Synthesis and evaluation of C-11, F-18 and I-125 small molecule radioligands for detecting oxytocin receptors

Article abstract of DOI:10.1016/j.bmc.2012.02.019

Compounds 1-4 were synthesized and investigated for selectivity and potency for the oxytocin receptor (OTR) to determine their viability as radioactive ligands. Binding assays determined 1-4 to have high binding affinity for both the human and rodent OTR

Full text of DOI:10.1016/j.bmc.2012.02.019

Bioorganic & Medicinal Chemistry 20 (2012) 2721–2738
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of C-11, F-18 and I-125 small molecule radioligands
for detecting oxytocin receptors
Aaron L. Smith a,b, Sara M. Freeman b, Jeffery S. Stehouwer a, Kiyoshi Inoue b, Ronald J. Voll a,
a,
Larry J. Young b, , Mark M. Goodman
⇑
⇑
a Department of Radiology and Imaging Sciences, Emory University, Atlanta, GA 30322, USA
b Center for Translational Social Neuroscience, Department of Psychiatry and Behavioral Sciences, Yerkes National Primate Research Center, Atlanta, GA 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds 1–4 were synthesized and investigated for selectivity and potency for the oxytocin receptor
(OTR) to determine their viability as radioactive ligands. Binding assays determined 1–4 to have high
binding affinity for both the human and rodent OTR and also have high selectivity for the human OTR
over human vasopressin V1a receptors (V1aR). Inadequate selectivity for OTR over V1aR was found for
rodent receptors in all four compounds. The radioactive (C-11, F-18, and I-125) derivatives of 1–4 were
synthesized and investigated for use as autoradiography and positron emission tomography (PET)
ligands. Receptor autoradiography performed with [125I]1 and [125I]2 on rodent brain slices provided
the first small molecule radioligand images of the OTR and V1aR. Biodistribution studies determined
Received 15 November 2011
Revised 28 January 2012
Accepted 6 February 2012
Available online 25 February 2012
Keywords:
Oxytocin
Vasopressin
Receptors
Iodine-125
Autoradiography
[
125I]1 and [125I]2 were adequate for in vivo peripheral investigations, but not for central investigations
due to low uptake within the brain. A biodistribution study with [18F]3 suggested brain uptake occurred
slowly over time. PET imaging studies with [18F]3 and [11C]4 using a rat model provided insufficient
uptake in the brain over a 90 and 45 min scan times respectively to merit further investigations in
non-human primates.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
noise ratios; however the selectivity of the peptide radiotracers
has not always been sufficient for distinguishing the densities of
Investigations of the physiology of oxytocin (OT) and vasopres-
sin (AVP) as neurotransmitters or neuromodulators in the central
nervous system (CNS) via their respective receptors in specific
areas of the brain has led to conclusive evidence implicating these
peptide hormones as modulators of social behaviors ranging from
maternal attachment to aggression. Furthermore, the OT system
has been proposed as a potential therapeutic target for enhancing
social functioning in autism spectrum disorders.1,2 The application
of receptor autoradiography for the in vitro localization and quan-
tification of brain tissue receptor densities has been a valuable
experimental technique in exploring the mechanisms by which
these peptides affect behavior.3–7 Autoradiographic studies investi-
gating the oxytocin receptor (OTR) and the vasopressin 1a receptor
(V1aR) densities have most commonly been conducted using the
radiolabeled peptides [125I]-ornithine vasotocin (125I-OVTA) and
homological receptors in other mammalian species, specifically
non-human primates and humans.8–10 Due to the variation in the
selectivity for the respective receptors across species, we thought
it would be useful to develop an alternative radioligand to the la-
beled peptides that are currently available for investigations. Fur-
thermore, reliable radioligands for in vivo PET neuroimaging are
not yet available for OTR or V1aR. In order to make progress toward
more accurate localization techniques for the OT and AVP receptor
systems in primates, this study aims to synthesize and characterize
novel molecules with high affinity and binding selectivity to pri-
mate OTR as well as to investigate the ability to incorporate radio-
active isotopes into their structure. A non-peptide small molecule
agonist or antagonist for the OTR could further be developed into
a potential PET imaging tracer, a primate-selective radioligand
for receptor autoradiography, or a pharmacological tool to manip-
ulate the OT system uniquely designed for use in primates. The
development of tools such as these could be used to finally eluci-
date the neuroanatomical distribution of OTR and V1aR in primate
brain and to determine whether compromised OTR expression is
associated with psychiatric disorders characterized by deficits in
social behavior, such as autism. We report here the synthesis and
radiolabeling of four compounds bearing the desired characteriza-
tions herein described, their biological evaluation in both rodent
[
125I]-linear vasopressin (125I-LVA), respectively. These radiola-
beled peptides have proven useful in the distinction of the respec-
tive OTR and V1aR densities in rodents, providing high signal to
⇑
Corresponding authors. Tel.: +1 404 727 8272; fax: +1 404 727 8070 (L.Y.); tel.:
+1 404 727 9366; fax: +1 404 712 5689 (M.G.)
E-mail addresses: lyoun03@emory.edu (L.J. Young),
mgoodma@emory.edu
(M.M. Goodman).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.019

2722
A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
and human OT systems, and PET imaging studies using rodent
models. In order to identify a suitable structure for labeling, our
search led us to modify the structure of a molecule developed by
droquinolin-2(1H)-one (2), 1-(4-(3,4-dihydroquinolin-1(2H)-yl)
piperidin-1-yl)-2-(2-(2-fluoroethoxy)-4-((1-(methylsulfonyl)piperi-
din-4-yl)oxy)phenyl)ethanone (3), and 1-(1-(2-(2-methoxy-4-((1-
(methylsulfonyl)piperidin-4yl)oxy)phenyl)acetyl)piperidin-4-yl)-3,
4-dihydroquinolin-2(1H)-one (4) are shown in Figure 1. The
approach to our investigation was simplified by making modifica-
tions only to the trifluoroethoxy chain in order to minimize
changes in the previously reported biological properties of 5. The
goal was to make a more practical target in which I-125 or F-18
could be incorporated directly from the commercially available
Merck Research Laboratories which was derived from
a
vasopressin receptor binding ligand discovered in a screen by
Otsuka Pharmaceuticals, 1-(1-(2-(4-(1-(methylsulfonyl)piperidin-
4-yloxy)-2-(2,2,2-trifluoroethoxy)phenyl)acetyl)piperidin-4-yl)-3,
4-dihydroquinolin-2(1H)-one (5), Figure 1.11,12 Having fluorine
atoms in a location suitable for generating a labeling precursor
and having high potency and selectivity for the human OTR over
all vasopressin receptor subtypes, the structural and biological
properties of 5 were favorable for our purpose. Although 5 was la-
beled and investigated using S-35, the methodology and expenses
involved with production of [35S] methane sulfonyl chloride were
not appealing for receptor autoradiographic studies.13 Our target
structures, (E)-1-(1-(2-(2-(3-iodoallyloxy)-4-(1-(methylsulfonyl)
piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydroquin-
olin-2(1H)-one (1), (Z)-1-(1-(2-(2-(3-iodoallyloxy)-4-(1-(methyl-
sulfonyl)piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihy-
[
125I]-sodium iodide or cyclotron produced F-18 using SN2 substi-
tution techniques. We included the E and Z iodopropenyl moieties
in our targets to mimic the trifluoroethoxy side chain, but enable
the incorporation of the I-125 isotope more suitable for autoradi-
ography. Additionally, if 1 or 2 proved to be viable candidates for
PET studies, the positron emitting I-124 isotope could be incorpo-
rated using the same synthetic pathway. The differences in electro-
negativity and molecular weight (the iodopropenyl moiety has a
molecular weight double that of the trifluoroethoxy group) are
the main variables in their structure activity relationships. To gen-
erate our first selective PET ligand for investigation of the OTR, we
eliminated two of the fluorine atoms in the trifluoroethoxy chain of
5 to give our candidate ligand 3. Having only a single fluorine atom
reduces the bulk of the side chain and reduces the electronegativ-
ity of 5, but it also eliminates the possibility for isotopic exchange
of fluorine atoms during an F-18 radiolabeling—thus preserving the
likelihood of desired high specific activity. Additionally, the reduc-
tion of electronegative pull on the side chain would reduce the
inductive polarization of the side chain, thus strengthening its O–
C bond. Nevertheless, a potential method for generating [18F]5 is
theoretically possible by generating a 1,1-difluoropropoxy moiety
and following methods outlined by Riss et al., but it was antici-
pated that the reduction of molecular weight involved in eliminat-
ing the two fluorine atoms increased the potential of 4 to breach
the blood–brain barrier.14 To investigate a side chain moiety with
a much lower molecular weight, we chose to investigate the meth-
oxy side chain due to its ability to incorporate a [11C]methyl group
via [11C]iodomethane. We anticipated the reduction in molecular
weight and bulk might make 4 the best candidate of this family
of compounds to breach the blood–brain barrier.
I
O
N
O
O
O
N
N
S
S
N
O
O
O
O
O
1
O
I
N
O
N
O
F
2
In addition to the synthesis of 1–4 and their radioactive deriva-
tives, we report in vitro competitive binding assay results in which
1–4 were used in competition with 125I-OVTA and 125I-LVA using
prairie vole brain sections. We also include competitive binding as-
say results derived from cell lines expressing the human OTR and
V1aR courtesy of Bryan Roth et al. The first small molecule receptor
autoradiography images of the OTR and V1aR were generated from
O
O
N
N
O
O
O
N
N
S
S
N
O
O
O
O
O
3
[
125I]1 and [125I]2 using prairie vole brain slices and are presented
in direct comparison to the images generated with 125I-OVTA and
125I-LVA. We report the lipophilicity of all radiligands and in vivo
biodistribution results of [125I]1, [125I]2, and [18F]3. Finally, we re-
port image data generated with [18F]3 and [11C ]4 in a PET scan
using a rat model.
O
N
O
F
4
2. Materials and methods
2.1. General
F
O
F
N
All reagents used for experiments were obtained from commer-
cially available sources unless otherwise noted. Solvents used for
reactions were obtained from either Aldrich or EMD Chemicals
Inc. Solvents used for chromatography were obtained from VWR.
1H and 13C NMR were recorded on a Varian spectrometer at 300
or 400 MHz and referenced to solvent or internal TMS standard.
Mass spectra were determined on a JEOL JMS-SX102/SX102 double
O
O
N
S
N
O
O
O
5
Figure 1. Structures of synthetic targets 1–4 and previously reported compound 5.

A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
2723
focusing mass spectrometer using electrospray ionization (ESI).
Silica gel column chromatography was performed using
Whatman’s Purasil 60A Silica gel (230–400 mesh). Thin layer chro-
matography was performed using 250 lm layers of F254 silica on
alumina plates obtained from EMD Chemicals Inc. Unless described
1.5 mmol) was then added dropwise at room temperature while
stirring. The reaction stirred for 16 h under argon at room temper-
ature. The DMF was then removed under high vacuum and the res-
idue was extracted into EtOAc (100 ml) and washed with water
(100 ml) and brine (100 ml). The organic layer was dried over
anhydrous magnesium sulfate, filtered, and concentrated. The
residual solid was dissolved in a minimal amount of EtOAc and
added to a silica gel column (25 mm in diameter and 255 mm in
height) packed with hexanes. The product was eluted via a gradi-
ent of hexanes, 4:1 hexanes: EtOAc, 1:1 hexanes: EtOAc. The frac-
tions containing product were concentrated to give 0.21 g (82%) of
a white solid. 1H NMR (CDCl3) d 7.71 (d, J = 8.8 Hz, 1H), 6.64 (d,
J = 2.0 Hz 1H), 6.50 (dd, J = 8.8, 2.4 Hz, 1H), 5.20 (s, 2H), 4.50–4.46
(m, 1H), 3.63–3.57 (m, 2H), 3.44 (s, 3H), 3.34–3.27 (m, 2H), 2.52
(s, 3H), 1.88–1.83 (m, 2H), 1.72–1.66 (m, 2H), 1.39 (s, 9H); 13C
NMR (CDCl3) d 197.5, 162.0, 158.7, 154.7, 132.4, 121.6, 107.9,
102.6, 94.4, 79.6, 72.1, 56.4, 31.7, 30.3, 28.4; HRMS (ESI) m/z
402.1892 (MNa+ [C20H29NO6Na] = 402.1887).
otherwise, analytical HPLC retention times were determined on a
Novapak 4
lm C-18 column measuring 3.9 Â 150 mm on a Waters
pump and 2487 UV detector set at 254 nm using a solvent system
A (50:50:0.1 ethanol:water:triethylamine) or solvent system B
(70:30:0.1 methanol:water:triethylamine). Radiochemical purity
was determined on a Bioscan AR-2000 radio-TLC for I-125 com-
pounds or a Waters HPLC equipped with UV and SAT/IN radioactiv-
ity detector module for C-11 and F-18 compounds. Both F-18 and
C-11 were obtained from a Siemens Eclipse 111 cyclotron after
proton beam bombardment of the respective O-18 or N-14 targets.
MicroPET images were obtained using a Siemens Inveon DPET
scanner with a cobalt-57 source for attenuation correction. Scans
were performed in a 12.5 cm field of view starting with the nose
of the rodent. A 7 min transmission scan was performed with the
cobalt-57 source prior to the emission scans. Scan times were
90 min for [18F]3 and 45 min for [11C ]4. Further details of the
Inveon scanner are as published by Bao et al.15
2.2.3. 2-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yloxy)-2-
(methoxymethoxy)phenyl)acetic acid (9)
Compound 8 (5.53 g, 14.6 mmol) and sulfur (0.7 g, 21.9 mmol)
were added to a round bottom flask with 22 ml of morpholine.
The flask was equipped with a reflux condenser and heated to
135–140 °C for 5 h. The excess morpholine was removed under
high vacuum. The residue was dissolved in a minimal amount of
dichloromethane and added to a silica gel column (40 mm in diam-
eter and 300 mm in height) packed with hexanes. The unreacted
starting material was separated from the intermediate product
via a gradient elution of hexanes, 2:1 hexanes: EtOAc, and 1:1 hex-
anes: EtOAc. The fractions containing unreacted product were con-
centrated and subjected to morpholine addition and separation
again. The combined intermediate product fractions were concen-
trated and then dissolved in methanol (75 ml) and 2 N NaOH
(75 ml). The mixture was heated to reflux for 24 h. The methanol
was then evaporated under reduced pressure and the product
was extracted into dichloromethane (150 ml  2) and washed with
water (150 ml  2). The organic layer was dried over anhydrous
magnesium sulfate, filtered, and concentrated to give 3.0 g (53%)
of product having purity adequate enough for the next step in
the synthesis. 1H NMR (CDCl3) d 7.08 (d, J = 8.4 Hz, 1H), 6.70 (d,
J = 2.1 Hz, 1H), 6.51 (dd, J = 8.4, 2.4 Hz, 1H), 5.16 (s, 2H), 4.47–
4.40 (m, 1H), 3.70–3.61 (m, 4H), 3.44 (s, 3H), 3.92–3.31 (m, 2H),
1.93–1.86 (m, 2H), 1.79–1.68 (m, 2H), 1.47 (s, 9H); 13C NMR
(CDCl3) d 177.6, 158.1, 156.4, 155.1, 131.5, 115.8, 108.2, 103.5,
94.5, 79.9, 72.3, 56.3, 35.4, 30.7, 28.7; HRMS (ESI–negative mode)
m/z 394.1880 (M+ [C20H28NO7] = 394.1871).
All animal experiments were carried out according to protocols
approved by the Institutional Animal Care and Use Committee (IA-
CUC) and Environmental Health and Safety Office of Emory
University.
2.2. Synthesis and radiolabeling
The synthesis methodology of the target compounds, their radi-
olabeled analogues, and precursors are reported in the following
procedures.
2.2.1. tert-Butyl 4-(4-acetyl-3-hydroxyphenoxy)piperidine-1-
carboxylate (7)
To a triple neck flask was added 2,4-dihydroxyacetophenone, 6
(2.73 g, 17.9 mmol), triphenylphosphine (7.05 g, 26.9 mmol), and
60 ml of tetrahydrofuran (THF). The flask was equipped with an
addition funnel and a premixed solution of diethyl azodicarboxyl-
ate (DEAD) (40% by weight in Toluene, 11.7 g, 26.9 mmol) and 4-
hydroxypiperidinecarboxylate in 40 ml THF was transferred to
the addition funnel via cannula. The flask was cooled to 0 °C and
the DEAD—triphenylphosphine mixture was added dropwise over
a period of 2 h. After addition of the mixture was complete, the
addition funnel was rinsed with 5 ml THF which was added to
the stirring mixture immediately. The mixture was allowed to
warm to room temperature and stir for an additional 18 h. The sol-
vents were removed under reduced pressure and high vacuum. The
solid residue was suspended in ether to separate triphenylphos-
phine oxide. The solid triphenylphosphine oxide was filtered and
the filtrate was concentrated. The residue was dissolved in a min-
imal amount of DCM and added to a silica gel column (40 mm in
diameter and 310 mm in height) packed with hexanes. The product
was eluted via a gradient of hexanes, 8:1 hexanes: EtOAc, and 4:1
Hexanes: EtOAc. Fractions containing product were concentrated
to give 4.91 g (77%) of a white solid. 1H NMR (CDCl3) d 7.63 (d,
J = 8.8 Hz, 1H), 6.45–6.39 (m, 2H), 4.56–4.49 (m, 1H), 3.73–3.65
(m, 2H), 3.38–3.30 (m, 2H) 2.55 (s, 3H), 1.99–1.89 (m, 2H), 1.78–
1.70 (m, 2H), 1.46 (s, 9H); 13C NMR (CDCl3) d 202.7, 165.3, 164.0,
155.0, 132.7, 114.1, 108.9, 102.3, 80.0, 72.6, 40.7, 30.5, 28.6, 26.4.
2.2.4. tert-Butyl 4-(3-(methoxymethoxy)-4-(2-oxo-2-(4-(2-oxo-
3,4-dihydroquinolin-1(2H)-yl)piperidin-1-yl)ethyl)phenoxy)
piperidine-1-carboxylate (11)
To a round bottom flask was mixed 1-hydroxybenzotriazole hy-
drate (0.26 g, 1.9 mmol), compound 9 (0.75 g, 1.9 mmol), quinoli-
none 10 (0.51 g, 1.9 mmol), 1-ethyl-3-(dimethylaminopropyl)
carbodiimide hydrochloride (0.58 g, 3.1 mmol) and DMF (10 ml).
To the stirring mixture was added diisopropylethylamine
(0.63 ml, 3.6 mmol). The mixture stirred for 19 h at room temper-
ature under argon. The DMF was removed under high vacuum and
the residue was extracted into EtOAc (125 ml). The organic layer
was washed with water (100 ml) and dried over anhydrous mag-
nesium sulfate, filtered, and concentrated. The residue was dis-
solved in a minimal amount of EtOAc and added to a silica gel
column (63 mm in diameter and 178 mm in height) packed with
EtOAc. The product was eluted with EtOAc. Fractions containing
product were concentrated to give 1.1 g of a white solid (80%).
1H NMR (CDCl3) d 7.18–7.15 (om, 3H), 7.03–6.96 (om, 2H), 6.69
2.2.2. tert-Butyl 4-(4-acetyl-3-(methoxymethoxy)phenoxy)
piperidine-1-carboxylate (8)
Compound 7 (0.23 g, 0.7 mmol) was added to a round bottom
flask with cesium carbonate (0.44 g, 1.5 mmol) and 3 ml of N,N-
dimethylformamide (DMF). Chloromethyl methyl ether (0.1 ml,

2724
A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
(d, J = 2.4 Hz, 1H), 6.56 (dd, J = 8.4, 2.4 Hz, 1H), 5.19 (s, 2H), 4.82 (br
d, J ꢀ11.7 Hz, 1H), 4.54–4.38 (m, 2H), 4.05 (br d, J ꢀ12.6 Hz, 1H),
3.73–3.62 (m, 4H), 3.46 (s, 3H), 3.36–3.28 (m, 2H), 3.06 (br t, J ꢀ
11.4 Hz, 1H), 2.83–2.79 (om, 2H), 2.68–2.29 (om, 5H),1.93–1.84
(m, 2H), 1.77–1.67 (m, 4H), 1.47 (s, 9H); 13C NMR (CDCl3) d
171.8, 170.4, 157.5, 155.6, 155.1, 140.3, 130.6, 128.9, 128.1,
127.3, 123.5, 117.2, 116.6, 108.6, 103.7, 94.8, 79.8, 72.4, 56.4,
55.5, 46.2, 42.4, 40.8, 34.3, 33.7, 30.7, 28.7, 25.9; HRMS (ESI) m/z
608.3338 (MH+ [C34H46N3O7] = 608.3330).
amount of DCM and added to a silica sep-pak plus cartridge. The
cartridge was rinsed with 12 ml of DCM and the product was then
eluted with EtOAc. The fractions containing product were concen-
trated to give 38 mg (95%) of a tan waxy solid. 1H NMR (CDCl3) d
7.27–7.15 (om, 3H), 7.03–6.96 (om, 2H), 6.74 (dt, Jd = 12.9 Hz,
Jt = 6.3 Hz, 1H), 6.52 (dd, J = 9.0, 2.1 Hz, 1H), 6.42 (d, J = 2.1 Hz
1H), 6.24 (d, J = 12.9 Hz, 1H), 4.82 (br d, ꢀ11 Hz, 1H), 4.52–4.38
(d, om, J = 6.3 Hz, 4H), 4.05 (br d, J ꢀ13.5 Hz, 1H), 3.69 (AB q,
J = 15.6 Hz, 2H), 3.34 (t, J = 5.4 Hz, 4H), 3.03 (br t, J ꢀ12 Hz, 1H),
2.81 (s, om, 5H), 2.66–2.41 (om, 4H), 2.29 (dq, Jq = 12.9 Hz,
Jd = 4.2 Hz, 1H), 2.09–1.94 (m, 4H), 1.72 (br t, J ꢀ13 Hz, 2H),
1.55–1.4 (m, 6H), 1.28 (sxt, J = 7.5 Hz, 6H), 0.94 (t, J = 7.8 Hz, 6H),
0.867 (t, J = 7.2 Hz, 9H); 13C NMR (CDCl3) d 171.8, 170.5, 157.1,
156.8, 142.9, 140.2, 134.7, 130.6, 128.9, 128.1, 127.2, 123.4,
117.2, 116.6, 107.1, 102.9, 101.7, 71.7, 70.46, 55.5, 46.3, 42.5,
42.3, 34.9, 33.9, 33.6, 30.1, 29.4, 29.3, 28.7, 25.9, 13.9, 10.5; HRMS
(ESI) m/z 872.3692 (MH+ [C43H66N3O6SSn] = 872.3689)
2.2.5. 1-(1-(2-(2-Hydroxy-4-(piperidin-4-yloxy)phenyl)acetyl)
piperidin-4-yl)-3,4-dihydroquinolin-2(1H)-one hydrochloride
(12)
Compound 11 (0.75 g, 1.2 mmol) was dissolved in DCM (ꢀ3 ml)
and 5 ml of 1 M HCl in ether was added while stirring rapidly. The
reaction was allowed to stir at room temperature for 12 h under ar-
gon. The solvents were removed and 20 ml of ether was added
back into the flask. The flask was sealed under argon and placed
in a À20 °C freezer for 4 h. The solid was filtered and rinsed with
ice cold ether and then dried under high vacuum and mild heat
to give a tan solid (quantitative yield). 1H NMR (CDCl3) d 9.84 (br
s, 1H), 7.20–7.15 (om, 3H), 7.04–6.98 (om, 2H), 6.96–6.91 (om,
2H), 6.59 (d, J = 2.1 Hz, 1H), 6.39 (dd, J = 8.7, 2.7 Hz, 1H), 4.77 (br
d, J = 13.5 Hz, 1H), 4.45–4.40 (m, 2H), 4.28, (br d, J = 14.4 Hz, 1H),
3.73 (s, 2H), 3.24–3.17 (m, 2H), 2.95–2.8 (om, 5H), 2.7–2.5 (om,
5H), 2.10–2.05 (m, 2H), 1.91–1.79 (m, 4H); 13C NMR (CDCl3) d
171.9, 158.4, 158.0, 140.2, 131.1, 128.9, 128.2, 127.4, 123.6,
116.4, 113.7, 108.5, 105.4, 55.2, 47.2, 42.8, 42.1, 36.1, 33.7, 30.0,
29.5, 28.5, and 25.9; HRMS (ESI) m/z 464.2548 (MH+
[C27H34N3O4] = 464.2544).
2.2.8. (Z)-1-(1-(2-(4-(1-(Methylsulfonyl)piperidin-4-yloxy)-2-(3-
(tributylstannyl)allyloxy)phenyl)acetyl)piperidin-4-yl)-3,4-
dihydroquinolin-2(1H)-one (14b)
The procedure was identical to that described above for com-
pound 14a except (Z)-tributyl(3-chloroprop-1-enyl)stannane was
used to give 97% of a waxy solid. 1H NMR (CDCl3) d 7.24–7.15
(om, 3H), 7.02–6.95 (om, 2H), 6.52 (dd, J = 8.4, 2.4 Hz, 1H), 6.44
(d, J = 2.4 Hz, 1H), 6.33 (d, J = 19.2 Hz, 1H), 6.14 (dt, Jd = 19.2 Hz,
Jt = 4.5 Hz, 1H), 4.83 (br d, J ꢀ11 Hz, 1H), 4.54 (d, J = 4.2 Hz, 2H),
4.50–4.38 (om, 2H), 4.07 (br d, J ꢀ13 Hz, 1H), 3.73 (AB q,
J = 15 Hz, 2H), 3.34 (t, J = 5.4 Hz, 4H), 3.03 (br t, J ꢀ12 Hz, 1H),
2.81 (s, om, 5H), 2.62 (br t, J ꢀ12 Hz, 1H), 2.57–2.46 (om, 3H),
2.29 (dq, Jq = 12.0 Hz, Jd = 3.6 Hz, 1H), 2.05–1.96 (m, 4H), 1.72 (br
t, J ꢀ14 Hz, 2H), 1.54–1.38 (m, 6H), 1.29 (sxt, J = 7.5 Hz, 6H),
1.01–0.79 (om, 15H); 13C NMR (CDCl3) d 171.7, 170.5, 157.1,
156.8, 142.5, 140.2, 132.2, 130.5, 128.9, 128.1, 127.2, 123.4,
117.1, 116.5, 107.1, 101.8, 71.8, 70.3, 55.5, 46.3, 42.5, 42.3, 34.9,
33.9, 33.6, 30.1, 29.4, 29.2, 28.7, 27.4, 25.9, 13.9, 9.7; HRMS (ESI)
m/z 872.3691 (MH+ [C43H66N3O6SSn] = 872.3689).
2.2.6. 1-(1-(2-(2-Hydroxy-4-(1-(methylsulfonyl)piperidin-4-
yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydroquinolin-2(1H)-
one (13)
To a stirring solution of compound 12 (0.44 g, 0.9 mmol) and
triethylamine (0.14 ml, 1.1 mmol) in anhydrous DCM (200 ml) at
room temperature was added methane sulfonyl chloride (69 ll,
0.9 mmol). The reaction stirred for 14 h at room temperature and
a TLC in 100% EtOAc showed one product spot (Rf = 0.45) and a spot
at the origin. The mixture was poured directly onto a column con-
taining a plug of silica gel, rinsed with 100 ml DCM, and then prod-
uct was eluted with 250 ml EtOAc. Fractions containing product
were combined and concentrated to give 0.25 g of a white solid
(51%). 1H NMR (CDCl3) d 9.98 (br s, 1H), 7.18–7.13 (om, 2H), 7.00
(t, J = 7.5 Hz, 1H), 6.92 (dd, J = 8.1, 2.7 Hz, 2H), 6.57 (d, J = 2.4 Hz,
1H), 6.39 (dd, J = 8.4, 2.4 Hz, 1H), 4.77 (br d, J ꢀ13.8 Hz, 1H),
4.50–4.37 (om, 2H), 4.28 (br d, J ꢀ13.8 Hz, 1H), 3.73 (s, 2H),
3.40–3.21 (om, 5H), 2.81(s, om, 5H), 2.72–2.46 (om, 5H), 2.02–
1.97 (m, 4H), 1.90–1.78 (m, 2H); 13C NMR (CDCl3) d 172.0, 171.9,
158.5, 157.9, 140.2, 131.1, 128.9, 128.2, 127.4, 123.6, 116.3,
113.8, 108.4, 105.7, 70.1, 55.3, 47.3, 42.8, 42.5, 36.1, 34.9, 33.7,
30.0, 29.5, 28.4, 28.9; HRMS (ESI) m/z 542.2325 (MH+
2.2.9. 2-(5-((1-(Methylsulfonyl)piperidin-4-yl)oxy)-2-(2-oxo-2-
(4-(2-oxo-3,4-dihydroquinolin-1(2H)-yl)piperidin-1-yl)ethyl)
phenoxy)ethyl 4-methylbenzenesulfonate (15)
Compound 13 (37 mg, 7 Â 10À2 mmol), cesium carbonate
(46 mg, 0.14 mmol), and ethane-1,2-diyl bis(4-methylbenzene-
sulfonate) (51 mg, 0.14 mmol) were mixed in 1.5 ml DMF under
argon. The mixture stirred for 16 h at room temperature. The
DMF was then removed under high vacuum at 70 °C. The residue
was dissolved in DCM and water (20 ml) and product was ex-
tracted into DCM (2 Â 20 ml). The organic layer was dried over
magnesium sulfate, filtered, and concentrated. The residue was
dissolved in a minimal amount of DCM and added to a silica
sep-pak plus cartridge. The cartridge was rinsed with 25 ml of
DCM and the product was then eluted with EtOAc. The fractions
containing product were concentrated to give 35 mg (70%) of a
tan solid. 1H NMR (CDCl3) d 7.79 (d, J = 8.4 Hz, 2H), 7.35 (d,
J = 7.8, 2H), 7.23–7.14 (m, 3H), 7.02–6.97 (om, 2H), 6.53 (dd,
J = 8.5, 2.1 Hz, 1H), 6.38 (d, J = 2.1 Hz, 1H), 4.79 (br d, J = 11.7,
1H), 4.48–4.46 (m, 1H), 4.37–4.30 (om, 3H), 4.16 (t, J = 4.3 Hz),
4.01 (br d, J = 15.3 Hz, 1H), 3.60 (AB q, J = 15.9 Hz, 2H), 3.49–3.31
(om, 4H), 3.04 (br t, J = 12.9 Hz, 1H), 2.83–2.66 (om, 5H), 2.62–
2.48 (om, 4H), 2.44 (s, 3H), 2.39–2.27 (m, 1H), 2.04–1.92 (om,
4H), 1.72 (br t, J = 12.9 Hz, 2H); 13C NMR (CDCl3) d 171.7, 170.1,
157.1, 156.3, 145.4, 140.5, 133.0, 131.1, 130.2, 128.8, 128.1,
127.3, 123.4, 117.6, 116.5, 107.8, 101.7, 70.5, 68.3, 66.3, 55.9,
46.2, 42.5, 42.3, 35.0, 33.8, 30.1, 29.3, 28.7, 26.0, 21.9; HRMS
(ESI) m/z 778.22491 (MK+ = [C47H45N3O9KS2] = 778.22288).
[C28H36O6N3S] = 542.2319)
m/z
564.2144
(MNa+
[C28H35O6N3NaS] = 564.2139).
2.2.7. (E)-1-(1-(2-(4-(1-(Methylsulfonyl)piperidin-4-yloxy)-2-(3-
(tributylstannyl)allyloxy)phenyl)acetyl)piperidin-4-yl)-3,4-
dihydroquinolin-2(1H)-one (14a)
Compound 13 (25 mg, 5 Â 10À2 mmol), (E)-tributyl(3-chloro-
prop-1-enyl)stannane (34 mg, 9 Â 10À2 mmol), and cesium car-
bonate (30 mg, 9 Â 10À2 mmol) were mixed in 1 ml DMF. The
mixture was stirred for 19 h at room temperature. The mixture
was then poured into a separatory funnel and extracted into EtOAc
(2 Â 25 ml), washing each extraction with 25 ml of water three
times. The organic layer was dried over magnesium sulfate, fil-
tered, and concentrated. The residue was dissolved in a minimal

A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
2725
2.2.10. (E)-1-(1-(2-(2-(3-Iodoallyloxy)-4-(1-(methylsulfonyl)
piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydro-
quinolin-2(1H)-one (1)
each extraction twice with 25 ml of water. The organic layer was
dried over magnesium sulfate, filtered, and concentrated. The res-
idue was dissolved in a minimal amount of DCM and added to a sil-
ica sep-pak plus cartridge. The cartridge was rinsed with 12 ml of
DCM and the product was then eluted with EtOAc. The fractions
containing product were concentrated to give 74 mg (77%) of a
white solid.
Compound 14a (40 mg, 5 Â 10À2 mmol) was dissolved in 10 ml
anhydrous DCM under argon. Iodine (12 mg, 5 Â 10À2 mmol) was
added and the mixture was purged with argon and stirred for 2 h
at room temperature. The mixture was diluted with 15 ml of
DCM and quenched with 10% aqueous sodium thiosulfate. The or-
ganic layer was separated and dried over anhydrous magnesium
sulfate, filtered, and concentrated. The residue was dissolved in a
minimal amount of DCM and added to a Waters silica Sep-Pak plus
cartridge (WAT051900) previously treated with 5 ml DCM. The
cartridge was rinsed with 12 ml of DCM and the product was then
eluted with EtOAc. The concentrated fractions produced 27 mg
(83%) of a yellow solid. 1H NMR (CDCl3) d 7.23–7.17 (m, 3H),
7.04–7.00 (m, 2H), 6.75 (dt, Jd = 15 Hz, Jt = 5.1 Hz, 1H), 6.55–6.50
(m, 2H), 6.41 (d, J = 2.4 Hz, 1H), 4.82 (br d, J = 11.1 Hz, 1H), 4.52–
4.64 (m, 3H), 4.43–4.35 (m, 1H), 4.05 (br d, J = 14.4 Hz, 1H), 3.68
(AB q, J = 15.6 Hz, 2H), 3.40–3.34 (m, 4H), 3.08 (br t, J = 12.6, 1H),
2.85–2.80 (m, 5H), 2.69–2.33 (m, 5H), 2.03–1.99 (m, 4H), 1.75 (br
t, J = 15.9 Hz, 2H); 13C NMR (CDCl3) d 171.8, 170.0, 157.2, 156.4,
140.6, 140.4, 131.1, 128.9, 128.1, 127.3, 123.4, 117.5, 116.4,
107.4, 101.8, 79.9, 70.4, 70.1, 55.8, 46.3, 42.5, 35.0, 34.2, 33.7,
30.1, 29.5, 28.8, 26.0; HRMS (ESI) m/z 708.1605 (MH+ [C31H39I-
N3O6S] = 708.1599); HPLC retention time = 6.65 (solvent system
A); Anal. Calcd for C31H38IN3O6S: C, 52.62; H, 5.41; N, 5.94. Found:
C, 53.47; H, 5.88; N, 5.63.
1H NMR (CDCl3) d 7.24 (d, J = 8.0 Hz, 1H), 7.21–7.15 (m, 2H),
6.55 (dd, J = 8.4, 2.4 Hz, 1H), 6.45 (d, J = 2.4 Hz, 1H), 4.83–4.80 (m,
2H), 4.70 (t, J = 3.6 Hz, 1H), 4.50 (qui, J = 3.6 Hz, 1H), 4.43–4.37
(m, 1H), 4.21 (dt, Jd = 28.4 Hz, Jt = 4 Hz, 2H), 4.11 (br d,
J
ꢀ12.4 Hz, 1H), 3.71 (AB q, J ꢀ15 Hz, 2H), 3.35 (t, J = 5.6 Hz, 4H),
3.06 (br t, J ꢀ12 Hz, 1H), 2.82 (s, om, 5H), 2.63 (br t, J = 12.8 Hz,
1H), 2.57–2.48 (m, 3H), 2.37 (dq, Jq = 12.4 Hz, Jd = 4.4, 1H), 2.04–
1.95 (m, 4H), 1.73 (br t, J = 14 Hz, 2H). 13C NMR (CDCl3) d 171.7,
170.2, 157.2, 156.7, 140.3, 131.1, 128.8, 128.0, 127.2, 123.4,
117.6, 116.5, 107.6, 101.8, 82.8, 81.1, 70.4, 68.0, 67.8, 55.7, 46.2,
42.5, 42.3, 35.0, 33.8, 33.7, 30.1, 29.4, 28.7, 25.9. HRMS (ESI) m/z
588.25449 (MH+ [C30H39N3O6FS] = 588.25381); HPLC retention
time = 2.44 min (solvent system B); Anal. Calcd for C30H38N3O6FS:
C, 61.31; H, 6.52; N, 7.15. Found: C, 61.42; H, 6.51; N, 6.91.
2.2.13. 1-(1-(2-(2-Methoxy-4-((1-(methylsulfonyl)piperidin-4-
yl)oxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydroquinolin-
2(1H)-one (4)
To a stirring solution of compound 13 (24 mg, 5 Â 10À2 mmol)
and cesium carbonate (30 mg, 9 Â 10À2 mmol) in DMF (1 ml) was
added iodomethane (6
l
l, 9 Â 10À2 mmol). The flask was purged
2.2.11. (Z)-1-(1-(2-(2-(3-Iodoallyloxy)-4-(1-(methylsulfonyl)
piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydro-
quinolin-2(1H)-one (2)
with argon and sealed. The mixture was allowed to stir for 12 h at
room temperature. The mixture was poured into a separatory fun-
nel with 50 ml of EtOAc and washed 3 times with 40 ml of water.
The water layers were sequentially mixed with another 50 ml of
EtOAc for further extraction. The organic layer was dried over mag-
nesium sulfate, filtered, and concentrated. The residue was dis-
solved in a minimal amount of EtOAc and added to a silica gel
column 10 mm in diameter and 275 mm in height. The product
was eluted with 75 ml of pure EtOAc followed by 25:1 EtOAc:
MeOH. The fractions containing product were concentrated to give
17 mg (68%) of a white solid. 1H NMR (CDCl3) d 7.21–7.16 (m, 3H),
7.03–6.98 (om, 2H), 6.50 (dd, J = 8.1, 2.4 Hz, 1H), 6.46 (d, J = 2.1 Hz,
1H), 4.82 (br d, J ꢀ 11.7, 1H), 4.52 (qui, J = 4.2 Hz, 1H), 4.45–4.37 (m,
1H), 4.06 (br d, J ꢀ10.2 Hz, 1H), 3.82 (s, 3H), 3.67 (AB q, J ꢀ15 Hz,
2H), 3.35 (t, J = 6 Hz, 4H), 3.06 (br t, J ꢀ11 Hz, 1H), 2.82(s, om,
5H), 2.63 (br t, J ꢀ 12 Hz, 1H), 2.58 (om, 3H), 2.37 (dq, Jq = 12.6 Hz,
Jd = 4.2 Hz, 1H), 2.06–1.98 (m, 4H), 1.73 (br t, J ꢀ 15 Hz, 2H). 13C
NMR (CDCl3) d 171.8, 170.3, 158.0, 157.3, 140.3, 130.8, 128.9,
128.1, 127.2, 123.5, 117.1, 116.5, 106.7, 100.6, 70.3, 55.8, 55.7,
46.3, 42.5, 42.4, 35.0, 34.0, 33.7, 30.1, 29.5, 28.8, 26.0. HRMS (ESI)
m/z 546.24811 (MH+ [C29H38N3O6S] = 556.24758); HPLC retention
time = 4.87 min (solvent system B); Anal. Calcd for C29H37N3O6S:
C, 62.68; H, 6.71; N, 7.56. Found: C, 61.47; H, 6.69; N, 7.02.
Compound 14b (20 mg, 2 Â 10À2 mmol), sodium iodide (11 mg,
excess), and 3 ml ethanol were mixed in air. Hydrogen peroxide
(85 ll of 3% weight aqueous solution) was then added while stir-
ring. The mixture stirred at room temperature for 3 h and was then
stopped with 10% aqueous sodium thiosulfate. The mixture was
then diluted with 30 ml DCM and washed with 30 ml of water
and 30 ml of brine. The organic layer was dried over anhydrous
magnesium sulfate, filtered, and concentrated. The residue was
dissolved in a minimal amount of DCM and added to a Waters sil-
ica Sep-Pak plus cartridge (WAT051900) previously treated with
5 ml DCM. The cartridge was rinsed with 12 ml of DCM and the
product was then eluted with EtOAc. The concentrated fractions
produced 15 mg (94%) of a yellow solid. 1H NMR (CDCl3) d 7.22–
7.15 (m, 3H), 7.02–6.97 (m, 2H), 6.62–6.49 (m, 3H), 6.42 (d,
J = 2.4 Hz, 1H), 4.81 (br d, J = 11.1 Hz, 1H), 4.52 (quin, J = 4.35 Hz,
1H), 4.37 (tt, J = 12 Hz, J = 3.9 Hz, 1H), 4.04 (br d, J = 11.7 Hz, 1H),
3.67 (AB q, J = 15 Hz,
2 H), 3.39–3.28 (m, 4H), 3.05 (td,
J = 13.5 Hz, J = 2.4 Hz, 1H), 2.81–2.78 (m, 5H), 2.66–2.47 (m, 4H),
2.37 (qd, J = 12.9, J = 3.6, 1H), 2.04–1.97 (m, 4H), 1.72 (br t,
J = 14.4 Hz, 2H); 13C NMR (CDCl3) d 171.8, 170.2, 157.2, 156.5,
140.4, 137.3, 131.0, 128.9, 128.1, 127.3, 123.5, 117.3, 116.5,
107.7, 101.6, 84.3, 71.3, 70.4, 55.8, 46.3, 42.5, 35.0, 34.1, 33.7,
30.1, 29.5, 28.8, 26.0; HRMS (ESI) m/z 708.1590 (MH+ [C31H39I-
N3O6S] = 708.1599); HPLC retention time = 6.84 min (solvent sys-
tem A); Anal. Calcd for C31H38IN3O6S: C, 52.62; H, 5.41; N, 5.94.
Found: C, 52.89; H, 5.69; N, 5.56.
2.2.14. (E)-1-(1-(2-(2-(3-[125I]Iodoallyloxy)-4-(1-(methylsul-
fonyl)piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihy
droquinolin-2(1H)-one ([125I]1) and (Z)-1-(1-(2-(2-(3-[125I]
Iodoallyloxy)-4-(1-(methylsulfonyl)piperidin-4-yloxy)phenyl)
acetyl)piperidin-4-yl)-3,4-dihydroquinolin-2(1H)-one ([125I]2)
To a sealed vial containing no-carrier-added Na[125I]I (MDS
2.2.12. 1-(4-(3,4-Dihydroquinolin-1(2H)-yl)piperidin-1-yl)-2-(2-
(2-fluoroethoxy)-4-((1-(methylsulfonyl)piperidin-4-yl)oxy)
phenyl)ethanone (3)
Compound 13 (69 mg, 0.13 mmol), 1-bromo-2-fluoroethane
(17 ll, 0.25 mmol), and cesium carbonate (83 mg, 0.25 mmol)
were mixed in 1.5 ml DMF. The mixture was stirred for 19 h at
room temperature. The DMF was removed under high vacuum at
60 °C. The residue was extracted into EtOAc (2 Â 25 ml), washing
Nordion—specific activity ꢀ2.52 Ci/
ide (ꢀ100 l) was equipped with a carbon filter vent. Approxi-
mately 100 g of tributyltin precursor 14a or 14b dissolved in
0.3 ml of ethanol was then added (300 l of a 1 mg/ml solution).
In rapid succession was added an excess of 0.4 N aqueous hydro-
chloric acid (100 l) and 3% hydrogen peroxide (50 l). The carbon
lmol) in 0.1 N sodium hydrox-
l
l
l
l
l
filter was removed from the reaction vial and it was placed on a
vortex and agitated vigorously for 1 min and then allowed to pro-

2726
A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
ceed for 45 min at room temperature. The reaction was then
quenched with aqueous sodium hydrogen sulfate (0.25 g/ml,
50 ll). The reaction mixture was drawn into a syringe containing
were diluted with twice their volume of water, loaded on a pre-
rinsed C18 sep-pak cartridge, rinsed with 25 ml saline, and eluted
with 1.5 ml ethanol into a sterile vial containing 13.5 ml saline. The
resulting mixture was then transferred under argon pressure over
an aqueous solution of saturated sodium bicarbonate (0.5 ml)
and injected on a C18 SepPak (preconditioned with 10 ml ethanol
followed by 15 ml water). The SepPak was then rinsed with
20 ml of water. The product was then eluted into sealed vials with
0.5 ml portions of ethanol (each portion was collected in a separate
vial). Using 100% EtOAc as solvent, an Rf of 0.38 was observed for
a Millipore filter (pore size of 1.0
lm) followed by a smaller Milli-
pore filter (pore size of 0.2 m). An HPLC analysis (Waters, Novap-
l
ak, 3.9 Â 150 mm) using both a UV detector and radioactivity
detector was conducted at a flow rate of 1 ml/min (70:30:0.1
methanol:water:triethylamine) retention time = 4.92 min. The
resulting analysis showed the formulated dose of [11C]4 to have
radiochemical purity greater than 99%. The total synthesis time
from end of bombardment was 65 min. The uncorrected radio-
chemical yield was 6.7%.
[
125I]1 and 0.33 for [125I]2 on a radioTLC. Using a 10:1 EtOAc: Hex-
anes solvent system, an Rf of 0.21 was found for [125I]2. There were
no radioactive side products. Radiochemical yields were 59% for
[
125I]1 and with of 42% for [125I]2. The product vials were then di-
luted to 10% ethanol with saline and used for the corresponding
autoradiography and biodistribution studies.
2.3. In vitro rodent OTR and V1aR binding assays: competitive
binding
2.2.15. 1-(4-(3,4-Dihydroquinolin-1(2H)-yl)piperidin-1-yl)-2-
(2-(2-[18F]fluoroethoxy)-4-((1-(methylsulfonyl)piperidin-4-yl)
oxy)phenyl)ethanone ([18F]3)
Prairie vole brains were collected immediately following eutha-
nasia and frozen on powdered dry ice. The brains were sectioned
from the prefrontal cortex (PFC) through the nucleus accumbens
Method A: A suspension containing 2 mg of compound 13 and
10 mg of cesium carbonate in 0.5 ml DMF was added to a V-tube
containing 2-[18F]fluoroethylbrosylate prepared according to pre-
viously reported methods.16,17 The mixture was heated to 110 °C
in an oil bath for 40 min and then cooled in an ice bath. The crude
mixture was diluted with 5 ml of HPLC mobile phase (50:50:0.1
ethanol:water:triethylamine) and purified via reverse phase HPLC
on a C18 xterra prep column (25 Â 100 mm). Product elution
peaked at 13 min with a flow rate of 6 ml/min. Fractions containing
product were diluted with twice their volume of water and loaded
on a pre-rinsed C18 sep-pak via vacuum and rinsed with 25 ml of
saline. Product was eluted with 1.5 ml of ethanol into a vial con-
taining 13.5 ml saline. The resulting mixture was then transferred
(NAcc) and ventral pallidum (VP) in seven series at 20 lm, on a
cryostat, and placed on Fisher Frost-plus slides. Slides were stored
at À80 °C until used in receptor autoradiography. Autoradiography
was performed as described previously with slight modifica-
tions.3,4,18,19 Brain sections of six different animals were used for
each study. The brain sections were removed from À80 °C storage,
allowed to thaw in air, dipped in 0.1% paraformaldehyde in PBS, pH
7.4, and rinsed twice in 50 mM Tris buffer, pH 7.4, to remove endog-
enous ligand. For competition studies, in order to generate a com-
petitive displacement curve, the seven adjacent series of brain
tissue sections were then incubated for 1 h in 50 pM of radioligand,
either 125I-OVTA (for detecting OTR) or 125I-LVA (for detecting
V1aR), with a gradient of seven different increasing concentrations
of competitor (either 1 or 2). Concentrations of the unlabeled com-
petitor included 0, 10À10, 10À9, 10À8, 10À7, 10À6, and 10À5 M. Un-
bound radioligand was removed by four washes in 50 mM Tris
buffer plus 2% MgCl2, pH 7.4, and then dipped into dd H2O and air
dried under a stream of cool air. Once dry, the slides were exposed
to BioMax MR film (Kodak) for 72 h with a set of 10 125I standards
(ARI 0133A from American Radiolabeled Chemicals, Inc.). Radioli-
gand tracers 125I-OVTA and 125I-LVA were obtained from Perkin El-
mer (NEX254010UC and NEX310010UC, respectively).
For the competitive binding assays, quantification of optical
binding density (OBD) was conducted on the resulting autoradio-
gram images in the following manner using AIS software (Imaging
Research, Inc.). After determining a flat field correction for lumi-
nosity levels, optical binding values from the set of standards were
loaded into the software and used to generate a standard curve,
from which binding density values from brain regions of interest
would be extrapolated. An average OBD for each region of interest
was calculated from three separate 30 Â 30 pixel circular area
‘punches’ through that brain region. An area of the brain with no
specific binding served as a background levels control for each
individual; an average of the OBD from three separate punches
in this background region was subtracted from the average value
for the specific binding in the region of interest. Specifically, for
the OTR autoradiograms labeled with 125I-OVTA, a final specific
binding value was calculated for the PFC as our region of interest
for each animal, and the primary somatosensory cortex served as
the background/nonspecific binding reference point. For the
V1aR autoradiograms labeled with 125I-LVA, a final specific binding
value for each individual was calculated for the VP and used the
caudate putamen as the background levels reference. These final
values for each animal were averaged for each treatment and plot-
ted (using GraphPad Prism software) as a logarithmic competition
curve showing standard error of the mean. Prism was also used to
under argon pressure over a Millipore filter (pore size of 1.0
lm)
followed by a smaller Millipore filter (pore size of 0.2 m). An HPLC
l
analysis (Waters, Novapak, 3.9 Â 150 mm) using both a UV detec-
tor and radioactivity detector was conducted at a flow rate of 1 ml/
min (70:30:0.1 methanol:water:triethylamine). The resulting anal-
ysis showed the formulated dose of [18F]3 to have radiochemical
purity greater than 99%. The total synthesis time including gener-
ation of 2-[18F]fluoroethylbrosylate was 145 min. The uncorrected
radiochemical yield was 2.3%.
Method B: The tosylate 15 was mixed with dried K22218F via
CPCU in acetonitrile at 110 °C for 15 min. The resulting product
was transferred via argon pressure with 7 ml of HPLC mobile phase
and subjected to identical purification and analytical techniques
outlined in Method A. The radiochemical purity was found to be
greater than 99%. The total synthesis time was 100 min after initial
transfer of activity from the cyclotron. The uncorrected radiochem-
ical yield was 19%.
2.2.16. 1-(1-(2-(2-[11C]methoxy-4-((1-(methylsulfonyl)piperi
din-4-yl)oxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydroquin
olin-2(1H)-one ([11C]4)
A typical trial was performed by preparing a mixture of com-
pound 13 (1.3 mg) and tetrabutylammonium hydrogen carbonate
(25 ll of a 0.075 M aq. solution) in DMF (400 ll) in a sealed 1 ml
v-vial. A balloon to capture excess activity was attached to the vial
and a delivery needle was submerged in the mixture. Then
[
11C]methyliodide was transferred from a GE methyliodide synthe-
sis unit via helium gas and bubbled through the cooled mixture.
After measuring activity trapped in the vial, the sealed vial was
heated at 110 °C for 10 min. The mixture was rapidly cooled, di-
luted with 0.5 ml of mobile phase solution (50:50:0.1 etha-
nol:water:triethylamine) and subjected to HPLC purification on a
C18 xterra prep column (25 Â 100 mm). Product elution peaked at
8.5 min using a flow rate of 6 ml/min. Fractions containing product

A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
2727
calculate the binding affinity (Ki) values for each competitor
molecule.
phosphate buffer solution (pH = 7.4) and 10 ml of octanol. After
shaking for 3 min, the buffer layer was discarded and 2 ml aliquots
of the remaining octanol layer were placed in separate test tubes
containing 2 ml of fresh buffer. The tubes were vortexed for 7 min
and then centrifuged (20,000Âg) for 5 min. Samples (0.5 ml) of both
the buffer and octanol layer were then transferred to separated
tubes and counted on a Cobra scintillation counter. The average
2.4. In vitro rodent OTR and V1aR binding assays: receptor
autoradiography using [125I]1 and [125I]2
Tissue sections were prepared as described above for the com-
petition assays and incubated in 70 pM of either radioligand,
counts of the two layers were used to determine the logP7.4
.
[
125I]1 and [125I]2, using the same methodology described above,
but without unlabeled competitors. The concentration of 70 pM
was determined after repeated optimization experiments (data
not shown). After incubation for 1 h in the radioligand, unbound
tracer was removed with rinses and the slides were exposed to Bio-
Max MR film (Kodak) for 72 h and then developed for visualization.
2.7. Rodent biodistribution studies
Tissue distributions studies were performed in female Sprague–
Dawley rats (180–200 g) after intravenous injection of [125I]1,
[
125I]2, or [18F]3 in 0.1–0.2 ml of 10% ethanol/saline. The animals
were allowed food and water ad libitum before the experiments.
The animals were anesthetized with an intramuscular injection
of 0.1 ml/100 g of a 1/1 ketamine (500 mg/ml)/xylazine (20 mg/
ml) solution, and cannulas were placed in the tail veins. The ani-
mals were euthanized at the indicated time points of 15, 30, 60
or 120 min, tissue samples from the brain, liver, kidney, heart, lung,
thyroid, muscle, uterus, and blood were taken, weighed, and
2.5. In vitro human OTR and vasopressin receptor binding
assays: competitive binding
The binding characteristics of 1 and 2 for the human OTR and
V1aR were determined by NIMH’s Psychoactive Drug Screening
Program at the University of North Carolina Chapel Hill. The proto-
col used for the assays was adopted from a previously reported
methodology and is summarized here as found on their website
http://pdsp.med.unc.edu/.20 A solution of the compound to be
tested was prepared as a 1-mg/ml stock in DMSO. A similar stock
of a reference compound (either oxytocin or vasopressin as a posi-
tive control) was also prepared. Eleven dilutions (5Â assay concen-
tration) of the test and reference compounds were prepared in a
binding buffer solution (100 mM NaCl, 10 mM MgCl2, 0.1 mg/ml
bacitracin, 1 mg/ml BSA, 20 mM Tris–HCl, pH 7.4) by serial dilution:
counted along with dose standards in a Packard Cobra II auto-
c
counter. The raw counts were decay-corrected to a standard time,
and the counts were normalized as the percent of total injected
dose per gram of tissue (% ID/g).
2.8. PET Imaging with [18F]3 and [11C]4
Sprague–Dawley rats were subcutaneously administered estro-
gen in the form of estradiol benzoate (10–12 lg in 0.2 ml sesame
0.05, 0.5, 1.5, 5, 15, 50, 150, 500 nM, 1.5, 5, 50
sponding assay concentrations span from 10 pM to 10
clude semilog points in the range where high-to-moderate
affinity ligands compete with radioligand for binding sites). The
radioligand ([3H]vasopressin or [3H]oxytocin) is diluted to 5 nM
(five times the assay concentration) in the binding buffer solution.
l
M (thus, the corre-
oil) once a day for 3 days prior to the scan dates to up-regulate
the oxytocin receptor expression. On the day of imaging, the rats
were anesthetized with ketamine/xylazine cocktail (100 and
10 mg/kg, respectively, via ip), affixed with a tail vein catheter,
and then strapped in a prone position on a plastic board designed
lM and in-
for the microPET. Radioligand [18F]3 (200–300
l
Ci) or [11C]4 (200–
Aliquots (50
96-well plate containing 100
duplicate 50- l aliquots of the test and reference compound dilu-
tions are added. Crude membrane fractions of cells expressing re-
combinant target OTR or vasopressin receptors (either of the 3
subtypes) were prepared from 10-cm plates by harvesting PBS-
rinsed monolayers, resuspending and lysing in chilled, hypotonic
50 mM Tris–HCl, pH 7.4, centrifuging at 20,000Âg, decanting the
supernatant and storing at À80 °C. These thawed cell lines are
resuspended in 3 ml of chilled binding buffer solution and homog-
l
l) of radioligand are dispensed into the wells of a
300 lCi) were then injected into the subject and PET imaging of the
l
l of binding buffer solution. Then,
brain area was performed for 90 min ([18F]3) or 45 min ([11C]4).
Three rats were scanned in this manner using [18F]3 and four rats
were scanned using [11C]4. Animals were euthanized with com-
pressed C02 following the scan.
l
3. Results and discussion
3.1. Synthesis of target compounds and labeling precursors
enized by several passages through a 26 gauge needle and 50
l
l are
In order to obtain the target compounds 1–4 and their radiola-
beled analogues, a versatile intermediate, 13, was generated to en-
able facile synthesis of various O-alkylated derivatives. Our applied
synthetic pathway to 13 is outlined in Scheme 1. The dihydroxy-
acetophenone 6 was alkylated specifically to the para position
using Mitsonobu conditions with a commercially available tert-
butoxycarbonyl protected hydroxy piperidine to give 7. The ortho
hydroxyl group was protected with MOM in DMF to give 8. The
acetophenone of 8 was converted to phenyl acetic acid 9 using
the Wilgerodt-Kindler reaction followed by saponification.
Although this step in the synthesis afforded a modestly low yield,
unreacted starting material could be recovered from the reaction.
Additionally, this methodology eliminated the use of highly toxic
thallium nitrate which was included in a previously reported syn-
thetic pathway of a similar molecule.22 It should also be noted that
the use of thallium nitrate was originally implemented in our syn-
thetic pathway, but the resulting yields were extremely lower than
what literature reported—including complete failures in multiple
trials. Acetic acid 9 was then reacted with quinolinone 10 via
condensation in the presence of hydroxybenzotriazole, 1-ethyl-3-
dispensed into each well. The 250- l reactions are incubated at
l
room temperature and shielded from light for 1.5 h and harvested
by rapid filtration onto Whatman GF/B glass fiber filters pre-soaked
with 0.3% polyethyleneimine using a 96-well Brandel harverster.
Four rapid 500-ll washes were performed with chilled standard
binding buffer to reduce non-specific binding. Filters are placed in
6-ml scintillation tubes and allowed to dry overnight. The next
day, 4 ml of EcoScint scintillation cocktail (National Diagnostics)
was added to each tube. The tubes were capped, labeled, and
counted by liquid scintillation counting. Raw data (dpm) represent-
ing total radioligand binding were plotted as a function of the log-
arithm of the molar concentration of the competitor. Non-linear
regression of the normalized raw data was performed in Prism 4.0.
2.6. Lipophilicity determination
The octanol/water partition coefficient was measured via a pre-
viously reported methodology.21 Approximately 5–10
lCi of 1 or 2
were added to a separatory funnel containing 5 ml of 0.1 M sodium

2728
A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
O
O
O
OH
O
OH
O
b
O
O
O
O
O
a
N
O
N
O
O
O
HO
N
OH
O
O
6
7
8
O
O
N
d
c
O
O
N
HO
O
N
O
O
N
O
O
O
O
N
O
O
11
9
NH
10
O
N
N
f
OH
OH
e
N
S
N
N
NH
O
HCl
O
O
O
O
12
13
Scheme 1. Synthesis of the versatile precursor 13. Reagents and conditions: (a) DEAD, Ph3P, THF 0 °C to rt. (b) chloro(methoxy)methane, Cs2CO3, DMF, rt. (c) (1) Morpholine,
sulfur, 120 °C (2) 2 N NaOH, 110 °C. (d) HOBT, EDC, DMF, rt. (e) 1 M HCl in Ether, rt. (f) Methanesulfonyl chloride, Et3N, DCM, rt.
(3-dimethylaminopropyl)carbodiimide, and Hünig’s base to give
11 in good yields. This reaction was also performed using Castro’s
reagent (benzotriazole-1-yl-oxy-tris-(dimethylamino)-phospho-
nium hexafluorophosphate) in place of hydroxybenzotriazole
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide with simi-
lar yields. A double deprotection of 11 was then performed using
1 M HCl in ether to give the hydrochloride salt 12. The piperidine
amine of 12 was selectively sulfonylated with 1 equiv of meth-
anesulfonyl chloride by using a dilute mixture of 12 in the pres-
ence of an organic base to produce sulfonamide 13. Multiple
trials were performed in attempts to optimize the addition so
that it occurred specifically on the amine position and not the
phenol position. An NMR analysis confirmed a side product found
in the reaction to be a double sulfonylated product—suggesting
O-sulfonylation as well as N-sulfonylation. Instead of using a
cold, concentrated mixture and a short reaction time, it was
found that using a small scale, diluting the mixture, and extend-
ing the reaction time eliminated sulfonylation on the phenol po-
sition altogether and prevented a difficult separation from the
double addition side product.
Having synthesized phenol 13, a family of compounds could be
generated bearing a similar structure as the original model com-
pound 5 by performing a simple and efficient O-alkylation with ce-
sium carbonate in DMF at room temperature. The versatility of 13
is featured in Scheme 2 which shows the synthetic pathway to
compounds 1–4. Alkylation of the phenol with E or Z tributyl(3-
chloroprop-1-enyl)stannane (synthesized according to previously
reported methods) gave the tributylstannyl precursors 14a and
14b.23 Iodination was then performed on 14a successfully with io-
dine in DCM for generation of 1, but it was discovered through
NMR analysis that the use of 14b with these conditions resulted
in isomerization. Therefore, to generate 2, iodination was per-
formed using sodium iodide in the presence of hydrogen peroxide.
Figure 2a shows the stacked 1H NMR data of the iodopropenyl
chemical shifts generated from 1 (top spectra), 2 (bottom spectra),
and the resultant product from reacting 14b with iodine in DCM
(middle spectra). The integration suggested a 50% mixture of the
diastereomers. The radiosynthesis of [125I]1 and [125I]2 involved
the use of sodium [125I]iodide and hydrogen peroxide, therefore
this phenomenon was not a concern for radiochemical purity.
Synthesis of 3, 4, and 15 were performed similarly using 1-bro-
mo-2-fluoroethane, iodomethane, and ethane-1,2-diyl bis(4-meth-
ylbenzenesulfonate) respectively with cesium carbonate in DMF.
These O-alkylation syntheses were generally allowed to stir over-
night with no evidence of any side products observed. An interest-
ing aspect found in the 1H NMR of these compounds was the
restricted rotation that resulted around the methylene carbon con-
necting the piperidinyl amide and the benzene ring. Figure 2b
shows the stacked 1H NMR data of the chemical shifts generated
by the methylene hydrogen atoms of precursor 13 and O-alkylated
product 3. The methylene protons of 13 become diastereotopic
after O-alkylation due to the restriction of rotation; the chemical
shift of 13 morphs from a singlet to form an AB quartet upon alkyl-
ation. In theory, the addition of heat would provide enough energy
to enable free rotation, but experiments were not undertaken to
determine the exact amount of heat. Therefore, free rotation may
or may not occur in vivo with the addition of body heat. Neverthe-
less, to investigate if restriction of rotation played a role in potency
or selectivity of our compounds, 13 was included in human cell
line binding assays.
3.2. In vitro binding assays using rodent brain tissue
To investigate the selectivity of 1–4 for the OTR over V1aR as
well as to determine their binding affinity for these receptors in ro-
dents, in vitro competition assays were performed with the four
compounds on prairie vole brain sections via autoradiography
using a procedure similar to that previously reported.3,24 The prai-
rie vole OTR and V1aR system was used because it has been exten-
sively characterized and this species has been shown to express the
OTR more broadly than in a rat brain. Figure 3 shows a representa-
tive set of brain images generated from co-incubating the tissue
sections in seven increasing concentrations of 1 in competition
for the OTR with [125I]-OVTA or in competition for V1aR with

A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
2729
I
SnBu3
O
O
N
N
c, d
O
O
O
O
S
N
S
N
N
N
O
O
O
O
O
O
1 - E
2 - Z
14a - E
14b - Z
a, b
F
O
O
e
N
N
OH
O
S
O
O
N
N
S
N
O
N
O
O
O
O
O
13
3
g
f
OTs
O
O
O
N
N
O
O
O
O
N
N
S
S
N
O
N
O
O
O
O
4
15
Scheme 2. Syntheses implemented with precursor 13 to generate 1–4. (a) Cs2CO3, (E)-tributyl(3-chloroprop-1-enyl)stannane, DMF, rt for 14a (b) Cs2CO3,(Z)-tributyl(3-
chloroprop-1-enyl)stannane, DMF, rt for 14b (c) I2 in DCM for 1 (d) 3% H2O2 (aq), NaI, EtOH, rt for 2. (e) Cs2CO3, 1-bromo-2-fluoroethane, DMF, rt. (f) Cs2CO3, ethane-1,2-diyl
bis(4-methylbenzenesulfonate), DMF, rt. (g) Cs2CO3, iodomethane, DMF rt.
[
125I]-LVA, standard radioligands used for this type of receptor
the values determined in the study were from rat uterine OTR and
used different techniques which leaves any comparisons in
question.
autoradiography in rodents. The binding density was quantified
and plotted as a competition curve that was used to determine
the binding affinities for these compounds (Fig. 4A–D, Table 1).
Increasing concentrations of all four ligands resulted in greater dis-
placement of both radioligands from the respective receptors as
indicated by decreased signal. Compounds 1–3 all demonstrated
similar potency with Ki values around 3 nm which is adequate
for their purpose. This demonstrates the viability of the iodoprop-
oxy moiety in mimicking the function of the fluoroethyl group and
vice versa. Compound 4 was not quite as potent with a Ki value of
18 nm. There are several variables that could contribute to the de-
crease in potency including side chain length, electronegativity,
and orientation of the compound around the methylene bond
(Fig. 2). We will not speculate as to which variable contributed
to the change in potency as these molecules were selected for
application purposes and not for determining binding affinity
trends. The selectivity of 1–4 for the rodent OTR over the rodent
V1aR was poor for all candidates. The best selectivity was observed
with 2 and 4 which were four times more selective for the rodent
OTR over V1aR, but still not adequate for distinction. For example,
a multiple of at least 50 would be desirable for ensuring distinction
between the receptor density patterns in vivo using PET imaging. It
is important to note the lack of selectivity in rodent tissue for these
compounds was anticipated due to previously reported studies in
rats using the similar compound 5 as the competitor.11 We did
not include the results from this previously reported data because
3.3. In vitro binding assay using human receptors
Our unpublished data as well as published reports indicate that
the commercially available radiolabeled peptides (e.g., [125I]-OVTA
and [125I]-LVA), which have been used to reliably investigate OTR
and V1aR in binding assays with rodent tissues, have displayed
mixed affinity for these receptors in primates. Therefore, these
radiolabeled peptides have decreased utility in localizing these
receptors in brain tissue from humans as well as other primate
species of interest.8,25 To determine the binding characteristics of
our selected compounds for the human OTR and all human vaso-
pressin receptor subtypes (V1a, V1b, and V2), compounds 1–4
and 13 were subjected to competitive binding assays using mem-
brane preparations from two different cell lines that have been
engineered to stably express either the gene for the human OTR
or the human vasopressin receptor (either of the 3 subtypes).
The radioligands used for competition were [3H]-OT for OTR and
[3H]-AVP for vasopressin receptors, as described by the NIMH’s
Psychoactive Drug Screening Program at the University of North
Carolina Chapel Hill. The competitive binding curves for the human
receptor assays are shown in Figure 5A–D and the data is tabulated
in Table 1. As can be seen from the charts and tables, the potency
for 1–4 were not altered significantly when assaying the OTR

2730
A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
Figure 2. Stacked NMR signals demonstrating reaction phenomenon and structural phenomenon. (a) the alkenyl hydrogen atoms of 1, 2, and the mixture of diasteriomers
generated from reacting 14b with I2 in DCM. (b) the methylene hydrogen atoms (indicated by ⁄) of 13 and 1 showing the formation of an AB quartet.
homologues from two different species with a Ki values ranging
from 5.7–28 nM. However, the potency for the human V1aR homo-
logue was significantly diminished (Ki value range of 5.3 Â 102 to
8.8 Â 103 nM), thus making these compounds selective for human
OTR. There was virtually no potency observed for the V1b subtype
and no significant potency for the V2 subtype with the lowest Ki
value being 1.4 Â 10-3 M for 1. Having potency of at least 79 times
greater for the human OTR over all vasopressin receptor subtypes,
1–4 were all valid candidates for pursuing selective detection of
human OTR in autoradiography or PET imaging studies.
Despite a lower potency for OTR, 13 displayed superior selectiv-
ity with no detectable affinity for any vasopressin receptor subtype
in the human OTR assays. We investigated 13 primarily because it
was a precursor in two of our radiosyntheses, but we were also
curious about structure allowing free rotation around its methy-
lene bond (Fig. 2b). In addition to free rotation, the hydroxyl group
acts as a hydrogen bond donor, a structural feature which is not
present in 1–4. Further structure activity studies will need to be
conducted to conclude if free rotation or the hydrogen bond donor
is responsible for the superior selectivity, perhaps by investigating
the unsubstituted moiety.
3.4. Radiolabeling of [125I]1 and [125I]2
Both [125I]1 and [125I]2 were obtained using the same method-
ology (reaction A of Scheme 3). The tributylstannyl compounds 14a
and 14b were treated with 125I+ for 45 min via in situ oxidation of
no-carrier-added Na125I with hydrogen peroxide. Both compounds
afforded their respective compounds in yields of 42% for the Z iso-
mer and 59% for the E isomer after sep-pak purification. No at-
tempts were made to improve these yields because ample
amounts of product were generated for our purpose; a diminutive
amount of [125I]1 and [125I]2 was required for autoradiography.
Due to the 60 day half-life of I-125, decay correction was not ap-
plied to the yields. Radiochemical purity was high for both diaste-
reomers at 95–96% pure as determined by radio-TLC. Thus, the use
of sep-pak purification was deemed adequate for acquiring accept-
able radiochemical purity. The radiolabeling was repeated a second
time and a radio-HPLC became available to accommodate the con-
tamination of the long half-life of I-125 45 days after these trials
(75% of a half-life). We investigated for UV traces to estimate spe-
cific activity and purity. It should be noted that decomposition of
both [125I]1 and [125I]2 was observed after being stored in the

A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
2731
A
C
B
D
0
10-10
10-9
10-8
10-7
10-6
10-5
Figure 3. Representative set of brain images generated from competitive receptor autoradiography. Columns A and B: tissue sections were co-incubated in seven increasing
concentrations of 1 in competition for the OTR with 50 pM [125I]-OVTA (A) or in competition for V1aR with 50 pM [125I]-LVA (B). Columns C and D: tissue sections were co-
incubated in seven increasing concentrations of 2 in competition for the OTR with 50 pM [125I]-OVTA (C) or in competition for V1aR with 50 pM [125I]-LVA (D).
10% ethanol in saline solution for 45 days when comparing the
radio-HPLC with the radio-TLC from the day of synthesis (see sup-
porting information). Approximately 15–20% more radioactivity
was detected where sodium [125I]iodide would be expected to
elute in comparison with the radio-TLC. Therefore, the shelf life
is limited in this media, and [125I]1 and [125I]2 should be used as
soon as possible for receptor assays following our protocol, or
stored in another media. The specific activity as determined from
calibration curves was found to be 408 Ci/mmol for [125I]1 and
413 Ci/ mmol for [125I]2. There were trace UV impurities found.
We speculated the impurities to be residual precursor or a proto-
destannylated side product of the precursor. Since our procedure
for radiolabeling involved the use of a small amount of hydrochlo-
ric acid to neutralize sodium hydroxide contained in the delivered
Na125I, it was theoretically possible for the tin labeling precursors
to undergo protodestannylation, a commonly known issue for
iodination using a tin precursor.26 Although this phenomenon is
more readily observed when a heated reaction mixture is required
for a deprotection reaction post labeling, it was investigated fur-
ther for clarification. To a 3 ml reaction mixture during an experi-
mental trial of the synthesis of 2, 200 ll of 0.4 N HCl was added
and the mixture was concentrated in vacuo to a residue. Only de-
sired product was found when analyzed via NMR after work-up.
The reaction mixture volume during the radiosynthesis of [125I]1
and [125I]2 is 300ul, and the presence of sodium hydroxide greatly
reduces the actual concentration of HCl in the mixture. Therefore,
we believe it was very unlikely that protodestannylation occurred
during radiosynthesis and any observed UV trace from our HPLC
came from the starting material. We did not investigate the po-
tency of 14a or 14b. Due to the significant difference in retention

2732
A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
Table 1
In vitro Ki (nM) determinations of 1–4 and 13 in competition assays with human and
rodent OTR and vasopressin receptors (3 subtypes)
Compound
Rodenta
OTR
Humanb
OTR
V1aR
V1aR
V1b
V2
1
2
3
4
3.6
4.2
2.9
18
—
1.7
16
9.4
78
—
10
5.7
16
28
78
7.9 Â 102
5.3 Â 102
8.7 Â 103
4.8 Â 103
>1 Â 104
>1 Â 104
9.6 Â 103
>1 Â 104
>1 Â 104
>1 Â 104
1.4 Â 103
5.8 Â 102
1.8 Â 103
3.7 Â 103
>1 Â 104
13
a
Values were determined from six sets of autoradiography data.
Ki values for human receptors were generously provided by the National
b
Institute of Mental Health’s Psychoactive Drug Screening Program, Contract #
HHSN-271-2008-00025-C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L.
Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer
Jamie Driscol at NIMH, Bethesda MD, USA. For experimental details please refer to
the PDSP web site http://pdsp.med.unc.edu/ and click on ‘Binding Assay’ or ‘Func-
tional Assay’ on the menu bar.
functional, as was demonstrated by our studies. The trace impuri-
ties did not hinder the binding of [125I]1 and [125I]2 to prairie vole
brain receptors during autoradiography (see Fig. 6).
3.5. Autoradiograpy with [125I]1 and [125I]2
To begin to assess the utility of [125I]1 and [125I]2 as adequate
radioligands for localizing brain receptors with in vitro autoradiog-
raphy, we subjected these radioligands for receptor autoradiogra-
phy on brain slices from prairie voles, whose OTR and V1aR
distributions are well established. Figure 6 shows the images gen-
erated from our assay demonstrating that both [125I]1 and [125I]2
bind to receptors in the NAcc and VP with a slightly lower signal
to noise ratio than the peptide radioligands. It has been established
previously that the NAcc in prairie voles contains a high density of
OTR and the VP contains a high density of V1aR; reference images
generated from [125I]-LVA (showing V1aR) and [125I]-OVTA (show-
ing OTR) are also reproduced from images previously reported by
Lim et al4 These results are consistent with our competition bind-
ing data described above and suggest the radioligands label both
the prairie vole OTR and V1aR in autoradiography. Therefore, with
the lower signal to noise ratio and lack of selectivity, [125I]1 and
[
125I]2 would not be ideal for rodent tissue autoradiography in
comparison to the peptide radioligands. Nevertheless, these auto-
radiography images are very encouraging as they demonstrate
the capability of these small molecules bind in a manner that en-
ables distinct OTR receptor density patterns to be recognized.
Therefore, based on these expected results in rodent tissue and
the binding data from cell culture which shows much higher selec-
tivity between the human OTR and human V1aR, we would predict
that these compounds would preferentially bind to the OTR over
the V1aR in primate tissue, thus distinguishing between the two.
Additionally, we would anticipate similar patterns to be formed
in vivo if these compounds were investigated via PET imaging with
the positron emitting I-124 analogues and they manage to breach
the blood–brain barrier (BBB). Future studies are planned to assess
the utility of these ligands to selectively label OTR in primate tissue
using autoradiography.
Figure 4. Competitive binding graphs generated from varying concentrations of 1–
4 in competition for OTR and V1aR on prairie vole brain slices with [125I]-OVTA and
[
125I]-LVA respectively. (A) Curves generated using 1. (B) Curves generated using 2.
3.6. Radiolabeling of [18F]3 and [11C ]4
(C) Curves generated using 3. (D) Curves generated using 4.
Reactions B and C in Scheme 3 present the two methodologies
used in attaining [18F]3. The first method involved synthesis of 2-
times in UV impurity traces and [125I]1 and [125I]2, the labeled
products could be separated completely from all detected trace
impurities via prep-HPLC. However, to avoid a lengthy contamina-
tion of an HPLC system, a sep-pak procedure may prove to be
[
18F]fluoroethylbrosylate according to previously reported meth-
odologies.16,17 Alkylation of phenol 13 with 2-[18F]fluoroethylbro-
sylate was then conducted in DMF at 110 °C for 40 min. This
method was successful in obtaining [18F]3 in high radiochemical

A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
2733
purity and enough product to generate a human dose, the yield
(2.3% uncorrected) was not ideal for routine production. Therefore,
to eliminate the use of 2-[18F]fluoroethylbrosylate and make the
process a one-step synthesis, 15 was subjected to SN2 substitution
with K22218F in acetonitrile at 110 C for 15 min (reaction C in
Scheme 3). The result was an 8 fold improvement in yield which
generated ample product for multiple studies. The radiochemical
purity was more than adequate as well. There were no UV impuri-
ties found in either method as HPLC separation was efficient in
both cases.
The conditions for radiolabeling [11C ]4 are outlined in reaction
D of Scheme 1. Several bases were tried unsuccessfully prior to the
use of tetrabutylammonium hydrogen carbonate (TBAHC). Sodium
hydroxide in various molarities was found to be too harsh for 13 as
decomposition was observed. Cesium carbonate did not generate
adequate yields even with a brosylate leaving group and 40 min
reaction time as was discussed for the synthesis of [18F]3. The
20 min half-life compounded the need to alter the conditions.
The use of TBAHC as purchased from ABX was found to be mild en-
ough to avoid decomposition of 13 and function well enough to in-
crease the reaction time. We were able to obtain uncorrected
yields of 6.7% with radiochemical purity >99% with only a 10 min
reaction time. No significant UV impurities were observed.
3.7. Lipophilicity
The lipophilicity of raiolabeled analogues of 1–4 were deter-
mined in efforts to predict their ability to cross the BBB and for fu-
ture reference if further structure–activity studies were needed.
The optimal range of lipophilicity desired of radioligands to
achieve BBB penetration has been reported to be logP7.4 = 2.0–3.5
by one source (Waterhouse) and 2.0 0.7 by another (Hansch).27,28
Our measured values determined from a previously reported
method are tabulated in Table 2.21 According to both Waterhouse
and Hansch, 1–4 were all in optimal range for penetrating the
BBB (values ranged 2.02–2.36). There are other factors that will
influence the ability of these molecules to penetrate the BBB
including size, hydrogen bonding donor and acceptors, functional
groups, rotatable bonds, and others.29 Our primary concern with
1–4 was their size and lack of hydrogen bond donor groups. Com-
putational studies have predicted a molecular weight below 500
should be a requirement for breaching the BBB.30 As can be seen
from Table 2, 1–4 are all over 500 g/mol with 4 being the smallest
at 555. We limited our investigations to of BBB permeability to cal-
culating logP7.4 for future reference as we planned to conduct
in vivo studies to further evaluate the radioactive analogues of
1–4.
3.8. Biodistribution studies
The distribution of radioactivity expressed as the mean percent
of total injected dose per gram (% ID/g) of tissue in female Spra-
gue–Dawley rats at 15, 30, 60, and 120 min after injection of either
[
125I]1, [125I]2, and [18F]3 is shown in Table 3a–c. The primary focus
of our study was brain penetration and uptake of neural OTR.
Having a measured ID/g of 0.05 0.01 or less across all time points
for [125I]1, [125I]2, and [18F]3, we believed our radioligands failed to
penetrate into the rodent brain efficiently. Considering the expres-
sion of the OTR in the rat brain is limited, we dissected and mea-
sured specific areas of the brain known to express OTR, including
the amygdala, basal hypothalamus, and dorsal striatum.31 As a com-
parison control, we examined the cerebellum and tabulated these
results in Table 4. The remaining brain was also counted to provide
the total brain uptake values in Table 3. The distribution within the
brain of all the radioligands was not very evident—suggesting lack
Figure 5. Curves generated from varying concentrations of 1–4 in competition with
[3H]-OT and [3H]-AVP for OTR and V1aR on membrane preparations from cells
stably transfected with the human OTR or V1aR gene. a(A) Curves generated using 1.
(B) Curves generated using 2. (C) Curves generated using 3. (D) Curves generated
using 4. aCompetitive binding plots of human receptors were generously provided
by the National Institute of Mental Health’s Psychoactive Drug Screening Program,
Contract # HHSN-271-2008-00025-C (NIMH PDSP). The NIMH PDSP is Directed by
Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project
Officer Jamie Driscol at NIMH, Bethesda MD, USA. For experimental details please
refer to the PDSP web site http://pdsp.med.unc.edu/ and click on ‘Binding Assay’ or
‘Functional Assay’ on the menu bar.

2734
A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
125I
SnBu3
O
O
N
N
O
a
O
O
O
A
N
S
N
S
N
N
O
O
O
O
O
O
[
[
125I]1 - E
12a - E
12b - Z
125
2
I] - Z
18F
O
O
N
N
b
OH
O
O
O
B
N
S
N
S
N
N
O
O
O
O
O
O
13
[18F]3
18F
O
OTs
O
c
N
N
O
O
O
O
C
D
N
S
N
S
N
N
O
O
O
O
O
O
15
13
[18F]3
O
O
11CH3
O
N
N
d
OH
O
O
N
S
N
S
N
N
O
O
O
O
O
O
[11C]4
Scheme 3. Radiosynthesis of [125I]1, [125I]2, [18F]3, and [11C]4. Reagents and conditions: (A) Na125I, 3% H2O2 (aq), 0.4 N HCl, EtOH, rt. (B) 2-[18F]fluoroethylbrosylate, Cs2CO3,
acetonitrile, 110C, 40 min. (C) K22218F, Acetonitrile, 110 °C, 15 min. (D) [11C]methyliodide, tetrabutylammonium hydrogen carbonate, DMF, 100 °C, 10 min.
125I]-OVTA
[
125I]-LVA
[
125I] 2
[
125I] 1
[
Nucleus
Accumbens
Caudate
Putamen
Ventral
Pallidum
Figure 6. Autoradiography images generated from [125I]1, [125I]2, [125I]-OVTA, and [125I]-LVA on prairie vole brain sections. Both [125I]1 and [125I]2 bound as expected to
regions of the brain where OTR and V1aR are expressed in prairie voles. The images from [125I]-LVA and [125I]-OVTA were provided with permission by Lim et al. 2004.

A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
2735
Table 2
was viable for PET imaging OTR of the CNS, a PET scan was per-
formed using a rat model.
Determined logP7.4 values and molecular weights of 1–4
Considering the periphery, the increase in I-125 uptake in the
thyroid over time suggests [125I]1 and [125I]2 both were broken
down readily in vivo at the iodine position with the cis-isomer
being slightly more stable. In contrast, [18F]3 appeared to remain
intact as there was no significant increase in F-18 bone uptake over
time. This is to be expected as iodine is a much better leaving
group than fluorine. There was significant uptake of [125I]1 and
Compound
logP7.4
MW
1
2
3
4
2.36
2.18
2.02
2.25
707.62
707.62
587.70
555.69
[
125I]2 observed within the uterus as was expected due to the
uterus being rich in OTR. The data generated from the uterus for
18F]3 was deleted from Table 3c due to a discovered experimental
of penetration. It is possible that BBB penetration occurred rapidly,
well before the first time point of 15 min and were then ejected
immediately via the P-glycoprotein efflux pump, a common obsta-
cle in developing drugs targeting the brain.32 Although it was not
performed in this study, a 5 min time point would help in determin-
ing if the P-glycoprotein pump or simply lack of retention was
responsible for inconsistent BBB penetration. A P-glycoprotein
pump assay would also determine if our targets were susceptible,
but we did not have the cell cultures readily available. We did, how-
ever, investigate this phenomenon with [18F]3 and [11C]4 via PET
imaging as described in the following section.
When comparing the regions of higher rat OTR densities with
the low OTR density region of the cerebellum, there was not a sig-
nificant difference as most activity measurements for the brain
samples were very near background readings. However, there
was a slightly noticeable increase in activity at the 60 and
120 min time points of [18F]3 in the amygdala and basal hypothal-
amus. This data could account for penetration of [18F]3 to the brain,
but at a rate not ideal for PET imaging. To further clarify if [18F]3
[
error. The liver was the region with the most uptake of all radiotra-
cers. The physiology of the liver involves breaking down small and
complex molecules; therefore the uptake in the liver indicates the
tracers were being metabolized. Elevated uptake in the kidney con-
firms this. In general, an overall lower amount of activity was de-
tected with [18F]3 in comparison to [125I]1 and [125I]2, especially
in the heart, blood, muscle, and lung. We speculate, due to the in-
creased stability of [18F]3 mentioned previously, [18F]3 saturates
OTR and V1aR sites (or possibly other receptors) more rapidly as
its radioactive isotope stays intact with the core molecule and,
therefore, less of it reaches the liver and kidney. The increased
activity measured for [125I]1 and [125I]2 in the heart, blood, muscle,
and lung was more likely free I-125 than [125I]1 or [125I]2 that did
not reach the OTR or V1aR.
3.9. PET imaging of [18F]3 and [11C]4 using rat models
Determining OTR densities non-invasively would be ideal for
correlating OTR density to social behavior patterns, normal and
abnormal, without incurring physical harm to subjects. We antici-
pated the use of [18F]3 and [11C]4 in PET imaging studies would
provide a good base in our path to develop such a tool. Although
Table 3
Biodistribution of radioactivity in female Sprague–Dawley rats after injection of
[
125I]1(a), [125I]2 (b), and [18F]3 (c)a
(a)
[
18F]3 and [11C]4 would not be ideal for use in rodent subjects
[
125I]1
15 minb
30 min
60 min
120 min
unless the distribution of both OTR and V1aR were desired, their
selectivity in homologous human receptors should provide a
distinctive pattern once the technology is proven viable. To
Blood
Heart
Lung
0.58 0.51
0.82 0.70
1.86 1.60
5.67 5.08
0.96 0.84
0.19 0.16
0.28 0.24
0.43 0.38
0.05 0.04
0.58 0.07
0.61 0.18
1.41 0.72
3.90 0.15
0.75 0.07
0.53 0.23
0.26 0.02
0.41 0.07
0.05 0.02
0.55 0.27
0.54 0.28
1.45 1.04
3.09 1.34
0.69 0.30
0.81 0.53
0.23 0.11
0.45 0.23
0.04 0.02
0.38 0.14
0.38 0.06
1.04 0.75
1.81 0.30
0.42 0.10
1.05 0.70
0.14 0.03
0.30 0.10
0.02 0.01
Liver
Kidney
Thyroidc
Muscle
Uterus
Brain
Table 4
Biodistribution of radioactivity in specific brain regions of female Sprague–Dawley
a
rats after injection of [125I]1 (a), [125I]2 (b), and [18F]3 (c)
(a)
(b)
[
[
125I]1
15 min
30 minb
60 min
120 min
125I]2
Basal Hypothalamus
Amygdala
Dorsal Striatum
Cerebellum
0.08 0.01
0.09 0.02
0.05 0.01
0.08 0.01
0.07 0.01
0.06 0.01
0.07 0.03
0.05 0.01
0.05 0.01
0.05 0.01
0.06 0.01
0.09 0.02
0.04 0.01
0.05 0.01
0.05 0.01
0.05 0.04
0.06 0.01
0.03 0.01
0.03 0.01
0.03 0.01
Blood
Heart
Lung
0.22 0.14
0.51 0.41
1.79 1.78
2.12 1.73
0.38 0.20
0.05 0.04
0.10 0.09
0.22 0.16
0.02 0.01
0.23 0.23
0.39 0.38
1.02 1.13
1.91 1.88
0.31 0.27
0.20 0.22
0.09 0.08
0.16 0.16
0.02 0.01
0.32 0.06
0.39 0.12
0.66 0.32
3.01 0.50
0.46 0.04
0.32 0.05
0.15 0.03
0.31 0.08
0.02 0.01
0.31 0.17
0.19 0.09
0.41 0.27
1.91 1.02
0.29 0.15
0.67 0.56
0.10 0.05
0.22 0.13
0.02 0.01
Liver
Remaining Brain
Kidney
Thyroidc
Muscle
Uterus
Brain
(b)
[
125I]2
Basal hypothalamus
Amygdala
Dorsal striatum
Cerebellum
0.04 0.01
0.06 0.01
0.03 0.01
0.03 0.01
0.03 0.01
0.03 0.01
0.08 0.06
0.03 0.01
0.03 0.01
0.02 0.01
0.03 0.01
0.06 0.01
0.02 0.01
0.03 0.01
0.02 0.01
0.04 0.01
0.05 0.02
0.02 0.01
0.03 0.01
0.02 0.01
(c)
[
18F]3
Remaining brain
Blood
Heart
Lung
Liver
Kidney
Bone
0.07 0.04
0.17 0.09
0.39 0.3
1.81 0.36
0.27 0.11
0.05 0.01
0.07 0.01
0.02 0.01
0.06 0.01
0.09 0.02
0.1 0.03
1.41 0.17
0.15 0.04
0.05 0.01
0.06 0.01
0.02 0.01
0.04 0.02
0.07 0.02
0.1 0.03
0.98 0.44
0.08 0.02
0.03 0.01
0.03 0.01
0.02 0.01
0.09 0.04
0.08 0.02
0.19 0.15
1.18 0.18
0.10 0.02
0.07 0.04
0.07 0.04
0.04 0.03
(c)
[
18F]3
Basal hypothalamus
Amygdala
Dorsal striatum
Cerebellum
0.03 0.01
0.05 0.01
0.02 0.01
0.02 0.01
0.02 0.01
0.03 0.01
0.10 0.03
0.03 0.01
0.05 0.04
0.02 0.01
0.06 0.01
0.11 0.07
0.03 0.01
0.02 0.01
0.02 0.01
0.11 0.04
0.13 0.02
0.05 0.03
0.04 0.03
0.03 0.03
Muscle
Brain
Remaining brain
a
Values are reported as the mean percent of total injected dose per gram (%ID/g)
of tissue standard deviation (n = 4 for all time points except where noted).
a
Values are reported as the mean percent of total injected dose per gram (%ID/g)
b
of tissue standard deviation (n = 4 for all time points except where noted).
n = 3.
b
c
n = 3.
Thyroid values are reported as percent dose per organ rather than % ID/g.

2736
A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
Figure 7. PET images of 295
l
Ci of [18F]3 in a 280 kg female Sprague-Dawley rat. (a) Sum of 90 min scan. (b) Sum of first 5 min of scan with red arrows pointing to suspected
uptake. (c) Sum of last 30 min of scan.
Figure 9. PET image of 277 l
Ci of [11C]4 in a 280 kg female Sprague-Dawley rat
over 45 min.
Figure 8. Time-activity curves showing uptake graphs over time of the rostral and
caudal halves of the brain, the whole brain, and muscle tissue from the periphery.
investigate the viability of [18F]3, three Sprague–Dawley rats were
investigated in 90 min PET imaging scans beginning at the time of
injection. Figure 7 shows three sagittal images of the brain region
which are a sum of the total 90 min of the scan (Fig. 7a), a sum of
the first 5 min of the scan (Fig. 7b), and a sum of the last 30 min of
the scan (Fig. 7c). A thorough investigation of the entire brain re-
gion from the sum of the total scan indicated that there was not
enough specific uptake of [18F]3 to merit regular investigative
use of OTR density mapping. To determine if [18F]3 was penetrat-
ing to the brain within the first 5 min post injection, we summed
the scan within this timeframe. As indicated by the red arrows in
Figure 7b, it appears there was some mild brain penetration of
may imply [18F]3 has affinity for the P-glycoprotein pump, but fur-
ther studies would need be conducted for validation. Additionally,
the TACs confirmed that [18F]3 or a metabolite of [18F]3 does, in
fact, gradually enter the brain over time. Thus, Figure 7c clearly
has a much higher background in the brain region than Figure
7b, yet no specific uptake was observed.
Although the potency of 4 was not quite as good as 3, we
wanted to investigate if a slightly smaller molecule would aid in
penetration of the BBB. Therefore, [11C]4 was investigated in four
Sprague–Dawley rats. The results were more disappointing than
those obtained from [18F]3. This was somewhat anticipated as a
compound bearing a similar structure, L-371257, is known for
being a very poor brain penetrant.33 Figure 9 has a representative
sagittal image from the sum of the 45 min scan and Figure 10 con-
tains a TAC curve generated from the entire brain region and an
area of muscle representing periphery uptake. The TAC curve sug-
gests that a gradual uptake of [11C]4 in the brain may occur as was
seen with [18F]3 as there was an uptick in activity near 2000 s, but
[
18F]3 immediately following injection.
A time-activity curve
(TAC) generated from our software (Fig. 8) confirmed that there ap-
peared to be a burst of activity into the brain immediately follow-
ing injection, followed a rapid decrease. Figure 8 includes the TACs
of the rostral and caudal halves of the brain, the whole brain, and
some muscle tissue representing periphery uptake. These TACs

A. L. Smith et al. / Bioorg. Med. Chem. 20 (2012) 2721–2738
2737
not viable for in vivo PET imaging of the OTR of the CNS although
visual evidence suggested [18F]3 did penetrate the brain temporar-
ily following injection. Therefore, the radioactive analogues of 1–4
are limited to in vitro studies following the procedures outlined
here. Perhaps an alternative approach, such as the use of mannitol
or adenosine, to help these compounds breach the BBB more read-
ily could increase their viability for in vivo purposes within the
CNS.34,35
The utility of [125I]1 and [125I]2 for localizing the OTR in primate
brain sections is yet to be determined. The successful localization
of primate OTR using these molecules would alleviate doubt that
has been shed on localizations generated with peptides.8 There-
fore, further studies are planned to assess the potential of these
I-125 ligands in validating the OTR localization of primate brain
sections.
Although having a slightly decreased potency for OTR, the syn-
thetic intermediate 13 was found to have achieved optimal selec-
tivity with no detectable affinity for any vasopressin receptor
subtype. This result suggests further structural modifications could
potentially lead to a radioligand candidate with the ability breach
the BBB and maintain retention. It is theoretically possible to label
13 using [11C]methanesulfonylchoride as has been prepared previ-
ously.36 The addition of a hydrogen bond donor to the structure
clearly improved selectivity despite allowing free rotation about
its methylene bond and further reducing its molecular weight.
Therefore, the potent and selective 13 may be a viable target for fu-
ture studies.
Figure 10. Time-activity curves showing uptake graphs over time of the whole
brain and muscle tissue from the periphery.
In conclusion, noteworthy progress has been made and is still
ongoing toward the development of small molecules bearing the
ability to localize the OTR using both in vitro and in vivo method-
ologies. The two novel PET ligands investigated here were found
unsuitable candidates for in vivo studies. However, two novel
I-125 molecules were proven viable for in vitro localization of
OTR via autoradiography and will be investigated further to
validate their utility with primate brain sections.
the scan was stopped shortly after two half-lives. It is also possible
that [11C]4 penetrated to the brain immediately following injection
as there was a spike in activity in the area, however, there was no
visual confirmation from the image itself.
4. Conclusion
Acknowledgements
The OTR ligands (E)-1-(1-(2-(2-(3-iodoallyloxy)-4-(1-(methyl-
sulfonyl)piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihy
droquinolin-2(1H)-one (1), (Z)-1-(1-(2-(2-(3-iodoallyloxy)-4-(1-
(methylsulfonyl)piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,
4-dihydroquinolin-2(1H)-one (2), 1-(4-(3,4-dihydroquinolin-1(2H)-
yl)piperidin-1-yl)-2-(2-(2-fluoroethoxy)-4-((1-(methylsulfonyl)
piperidin-4-yl)oxy)phenyl)ethanone (3), and 1-(1-(2-(2-methoxy-
4-((1-(methylsulfonyl)piperidin-4-yl)oxy)phenyl)acetyl)piperidin-
4-yl)-3,4-dihydroquinolin-2(1H)-one (4) have been synthesized,
radiolabeled, and evaluated via in vitro and in vivo methods. Com-
petitive binding assays performed using prairie vole brain slices
with 1–4 showed a high potency for both the OTR and V1aR; no
significant selectivity was observed for these compounds in rodent
tissue. Competitive binding assays using cell lines expressing the
homologous human OTR and vasopressin receptors (three sub-
types) revealed 1–4 displayed potency for the OTR comparable to
that of the rodent receptors, but showed significantly improved
selectivity for the OTR over V1aR. Potency for the human OTR
was at least 79-fold greater (1) than that of the V1aR and as much
as 500-fold greater (3).
We thank Larry Williams, Mel Camp, and Eugene Malveaux for
their contributions in the rodent distribution studies. We thank the
NIMH PDSP directed by Bryan L. Roth MD, PhD at the University of
North Carolina at Chapel Hill and Project Officer Jamie Driscol at
NIMH, Bethesda MD, USA for their contributions of the human cell
line studies. We also thank Dr. Fred Strobel at the Emory University
Mass Spectrometry Center for our mass spectrometry data. This re-
search was funded by the National Institute of Mental Health
through Grant 5 R21 MH090776-02. We also acknowledge NIH
MH064692 (LJY) and RR00165 to YNPRC.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.02.019.
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