed with 5% NH3 in N2
(v/v) gas stream at a flow rate of 400 mL/min and passed through Pt
furnace at 950 °C to give a [11C]HCN-containing gas, which was
absorbed to the reaction solution via a bubbling tube until the
radioactivity of the vessel reached saturation (45 s). The average of the
total radioactivity recovered in the reaction vessel was about 70%
based on [11C]CO2 at EOS. The synthesis of [11C]10a from
[11C]HCN via [11C]CuCN was successfully carried out.35,36
A freshly prepared solution of sodium metabisulfate (150 μL, 48
mM; 7.2 μmol) was added to a solution of copper(II) sulfate (100 μL,
44 mM; 6.6 μmol) at rt under N2 stream 10 min prior to EOB.
[11C]HCN gas was bubbled into the mixture at rt and a flow rate of
400 mL/min until the radioactivity reached saturation. The solution
was then heated to 80 °C for 2 min. A solution of 10b (3.5 mg, 6.8
μmol) in DMF (250 μL) was added to the reaction mixture at rt and
heated to 165 °C for 5 min. The reaction mixture was purified by
HPLC (YMC Pack Pro C18) using the mobile phase of MeCN/H2O/
TEA (5/5/0.01, v/v/v) at a flow rate of 5.0 mL/min to give 199.4
MBq of [11C]10a. The tR of [11C]10a was 11.0 min for purification
and 5.2 min for analysis on HPLC. The synthesis time from EOB, 33.8
min; radiochemical yield (decay-corrected), 2.8% based on [11C]CO2;
radiochemical purity, >99%; specific activity at EOS, 47 GBq/μmol.
4. Receptor Binding Assay for Bmax Evaluation. Preparation of
Brain Homogenate. Parts of the cerebral cortex which contained
mainly layer VI were collected from Sprague−Dawley (SD) rats (male,
7 weeks old, n = 11, Charles River, Yokohama, Japan). Wet cerebral
cortex tissues were weighed, homogenized in 0.32 M sucrose and 10
mM Tris-HCl (pH 8.0) on ice, and centrifuged (1000g, 4 °C, 10 min).
The supernatant was collected and centrifuged (30 000g, 4 °C, 20
min). The precipitate was suspended in 1 mM ethylene glycol bis(2-
aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and 50 mM
Tris-HCl (pH 8.0) on ice and centrifuged (30 000g, 4 °C, 20 min);
this procedure was conducted two times. Next the precipitate was
suspended in 2 mM EGTA, 10 mM MgCl2, and 20 mM 4-(2-
hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES, pH 7.4) on ice
and centrifuged (30 000g, 4 °C, 20 min); this procedure was repeated
three times. The precipitate was suspended in 2 mM EGTA, 10 mM
MgCl2, and 20 mM HEPES (pH 7.4). This microsomal fraction was
stored as a receptor stock solution at −80 °C until use.
(1R,2S)-2-(((2,4-Dimethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoro-
6 - [ 1 1 C ] m e t h o x y p y r i d i n - 2 - y l ) - 2 - ( 3 - fl u o r o p h e n y l ) -
cyclopropanecarboxamide ([11C]11a). [11C]CH3I was synthesized
from cyclotron-produced [11C]CO2 as described previously.32 Briefly,
[11C]CO2 was bubbled into 0.04 M LiAlH4 in anhydrous THF (300
μL). After evaporation of THF, the remaining complex was treated
with 57% hydroiodic acid (300 μL) to give [11C]CH3I, which was
distilled at 180 °C and transferred under helium gas into a solution of
11b (1.0 mg, 2.3 μmol) and 0.5 M K2CO3 (5 μL, 2.5 μmol) in
anhydrous DMF (300 μL) at −15 to −20 °C. After radioactivity
reached saturation, this reaction mixture was heated at 50 °C for 3
min. The reaction mixture was applied to a semipreparative HPLC
system. HPLC (YMC Pack Pro C18) purification was completed using
the mobile phase of MeCN/H2O/TEA (6/4/0.01, v/v/v) at a flow
rate of 5.0 mL/min. The radioactive fraction corresponding to the
desire product was collected in a sterile flask, evaporated to dryness in
vacuo, redissolved in 3 mL of sterile saline, and passed through a 0.22
μm Millipore filter to give 2.23 GBq of [11C]11a. The tR of [11C]11a
was 8.8 min for purification and 6.2 min for analysis on HPLC. The
specific activity of [11C]11a was calculated by comparison of the
assayed radioactivity to the mass associated with the carrier UV peak at
254 nm. The synthesis time from end of bombardment (EOB), 31.5
min; radiochemical yield (decay-corrected), 36% based on [11C]CO2;
radiochemical purity, > 99%; specific activity at end of synthesis
(EOS), 106 GBq/μmol.
(1R,2S)-2-(((2,4-Dimethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoro-
p y r i d i n - 2 - y l ) - 2 - ( 3 - fl u o r o - 4 - [ 1 1 C ] m e t h o x y p h e n y l ) -
cyclopropanecarboxamide ([11C]7a). Following the same procedure
as that described for [11C]11a, [11C]7a was obtained from 7b (1.60
mg, 3.8 μmol). The reaction mixture was purified by HPLC (YMC
Pack Pro C18) using the mobile phase of MeOH/H2O/TEA (7/3/
0.01, v/v/v) at a flow rate of 4.0 mL/min to give 1.27 GBq of [11C]7a.
The tR of [11C]7a was 9.6 min for purification and 5.0 min for analysis
on HPLC. The synthesis time from EOB, 28.1 min; radiochemical
yield decay-corrected), 28.0% based on [11C]CO2; radiochemical
purity, >99%; specific activity at EOS, 70 GBq/μmol.
(1R,2S)-2-(((2,4-Dimethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoro-
4 - [ 1 1 C ] m e t h o x y p y r i d i n - 2 - y l ) - 2 - ( 3 - fl u o r o p h e n y l ) -
cyclopropanecarboxamide ([11C]8a). Following the same procedure
as that described for [11C]11a, [11C]8a was obtained from 8b (1.33
mg, 3.1 μmol). The reaction mixture was purified by HPLC (YMC
Pack Pro C18) using the mobile phase of MeOH/H2O/TEA (5/5/
0.01, v/v/v) at a flow rate of 5.0 mL/min to give 1.05 GBq of [11C]8a.
The tR of [11C]8a was 9.7 min for purification and 8.4 min for analysis
on HPLC. The synthesis time from EOB, 27.5 min; radiochemical
yield decay-corrected, 10.7% based on [11C]CO2; radiochemical
purity, >99%; specific activity at EOS, 107 GBq/μmol.
(1R,2S)-2-(((2-Methyl-4-[11C]methoxymethylpyrimidin-5-yl)oxy)-
methyl)-N-(5-fluoropyridin-2-yl)-2-(3-fluorophenyl)-
cyclopropanecarboxamide ([11C]9a). [11C]CH3I was introduced to a
solution of 9b (1.87 mg, 4.4 μmol) and NaH (60% in oil, 600 μg, 15
μmol) in anhydrous DMF (250 μL) at −15 to −20 °C. The reaction
mixture was heated at 40 °C for 3 min and purified by HPLC to give
[11C]9a. The reaction mixture was purified by HPLC (YMC Pack Pro
C18) using the mobile phase of MeCN/H2O/TEA (4.5/5.5/0.01, v/v/
v) at a flow rate of 5.0 mL/min to give 1.55 GBq of [11C]9a. The tR of
[11C]9a was 7.3 min for purification and 11.5 min for analysis on
HPLC. The synthesis time from EOB, 33.5 min; radiochemical yield
decay-corrected), 21.6% based on [11C]CO2; radiochemical purity,
>99%; specific activity at EOS, 120 GBq/μmol.
Binding Assay. Receptor stock solution was resuspended in binding
buffer (1 mM CaCl2, 5 mM MgCl2, and 25 mM HEPES, pH 7.4) to a
final concentration of 5 mg tissue equiv/assay. The incubation time for
[3H]4 on OX2R was 60 min at 25 °C. After incubation, membranes
were filtered onto a GF/B filter and washed three times with ice−cold
wash buffer (same as the binding buffer). Each filter was placed in a
vial and 5 mL of liquid scintillator reagent (Atomlight; PerkinElmer
Life & Analytical Sciences) were added. Radioactivity was counted (2
min) in a liquid scintillation counter (2500, PerkinElmer Life &
Analytical Sciences).
Saturation isotherms were determined by addition of various
concentrations of [3H]4 (5−1280 nM). Nonspecific binding for [3H]4
was measured in the presence of 0.1 mM unlabeled 4. Bmax was
calculated by Scatchard analysis of the saturation isotherm experiment.
(1R,2S)-2-(((2-Methyl-4-[11C]methoxymethylpyrimidin-5-yl)oxy)-
methyl)-N-(5-fluoropyridin-2-yl)-2-phenylcyclopropanecarboxa-
mide ([11C]12a). Following the same procedure as that described for
K
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
5. Measurement of Lipophilicity. cLogP values of tested
compounds were determined computationally using Daylight Software
ver. 4.94 (Daylight Chemical Information Systems, Inc., Niguel, CA).
log D values of selected compounds were measured by mixing 11C-
labeled compound (radiochemical purity, 100%; about 200 000 cpm)
with n-octanol (3.0 g) and phosphate-buffered saline (PBS; 3.0 g, 0.1
M, pH 7.4) in a test tube. The mixture was vortexed for 3 min at rt,
followed by centrifugation at 2330g for 5 min. Aliquots of 1 mL of PBS
and 1 mL of n-octanol were removed and weighed, and their
radioactivity was quantified with a 1480 Wizard automatic γ counter
(Perkin-Elmer, Waltham, MA). Sample from the remaining organic
layer was removed and repartitioned until a consistent log D was
obtained. The log D value was calculated by the following formula: log
D = log[(cpm/g n-octanol)/(cpm/g PBS)]. All measurements were
performed in triplicate.
membrane suspension of OX1R or OX2R containing 5 μg protein was
mixed with 10 μL of compounds, Orexin A (100 μM, Peptide
Institute, Osaka, Japan) solution or vehicle and 10 μL of [125I]-orexin
A solution (2 nM, PerkinElmer, Waltham, MA) and 60 μL of assay
buffer (final volume: 100 μL) and the mixture incubated for 30 min at
rt on a 96-well Flashplate (PerkinElmer). All reaction mixtures were
discarded and washed with 200 μL of 25 mM HEPES buffer
containing 525 mM of NaCl. The radioactivity (disintegrations per
minute, DPM) of each well was measured using a TopCount device
(PerkinElmer). The inhibitory activities of test compounds were
calculated using the following formula:
inhibition % = 100 − 100 × (T − N)/(C − N)
T: DPM in the presence of compounds (test)
N: 10 μM orexin A (nonspecific binding)
6. P-gp Transcellular Transport Study.26,37 LLC-PK1 and
LLC-MDR1 cells were seeded at a density of 4.0 × 105 cells/cm2 onto
porous membrane filters of 24-well cell culture insert plates. Cells were
cultured in 5% CO2 at 37 °C and were used for the transport studies 5
days after seeding. For transcellular transport experiments, each cell
monolayer was preincubated at 37 °C for 2 h in Hank’s balanced salt
solution containing 10 mM HEPES (HBSS buffer).
C: DPM in the absence of compound (control)
All values were determined as averages of three wells in a single
measurement.
9. In Vitro Autoradiography. For Screening. Rat brain sections
(20 μm thick) were dried at rt and preincubated for 20 min in 25 mM
HEPES buffer (pH 7.4) containing 5 mM MgCl2 and 1 mM CaCl2 at
rt. After preincubation, these sections were incubated for 60 min at rt
in fresh buffer with [11C]7a (7.6 nM), [11C]8a (2.0 nM), [11C]9a (2.5
nM), [11C]10a (59.0 nM), [11C]11a (1.6 nM), [11C]12a (2.6 nM),
respectively. Unlabeled compounds 7a−12a (10 μM) were used to
assess the nonspecific binding of these radioligands in the brain. After
incubation, brain sections were rapidly washed with cold distilled water
three times and dried at rt. An imaging plate (BAS-IP MS2025,
Fujifilm, Tokyo, Japan) was exposed to the dried sections for 1 h.
Radioactive standards calibrated with known amounts of [11C]7a−
[11C]12a were induced in the exposure process. Quantitative
autoradiogram analysis was performed using a computer-assisted
image analyzer (Multi Gauge; Fujifilm). Optical density values were
converted to fmol/mg protein using a computer-generated regression
analysis which compared film densities produced by tissue sections and
radioactive standards.
Transcellular transport experiments were initiated by adding the
solution of test compounds to the apical or basal side of the cell
culture inserts. After incubation at 37 °C for 2 h, HBSS buffer was
sampled from the compartment opposite to that spiked with test
compounds and was analyzed for test compound concentration by LC-
MS. The apparent permeability coefficient (Papp) of the test
compounds were estimated using eq 1, where Q, t, C0, and A
represent permeated amount of test compounds, incubation time,
initial concentration of test compounds, and membrane area,
respectively.
P
= Q /t/C0/A
app
(1)
Both FRs across the monolayer, LLC-PK1 and LLC-MDR1, were
defined according to eq 2, where Papp, b to a and Papp, a to b represent the
Papp in the basal-to-apical direction and the apical-to-basal, and the
corrected FR was defined by eq 3.
Assessment of Specificity of 9a for OXRs. All procedures were
performed as in the above-mentioned screening method. [11C]9a (3.6
nM) and unlabeled compounds 3 (20 μM) and 4 (200 nM) were
used. Coronal slices were collected at +1.5 mm and −3.0 mm to
bregma. Representative autoradiograms were acquired from serial
brain sections, and the neocortical layer VI in each slice was encircled
based on the brain atlas. Radioactivity in this area was then measured
in eight slices (consisting of two slices generated at +1.5 mm to
bregma and six slices at −3.0 mm to bregma, n = 3).
10. Animal PET Studies. Wild-Type Rats. Prior to PET scans,
anatomical template images of the rat brain were generated by a high-
resolution MRI system. Briefly, a rat was anesthetized with sodium
pentobarbital (50 mg/kg, ip) and scanned with a 400 mm bore, 7 T
horizontal magnet (NIRS/KOBELCO, Kobe, Japan/Bruker BioSpin)
equipped with 120 mm diameter gradients (Bruker BioSpin). A 72
mm diameter coil was used for radiofrequency transmission, and
signals were received by a four-channel surface coil. Coronal T2-
weighted MRI images were obtained by a fast spin−echo sequence
with the following imaging parameters: repetition time = 8000 ms,
effective echo time = 15 ms, field of view (FOV) = 35 mm × 35 mm,
and slice thickness = 0.6 mm.
PET scans for anesthetized SD rats (male, 410−500 g) were
performed with a small animal-dedicated micro PET FOCUS 220
system (Siemens Medical Solutions, Malvern, PA), which yields a 25.8
cm (transaxial) × 7.6 cm (axial) FOV, and a spatial resolution of 1.3
mm full-width at half-maximum (fwhm) at the center of FOV.38
Subsequently, list-mode scans were performed for 90 min. All list-
mode data were sorted and Fourier-rebinned into two-dimensional
sinograms (frames, 1 min × 4 scans, 2 min × 8 scans, 5 min × 14
scans). Images were thereafter reconstructed using two-dimensional
filtered back-projection with a 0.5-mm Hanning filter. To inject [11C]
9a, a 24-gauge needle with catheter (Terumo, Tokyo, Japan) was
FR = Papp,btoa/P
app,atob
(2)
corrected FR = (FR in LLC‐MDR1cells)/(FR in LLC‐PK1cells)
(3)
7. Animals. SD rats at 9−11 weeks of age (male, 410−500 g) were
purchased from Japan SLC (Shizuoka, Japan), and P-gp-knockout rats
at 10 weeks of age (Mdr1a knockout rats, male, 410−500 g) were
purchased from SAGE Laboratories (St. Louis, MO). The animals
were housed under a 12 h light−dark cycle, allowed free access to food
pellets and water, and used for in vivo PET studies. A rhesus monkey
at 3 years and 8 months of age (male, 4.25 kg) was purchased from
Japan SLC (Shizuoka, Japan). The monkey was housed in an
individual cage and was supplied with a balanced diet and ad libitum
tap water from a feeding valve. The room was illuminated from 7 a.m.
to 9 p.m. Age and body weight of the monkey at that time of PET scan
was 3 years and 11 months and 4.65 kg, respectively. The animal
experiments were approved by the Animal Ethics Committee of
National Institute of Radiological Sciences (NIRS).
8. In Vitro Binding Assays for Ki Value Evaluation. Human
OX1R- or OX2R-expressing CHO cells (Chinese hamster ovary cells)
were homogenized in 20 mM HEPES buffer (pH = 7.4) containing 10
mM MgCl2 and 2 mM EGTA with a Polytron homogenizer
(Kinematica AG, Lucerne, Switzerland). The homogenate was
centrifuged at 1000g for 10 min at 4 °C, and the supernatant was
further centrifuged at 40 000g for 45 min at 4 °C (Beckman Coulter,
Fullerton, CA). The pellet was resuspended in the same buffer and
stored at −80 °C before use.
Cell homogenate was diluted to 250 μg/mL in 25 mM HEPES
buffer (pH = 7.5) containing 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA,
0.1% sodium azide, and 0.05% Tween 20. Twenty microliters of
L
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
placed into the rat tail vein. The injected dose of the radioligand was
78.7−189.8 MBq (127.6 43.1 MBq, mean SD). Body temperature
was maintained with a 37 °C plate heater (Bio Research Center Inc.,
Aichi, Japan). During the scan, the rat was maintained under
anesthesia with 1.5% (v/v) isoflurane. For the inhibition study,
unlabeled 9a (10 mg/kg, po, dissolved 9a with 5% DMSO and 10%
cremophore in 10 mM HCl, in H2O) was administered to the rat 1 h
before the radioligand injection.
Gi signaling to Ca2+ response.40 Thereafter, GPCRs+Gqi5/HEK293
and Gqi5/HEK293 cells were seeded separately at 5000/25 μL/well
into collagen type I-coated 384-well plates and cultured overnight in
Dulbecco’s Modified Eagle’s Medium. Subsequently, calcium-sensitive
fluorescent dye was added to cells and incubated for 1 h at 37 °C.
Measurement of Agonistic Activity. For quantification of agonistic
activity, 9a was added to cells and calcium response was recorded.
Solutions of agonistic reference ligands were diluted in wells of cell
plates with equal volumes (25 μL) of cell culture medium, calcium dye,
and assay buffer. Solutions of 9a were diluted in wells of cell plates
wells with equal volumes (25 μL each) of cell culture medium and
calcium dye, resulting in final concentrations of 3, 10, and 30 μM,
respectively.
Measurement of Antagonistic Activity against Low or High Dose
of Agonistic Reference Ligand. For quantification of antagonistic
activity, solutions of 9a were diluted in wells of cell plates with equal
volumes (25 μL each) of cell culture medium, calcium dye, and an
excess concentration of agonistic reference ligands resulting in final
concentrations of 3, 10, and 30 μM for 9a. Cells were first incubated
with 9a for 1 h at rt, and then reference ligands at a dose inducing
either 20% (EC20) or 80% (EC80) of maximal response were added to
detect inhibitory effects of 9a against these reference ligands. Ca2+
concentration was visualized in real time using a calcium indicator dye
(Calcium 4, Molecular Devices, Sunnyvale, CA), and fluorescence was
detected by the FDSS7000 device (Hamamatsu Photonics, Hama-
matsu, Japan).
P-gp-knockout Rats. PET scans for P-gp-knockout rats (male,
410−500 g) were performed in the same manner as in the scans for
SD rats.
Rhesus Monkey. MRI images of the monkey brain were generated
by an apparatus identical to that for the above-mentioned rat MRI.
Horizontal brain MRI images were obtained by a gradient echo pulse
sequence termed FLASH with the following imaging parameters:
repetition time = 481 ms, effective echo time = 7.6 ms, FOV = 110
mm × 110 mm, slice thickness = 1.5 mm. PET scans for a monkey was
performed using a high-resolution SHR-7700 PET camera (Hama-
matsu Photonics, Shizuoka, Japan) designed for laboratory animals,
which provides 31 transaxial slices 3.6 mm (center-to-center) apart
and a 33.1 cm (transaxial) × 11.16 cm (axial) FOV. The spatial
resolution for the reconstructed images was 2.6 mm fwhm at the
center of FOV.39 Prior to PET scans, the monkey was initially
anesthetized with thiamylal, and anesthesia was maintained using 1.5%
(v/v) isoflurane. Following a transmission scan for attenuation
correction using a 68Ge-68Ga source, dynamic emission scans were
conducted in a three-dimensional acquisition mode for 90 min
(frames, 1 min × 4 scans, 2 min × 8 scans, 5 min × 14 scans).
Emission scan images were reconstructed with a 4 mm Colsher filter.
[11C]9a was injected via the crural vein as a single bolus at the start of
emission scanning. Injected dose of the radioligand was 115.5 MBq.
An additional PET measurement was carried out by injecting a P-gp
inhibitor, tariquidar (8 mg/kg/5 mL of saline with 10% DMSO), via
the crural vein as a single slow bolus over 10 min at 0.25 h before
[11C]9a administration. Injected dose of the radioligand was 87.7
MBq. All PET scans were acquired in the same monkey.
ASSOCIATED CONTENT
* Supporting Information
■
S
Purities of 7a−12a and precursors 7b−12b determined by
HPLC. Spectrum data for 7a−12a and precursors 7b−12b.
HPLC analytic charts for 7a−12a and precursors 7b−12b;.
HPLC purification charts for [11C]7a−[11C]12a. HPLC
analytical charts for [11C]7a−[11C]12a. GPCR panel assays
to determine off-target activity of 10 μM of 9a. Rat PET study
of [11C]8a. This material is available free of charge via the
Internet at http://pubs.acs.org.
Image Acquisition and Data Analysis. Anatomical regions of
interest (ROIs) were manually defined on the striatum, thalamus,
pons, and cerebellum in the PET images coregistered with MRI images
using PMOD software (PMOD Technologies Ltd., Zurich, Switzer-
land). Regional radioactivity in the brain was decay-corrected to the
injection time and was expressed as the percent of injected dose (%
ID/mL = % injected dose/cm3 brain) in rat experiments and as the
percentage standardized uptake value [% SUV = % ID/mL × body
weight (g)] in monkey experiments.
AUTHOR INFORMATION
Corresponding Author
*Phone: +81 43 206 4700. Fax: +81 43 253 0396. E-mail:
mhiguchi@nirs.go.jp.
■
Notes
11. Metabolite Study for Rat Plasma and Brain. After the
intravenous injection of [11C]9a (330 MBq/0.1 mL), a P-gp-knockout
rat (male Mdr1a-knockout rat weighing 450 g, identical to one of the
individuals used for the PET imaging, male) was killed by decapitation
at 30 min. Blood and whole brain were quickly removed, and the
blood sample (0.5 mL) was centrifuged at 20 000g for 2 min.at 4 °C to
separate plasma. The supernatant (0.2 mL) was then collected in a test
tube containing MeCN (0.2 mL), and resulting mixture was vortexed
for 15 s and centrifuged at 20 000g for deproteinization. The resulting
supernatant was collected. Subsequently, the one-half of a rat brain was
homogenized with a Silent Crusher S homogenizer (Rose Scientific
Ltd., Edmonton, Canada) in ice-cold 50% aqueous MeCN (2.5 mL)
solution. The resulting homogenate was centrifuged at 20 000g for 2
min at 4 °C. The supernatant (0.5 mL) was collected and resuspended
with an equal volume of MeCN (0.5 mL) followed by centrifugation at
20 000g for 2 min for deproteinization. Supernatants (500 μL) were
analyzed by HPLC (Capcell Pak C18, S-5 μm, 4.6 mm ID × 250 mm,
Shiseido, Tokyo, Japan) combined to analytical radio-UPLC using the
mobile phase of MeCN/H2O/TEA (5/5/0.01, v/v/v) at a flow rate of
1.5 mL/min. Fraction of unmetabolized [11C]9a as the percentage of
total radioactivity (decay corrected) in the HPLC chromatogram was
calculated as: (peak area for [11C]9a/total peak area) × 100.
12. GPCR Panel Assay. Target GPCRs were stably expressed in
human embryonic kidney (HEK) 293 cells, a subset of which
coexpressed Gqi5 chimeric G-protein (Gqi5/HEK293), to convert its
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to the staff of NIRS and Eisai for
support with the cyclotron operation, radioisotope production,
radiosynthesis, and animal experiments, Mr. Yuichiro Yoshida,
Masanao Ogawa, and Nobuki Nengaki for manipulating the
semiautosynthetic machine in the hot cell, Dr. Masayuki
Fujinaga for log D measurement, Mr. Takashi Ueno for the P-
gp assay, Dr. Carsten Beuckmann for pharmacological support,
Mr. Kenji Furutsuka for HRMS, Dr. Yuji Nagai for monkey
PET scans, Ms. Kaori Yamada for rat PET scans, Dr. Toru Arai
for the GPCR panel assay, Mr. Tomoteru Yamasaki for
radiometabolite analysis, and Dr. Francois Bernier, Dr. Krause
Stephen, and Dr. Paul McCracken for their assistance in the
preparation of the manuscript.
ABBREVIATIONS USED
■
AUC, area under time−activity curve; BBB, blood−brain
barrier; Bmax, the maximum binding; CER, cerebellum; CHO
cells, Chinese hamster ovary cells; CNS, central nervous
system; CTX, neocortex; DCM, dichloromethane; DIAD,
M
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(12) Boss, C.; Brisbare-Roch, C.; Jenck, F. Biomedical application of
orexin/hypocretin receptor ligands in neuroscience. J. Med. Chem.
2009, 52, 891−903.
diisopropyl azodicarboxylate; DIPEA, N,N-diisopropylethyl-
amine; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; DPM, disintegrations per minute; EGTA, ethylene
glycol bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; EOB,
end of bombardment; EOS, end of synthesis; ESI-MS,
electrospray ionization mass spectra; FAB-MS, fast atom
bombardment mass spectra; FOV, field of view; FR, flux
ratio; fwhm, full-width at half-maximum; GPCR, G-protein-
coupled receptor; HATU, N,N,N′,N′-tetramethyl-O-(7-azaben-
zotriazol-1-yl)uronium hexafluorophosphate; HEPES, 4-(2-
hydroxyethyl)-1-piperazinethanesulfonic acid; HIP, hippocam-
pus; HOBt, 1-hydroxybenzotriazole monohydrate; HPLC,
high-performance liquid chromatography; HRMS, high-reso-
lution mass spectra; % ID/mL, percentage of injected
radioligand dose per unit volume of tissue; Kd, dissociation
constant; Ki, inhibition constant; MDR1, multidrug resistance
protein 1; MOM, methoxymethyl; MPFC, medial prefrontal
cortex; MRI, magnetic resonance imaging; NaHMDS, sodium
hexamethyldisilazane; OXR, orexin receptor; OX1R, orexin-1
receptor; OX2R, orexin-2 receptor; PBS, phosphate-buffered
saline; PET, positron emission tomography; Pd2(dba)3, tris-
(dibenzylideneacetone)dipalladium(0); P-gp, P-glycoprotein;
ROI, regions of interest; rt, room temperature; SD, standard
deviation; TEA, triethylamine; THA, thalamus; THF, tetrahy-
drofuran; tR, retention time; WSC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; Xant phos,
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(13) Cai, J.; Cooke, F. E.; Sherborne, B. S. Antagonists of the orexin
receptors. Curr. Opin. Drug. Discovery Dev. 2006, 16, 631−646.
(14) Roecker, A. J.; Coleman, P. J. Orexin receptor antagonists:
medicinal chemistry and therapeutic potential. Curr. Top. Med. Chem.
2008, 8, 977−987.
(15) Gatfield, J.; Brisbare-Roch, C.; Jenck, F.; Boss, C. Orexin
receptor antagonists: a new concept in CNS disorders? Chem. Med.
Chem. 2010, 5, 1197−1214.
(16) Winrow, C. J.; Gotter, A. L.; Cox, C. D.; Doran, S. M.;
Tannenbaum, P. L.; Breslin, M. J.; Garson, S. L.; Fox, S. V.; Harrell, C.
M.; Stevens, J.; Reiss, D. R.; Cui, D.; Coleman, P. J.; Renger, J. J.
Promotion of sleep by suvorexant - a novel dual orexin receptor
antagonist. J. Neurogenet. 2011, 25, 52−61.
(17) Malherbe, P.; Borroni, E.; Pinard, E.; Wettstein, J. G.; Knoflach,
F. Biochemical and electrophysiological characterization of almorexant,
a dual orexin 1 receptor (OX1)/orexin 2 receptor (OX2) antagonist:
comparison with selective OX1 and OX2 antagonists. Mol. Pharmacol.
2009, 76, 618−31.
(18) Malherbe, P.; Borroni, E.; Gobbi, L.; Knust, H.; Nettekoven, M.;
Pinard, E.; Roche, O.; Rogers-Evans, M.; Wettstein, J. G.; Moreau, J. L.
Biochemical and behavioural characterization of EMPA, a novel high-
affinity, selective antagonist for the OX(2) receptor. Br. J. Pharmacol.
2009, 156, 1326−1341.
(19) Langmead, C. J.; Jerman, J. C.; Brough, S. J.; Scott, C.; Porter, R.
A.; Herdon, H. J. Characterisation of the binding of [3H]-SB-674042, a
novel nonpeptide antagonist, to the human orexin-1 receptor. Br. J.
Pharmacol. 2004, 141, 340−346.
(20) Liu, F.; Majo, V. J.; Prabhakaran, J.; Castrillion, J.; Mann, J. J.;
Martinez, D.; Kumar, J. S. D. Radiosynthesis of [11C]BBAC and
[11C]BBPC as potential PET tracers for orexin2 receptors. Bioorg.
Med. Chem. Lett. 2012, 22, 2172−2174.
(21) Wang, C.; Moseley, C. K.; Carlin, S. M.; Wilson, C. M.;
Neelamegam, R.; Hooker, J. M. Radiosynthesis and evaluation of
[11C]EMPA as a potential PET tracer for orexin 2 receptors. Bioorg.
Med. Chem. 2013, 23, 3389−3392.
(22) Trivedi, P.; Yu, H.; MacNeil, D. J.; Van der Ploeg, L. H. T.;
Guan, X. -M. Distribution of orexin receptor mRNA in the rat brain.
FEBS Lett. 1998, 438, 71−75.
(23) Marcus, J. N.; Aschkenasi, C. J.; Lee, C. E.; Chemelli, R. M.;
Saper, C. B.; Yanagisawa, M.; Elmquist, J. K. Differential expression of
orexin receptors 1 and 2 in the rat brain. J. Comp. Neurol. 2001, 435,
6−25.
(24) Wright, G. J.; Cherwinski, H.; Foster-Cuevas, M.; Brooke, G.;
Puklavec, M. J.; Bigler, M.; Song, Y.; Jenmalm, M.; Gorman, D.;
McClanahan, T.; Liu, M. R.; Brown, M. H.; Sedgwick, J. D.; Phillips, J.
H.; Barclay, A. N. Characterization of the CD200 receptor family in
mice and humans and their interactions with CD200. J. Immunol.
2003, 171, 3034−3046.
(25) Terauchi, T.; Takemura, A.; Doko, T.; Yoshida, Y.; Tanaka, T.;
Sorimachi, K.; Naoe, Y.; Beuckmann, C.; Kazuta, Y. Cyclopropane
compound. WO 2012/039371 A1, 2010.
(26) Adachi, Y.; Suzuki, H.; Sugiyama, Y. Comparative studies on in
vitro methods for evaluating in vivo function of MDR1 P-glycoprotein.
Pharm. Res. 2001, 18, 1660−1668.
(27) Mintun, M. A.; Raichle, M. E.; Kilbourn, M. R.; Wooten, G. F.;
Welch, M. J. A quantitative model for the in vivo assessment of drug
binding sites with positron emission tomography. Ann. Neurol. 1984,
15, 217−227.
(28) Eckelman, W. C.; Mathis, C. A. Targeting proteins in vivo: in
vitro guidelines. Nucl. Med. Biol. 2006, 33, 161−164.
(29) Eckelman, W. C.; Kilbourn, M. R.; Mathis, C. A. Discussion of
targeting proteins in vivo: In vitro guidelines. Nucl. Med. Biol. 2006, 33,
449−451.
(30) Guo, Q.; Brady, M.; Gunn, R. N. A biomathematical modeling
approach to central nervous system radioligand discovery and
development. J. Nucl. Med. 2009, 50, 1715−1723.
REFERENCES
■
(1) Sakurai, T.; Amemiya, A.; Ishii, M.; Matsuzaki, I.; Chemelli, R.
M.; Tanaka, H.; Williams, S. C.; Richardson, J. A.; Kozlowski, G. P.;
Wilson, S.; Arch, J. R. S.; Buckingham, R. E. C.; Haynes, A. C.; Carr, S.
A.; Annan, R. S.; McNulty, D. E.; Liu, W.-S.; Terrett, J. A.;
Elshourbagy, N. A.; Bergsma, D. J.; Yanagisawa, M. Orexins and
orexin receptors: A family of hypothalamic neuropeptides and G
protein-coupled receptors that regulate feeding behavior. Cell 1998,
92, 573−585.
(2) van den Pol, A. N.; Gao, X.-B.; Obrietan, K.; Kilduff, T. S.;
Belousov, A. B. Presynaptic and postsynaptic actions and modulation
of neuroendocrine neurons by a new hypothalamic peptide,
hypocretin/orexin. J. Neurosci. 1998, 18, 7962−7971.
(3) Brisbare-Roch, C.; Dingemanse, J.; Koberstein, R.; Hoever, P.;
Aissaoui, H.; Flores, S.; Mueller, C.; Nayler, O.; van Gerven, J.; de
Haas, S. L.; Hess, P.; Qiu, C.; Buchmann, S.; Scherz, M.; Weller, T.;
Fischli, W.; Clozel, M.; Jenck, F. Promotion of sleep by targeting the
orexin system in rats, dogs and humans. Nat. Med. 2007, 13, 150−155.
(4) Thompson, J. L.; Borgland, S. L. A role for hypocretin/orexin in
motivation. Behav. Brain Res. 2011, 217, 446−453.
(5) Nishino, S. Chapter 47 - Hypothalamus, hypocretins/orexin, and
vigilance control. Handb. Clin. Neurol. 2011, 99, 765−782.
(6) Zhang, J.; Li, B.; Yu, L.; He, Y.-C.; Li, H.-Z.; Zhu, J.-N.; Wang, J.-
J. A role for orexin in central vestibular motor control. Neuron 2011,
69, 793−804.
(7) Okumura, T.; Nozu, T. Role of brain orexin in the
pathophysiology of functional gastrointestinal disorders. J. Gastro-
enterol. Hepatol. 2011, 26, 61−66.
(8) Sakurai, T. Orexins and orexin receptors: implication in feeding
behavior. Regul. Pept. 1999, 85, 25−30.
(9) Sharf, R.; Sarhan, M.; Dileone, R. J. Role of orexin/hypocretin in
dependence and addiction. Brain Res. 2010, 1314, 130−138.
(10) Coleman, P. J.; Renger, J. J. Orexin receptor antagonists: a
review of promising compounds patented since 2006. Expert. Opin.
Ther. Pat. 2010, 20, 307−324.
(11) Bingham, M. J.; Cai, J.; Deehan, M. R. Eating, sleeping and
rewarding: orexin receptors and their antagonists. Curr. Opin. Drug.
Discovery Dev. 2006, 9, 551−559.
N
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(31) Kazuta, Y.; Tsujita, R.; Yamashita, K.; Uchino, S.; Kohsaka, S.;
Matsuda, A.; Shuto, S. Synthesis of derivatives of (1S,2R)-1-phenyl-2-
[(S)-1- aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC)
modified at the 1-aromatic moiety as novel NMDA receptor
antagonists: The aromatic group is essential for the activity. Bioorg.
Med. Chem. 2002, 10, 3829−3848.
(32) Suzuki, K.; Inoue, O.; Hashimoto, K.; Yamasaki, T.; Kuchiki, M.;
Tamate, K. Computer-controlled large scale production of high
specific activity [11C]RO 15-1788 for PET studies of benzodiazepine
receptors. Int. J. Appl. Radiat. Isot. 1985, 36, 971−976.
́
(33) Batailler, M.; Mullier, A.; Sidibe, A.; Delagrange, P.; Prevot, V.;
Jockers, R.; Migaud, M. Neuroanataomical distribution of the orphan
GPR50 receptor in adult sheep and rodent brains. J. Neuroendocrinol.
2011, 23, 1365−2826.
(34) Yasuno, F.; Zoghbi, S. S.; Mccarron, J. A.; Hong, J.; Ichise, M.;
Brown, A. K.; Gladding, R. L.; Bacher, J. D.; Pike, V. W.; Innis, R. B.
Quantification of serotonin 5-HT1A receptors in monkey brain with
[11C](R)-(−)-RWAY. Synapse 2006, 60, 510−520.
(35) Iwata, R.; Ido, T.; Takahashi, T.; Nakanishi, H.; Iida, S.
Optimization of [11C]HCN production and no-carrier-added
[1-11C]amino acid synthesis. Int. J. Radiat. Appl. Instrum. A 1987,
38, 97−102.
(36) Mathews, W. B.; Monn, J. A.; Ravert, H. T.; Holt, D. P.;
Schoepp, D. D.; Dannals, R. F. Synthesis of a mGluR5 antagonist using
[11C]copper(I) cyanide. J. Labelled Compd. Radiopharm. 2006, 49,
829−834.
(37) Sugimoto, H.; Matsumoto, S.; Tachibana, M.; Niwa, S.;
Hirabayashi, H.; Amano, N.; Moriwaki, T. Establishment of in vitro
P-glycoprotein inhibition assay and its exclusion criteria to assess the
risk of drug−drug interaction at the drug discovery stage. J. Pharm. Sci.
2011, 100, 4013−4023.
(38) Tai, Y. C.; Ruangma, A.; Rowland, D.; Siegel, S.; Newport, D. F.;
Chow, P. L.; Laforest, R. Performance evaluation of the microPET
focus: A third-generation microPET scanner dedicated to animal
imaging. J. Nucl. Med. 2005, 46, 455−463.
(39) Watanabe, M.; Okada, H.; Shimizu, K.; Omura, T.; Yoshikawa,
E.; Kosugi, T.; Mori, S.; Yamashita, T. A high resolution animal PET
scanner using compact PS-PMT detectors. IEEE Trans. Nucl. Sci. 1997,
44, 1277−1282.
(40) Coward, P.; Wada, H. G.; Falk, M. S.; Chan, S. D.; Meng, F.;
Akil, H.; Conklin, B. R. Controlling signaling with a specifically
designed Gi-coupled receptor. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
352−357.
O
dx.doi.org/10.1021/jm400772t | J. Med. Chem. XXXX, XXX, XXX−XXX