Synthesis and pharmacological in vitro and in vivo profile of 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68), a selective antagonist of the neuropeptide S receptor

Article abstract of DOI:10.1124/jpet.107.135103

Neuropeptide S (NPS) has been shown to modulate arousal, sleep wakefulness, anxiety-like behavior, and feeding after central administration of the peptide agonist to mice or rats. We report here the chemical synthesis and pharmacological characterization of SHA 66 (3-oxo-1,1-diphenyl-tetrahydro- oxazolo[3,4-a]pyrazine-7-carboxylic acid benzylamide) and SHA 68 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide), two closely related bicyclic piperazines with antagonistic properties at the NPS receptor (NPSR). The compounds block NPS-induced Ca²⁺ mobilization, and SHA 68 shows displaceable binding to NPSR in the nanomolar range. The antagonistic activity of SHA 68 seems to be specific because it does not affect signaling at 14 unrelated G protein-coupled receptors. Analysis of pharmacokinetic parameters of SHA 68 demonstrates that the compound reaches pharmacologically relevant levels in plasma and brain after i.p. administration. Furthermore, peripheral administration of SHA 68 in mice (50 mg/kg i.p.) is able to antagonize NPS-induced horizontal and vertical activity as well as stereotypic behavior. Therefore, SHA 68 could be a useful tool to characterize physiological functions and pharmacological parameters of the NPS system in vitro and in vivo. Copyright

Full text of DOI:10.1124/jpet.107.135103

0022-3565/08/3253-893–901$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics
JPET 325:893–901, 2008
Vol. 325, No. 3
135103/3340636
Printed in U.S.A.
Synthesis and Pharmacological in Vitro and in Vivo Profile of
3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-
carboxylic Acid 4-Fluoro-benzylamide (SHA 68), a Selective
Antagonist of the Neuropeptide S Receptor
Naoe Okamura, Stephen A. Habay, Joanne Zeng, A. Richard Chamberlin, and
Rainer K. Reinscheid
Departments of Pharmaceutical Sciences (N.O., J.Z., A.R.C., R.K.R.), Pharmacology (N.O., J.Z., R.K.R.), Chemistry (S.A.H.,
A.R.C.), and Molecular Biology and Biochemistry (R.K.R.), University of California, Irvine, California
Received December 7, 2007; accepted March 11, 2008
ABSTRACT
Neuropeptide S (NPS) has been shown to modulate arousal, in the nanomolar range. The antagonistic activity of SHA 68
sleep wakefulness, anxiety-like behavior, and feeding after cen- seems to be specific because it does not affect signaling at 14
tral administration of the peptide agonist to mice or rats. We unrelated G protein-coupled receptors. Analysis of pharmaco-
report here the chemical synthesis and pharmacological char- kinetic parameters of SHA 68 demonstrates that the compound
acterization of SHA 66 (3-oxo-1,1-diphenyl-tetrahydro-ox- reaches pharmacologically relevant levels in plasma and brain
azolo[3,4-a]pyrazine-7-carboxylic acid benzylamide) and SHA after i.p. administration. Furthermore, peripheral administration
68 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7- of SHA 68 in mice (50 mg/kg i.p.) is able to antagonize NPS-
carboxylic acid 4-fluoro-benzylamide), two closely related bi- induced horizontal and vertical activity as well as stereotypic
cyclic piperazines with antagonistic properties at the NPS re- behavior. Therefore, SHA 68 could be a useful tool to charac-
ceptor (NPSR). The compounds block NPS-induced Ca2ϩ terize physiological functions and pharmacological parameters
mobilization, and SHA 68 shows displaceable binding to NPSR of the NPS system in vitro and in vivo.
Neuropeptide S (NPS) and its receptor, NPSR, are a re- expressed in only a few brainstem structures; in particular,
cently identified transmitter system that modulates a num- in a previously uncharacterized nucleus situated between the
ber of brain functions (Okamura and Reinscheid, 2007). NPS
is a small peptide of 20 amino acids that occurs in all tetrapod
vertebrates but is absent from fish (Reinscheid, 2007). Acti-
vation of NPSR produces transient increases in intracellu-
lar Ca2ϩ and cAMP and thus increases cellular excitability
(Reinscheid et al., 2005). Expression of NPS precursors and
receptors is found in specific brain areas that have been
associated with arousal, emotional processing, energy, and
hormonal homeostasis, as well as learning and memory (Xu
et al., 2004, 2007). In the rat, NPS precursor transcripts are
noradrenergic locus coeruleus and Barrington’s nucleus. Be-
sides the pericoerulear region, NPS mRNA is only found in
the lateral parabrachial nucleus and the principle sensory 5
nucleus of the rat brainstem. A few scattered cells expressing
NPS precursor transcripts are also detected in the amygdala
and hypothalamus. In the brainstem, the majority of NPS-
expressing neurons coexpress other excitatory transmitters,
such as glutamate, acetylcholine, or corticotropin-releasing
factor (Xu et al., 2007). NPSR mRNA is found at high levels
in hypothalamus, thalamus, amygdala, various cortical re-
gions, and the parahippocampal formation. Central admin-
istration of NPS was shown to produce profound arousal that
is independent of novelty (Xu et al., 2004). NPS is also able to
induce wakefulness by suppressing all stages of sleep, as
demonstrated by electroencephalographic recordings in rats.
In addition, NPS administration was shown to produce anx-
This study was supported in part by the National Institute of Mental Health
(Grant MH-71313; to R.K.R.) and by the National Institute of Neurological
Disorders and Stroke (Grant NS-27600; to A.R.C.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.135103.
ABBREVIATIONS: NPS, neuropeptide S; NPSR, neuropeptide S receptor; SHA 66, 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-
carboxylic acid benzylamide; SHA 68, 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide; ESI, elec-
trospray ionization; THF, tetrahydrofuran; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; GPCR, G protein-coupled receptor; PBS,
phosphate-buffered saline; PK, pharmacokinetic; BBB, blood-brain barrier; LC/MS/MS, liquid chromatography-coupled tandem mass spectrom-
etry; ANOVA, analysis of variance; HEK, human embryonic kidney; Veh, vehicle; Fmoc, 9H-fluoren-9-ylmethoxy carbonyl.
893

894
Okamura et al.
iolytic-like effects across various behavioral paradigms mea- to have affinities below 100 nM, but no primary data were
suring responses to stressful or unfamiliar environments in presented to support the claim. We therefore set out to syn-
mice (Xu et al., 2004; Leonard et al., 2005). Other studies thesize these compounds and validate their pharmacological
reported anorexic effects after central NPS administration properties as potential NPSR antagonists in vitro and in
(Beck et al., 2005; Smith et al., 2006) and modulation of vivo. The availability of synthetic NPSR antagonist com-
stress hormone levels (Smith et al., 2006), although a recent pounds will certainly facilitate further research on physio-
study challenged the initial findings on NPS-induced inhibi- logical functions of the NPS system.
tion of feeding behavior (Niimi, 2006).
A functional polymorphism in the human NPSR gene has
been identified that encodes an Asn-to-Ile amino acid ex-
change at position 107 of the receptor protein (A/T; single
Materials and Methods
Chemicals. All chemicals were of analytical grade or higher qual-
ity. NPS was synthesized by the Peptide Proteomic Centre, Brain
nucleotide polymorphism database accession no. rs324981).
Research Centre, University of British Columbia (Vancouver, BC,
NPSR Ile107 shows a 5- to 10-fold increased agonist sensi-
Canada), and stock solutions were dissolved in water. [125I]Tyr10-
tivity compared with NPSR Asn107 when measuring second
NPS was kindly provided as a gift from PerkinElmer Life and Ana-
lytical Sciences (Waltham, MA). Other peptides were purchased from
messenger responses, although binding affinity for the nat-
ural agonist NPS is unchanged at both receptor isoforms
Bachem (Torrance, CA) or the American Peptide Co., Inc. (Sunny-
(Reinscheid et al., 2005). This polymorphism had been orig-
inally associated with an increased risk of asthma and other
vale, CA).
Synthesis and Structure Verification of SHA 66 and SHA
allergic diseases (Laitinen et al., 2004), but the role of NPSR 68. 1H NMR and 13C NMR spectra were recorded at ambient tem-
perature at 400 and 100 MHz, respectively, using a Bruker DRX 400
spectrometer (Bruker, Billerica, MA). All spectra were acquired in
CDCl3 with chemical shifts reported as ␦ values in parts per million
and are calibrated according to internal CDCl3 (7.26 ppm) solvent
residual peak. The data are reported as follows: multiplicity (br,
broad; s, singlet; d, doublet; t, triplet; dd, double doublet; dt, double
triplet; m, multiplet), integration, and coupling constants (Hertz). IR
spectra were obtained on a PerkinElmer model 1600 series Fourier-
transform infrared spectrophotometer and are reported in wave
numbers (centimetersϪ1). High-resolution mass spectra were ac-
quired on a Waters LCT Premier (ESI) spectrometer. Synthesis and
characterization of previously unreported compounds are detailed
below. Structures are numbered (in bold type) according to the syn-
thesis scheme as depicted in Fig. 1. LogP values for SHA 66 and
SHA 68 were calculated with a web-based calculator (http://intro.
bio.umb.edu/111–112/OLLM/111F98/newclogp.html).
in airway function is currently unclear (Allen et al., 2006).
We have recently found evidence for a gender-specific asso-
ciation of the NPSR N107I polymorphism with panic disorder
(Okamura et al., 2007), and a genome-wide screen for mark-
ers of sleep behavior and circadian phenotypes identified the
NPSR Ile107 polymorphism strongly associated with mean
bedtime in a random population cohort (Gottlieb et al., 2007).
These early data indicate that the endogenous NPS system
might be involved in modulating sleep or circadian behaviors
as well as emotional processing.
Characterization of physiological functions modulated by
NPS is still at an early stage, and availability of a selective
NPSR antagonist is critically important for such research. A
series of synthetic compounds with presumed activity at
NPSR were published recently in a patent application by
Takeda Pharmaceuticals Inc. (Osaka, Japan), but no phar-
macological or biological data were presented (Fukatsu et al.,
2005). The patent disclosed that structures containing a sub-
stituted bicyclic piperazine scaffold possess NPSR antagonis-
7-Benzyl-1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-
one (Fig. 1, Structure 6). To a solution of 4 (1.00 g, 3.74 mmol) in
tetrahydrofuran (THF; 12 ml) and N,N,NЈ,NЈ-tetramethylethylene
diamine (1.20 ml, 8.23 mmol) was added sec-butyllithium (6.20 ml,
8.23 mmol) drop-wise at Ϫ78°C. The mixture was allowed to warm to
tic activity. Two structurally similar compounds were quoted Ϫ30°C over 2 h, at which point the reaction was cooled back to
Fig. 1. Chemical synthesis of SHA 66
and SHA 68. Intermediate compounds
are numbered (in bold type) and de-
scribed under Materials and Methods.

Characterization of NPSR Antagonist
895
Ϫ78°C. A solution of benzophenone (1.40 g, 7.48 mmol) in THF (8 ml) 60.4, 46.5, 44.8, 43.4, 41.2; HRMS (ESI) calculated for
was added drop-wise. The flask was allowed to warm to room tem-
perature overnight, and the reaction was quenched by the addition of
C
26H25N3O3Na(MϩNa) 450.1794, found 450.1783.
SHA 68. To a mixture of 7 (2.00 g, 3.87 mmol) and 4-fluorobenzyl
saturated aqueous NH4Cl (10 ml), extracted with ethyl acetate (3 ϫ isocyanate (986 ␮l, 7.74 mmol) in THF (20 ml) was added 1,8-
30 ml), washed with H2O, dried (Na2SO4), filtered, and concentrated. diazabicyclo[5.4.0]undec-7-ene (636 ␮l, 4.25 mmol) drop-wise. The
The residue was purified by column chromatography (10–50%
reaction was stirred for 15 min at room temperature, quenched by
EtOAc/hexanes) to yield 6 (1.06 g, 76%) as a clear oil that foamed addition of saturated aqueous NH4Cl (10 ml), extracted with ethyl
under vacuum. IR (neat): 3027, 2814, 1769, 1449, 1249, 1032, 995, acetate (3 ϫ 40 ml), dried (Na2SO4), filtered, and concentrated. The
917, 755, 699 cmϪ1; 1H NMR (400 MHz, CDCl3): ␦ 7.52–7.55 (m, 2H), residue was purified by column chromatography (40% EtOAc/hex-
7.23–7.40 (m, 13H), 4.58 (br d, 1H, J ϭ 9.0 Hz), 3.82 (dd, 1H, J ϭ 2.5, anes then 10% MeOH/CHCl3) to give SHA 68 (1.72 g, 100%) as a
13.2 Hz), 3.52 (d, 1H, J ϭ 13.1 Hz), 3.33 (d, 1H, J ϭ 13.1 Hz), 3.12 (br clear oil that foamed under vacuum. IR (neat): 3356, 1748, 1633,
t, 1H, J ϭ 12.1 Hz), 2.71 (br d, 1H, J ϭ 8.5 Hz), 2.58 (dd, 1H, J ϭ 2.6, 1538, 1508, 1449, 1222, 1156, 1032, 981, 832, 753, 701; 1H NMR (400
11.4 Hz), 1.96 (br t, 1H, J ϭ 8.6 Hz), 1.61 (t, 1H, J ϭ 11.1 Hz); 13C MHz, CDCl3): ␦ 7.49 (br d, 2H, J ϭ 7.2 Hz), 7.22–7.40 (m, 10H), 6.98
NMR (100 MHz, CDCl3): ␦ 156.1, 142.8, 138.8, 129.1, 128.7, 128.6, (t, 2H, J ϭ 8.6 Hz), 5.07 (t, 1H, J ϭ 5.4 Hz), 4.28–4.42 (m, 3H), 4.03
128.5, 128.4, 128.0, 127.5, 126.1, 125.9, 85.4, 63.0, 61.5, 55.9, 50.9,
41.8; HRMS (ESI) calculated for C25H24N2O2Na (MϩNa) 407.1736, 1H, J ϭ 12.1 Hz), 3.04 (dt, 1H, J ϭ 3.6, 12.6 Hz), 2.86 (dt, 1H, J ϭ 3.5,
found 407.1732.
13.0 Hz), 2.14 (dd, 1H, J ϭ 11.4, 13.1 Hz); 13C NMR (100 MHz,
(dd, 1H, J ϭ 2.8, 13.3 Hz), 3.79 (dd, 1H, J ϭ 3.0, 13.0 Hz), 3.67 (br d,
3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7- CDCl3): ␦ 157.2, 156.1, 141.9, 138.4, 135.0, 129.5, 129.4, 128.9, 128.8,
carboxylic acid 9H-fluoren-9-ylmethyl Ester (Fig. 1, Structure 128.7, 128.4, 126.0, 125.9, 115.7, 115.5, 85.9, 60.6, 46.6, 44.5, 43.8,
7). To a solution of 6 (3.48 g, 9.05 mmol) in acetonitrile (45 ml) was 41.4; HRMS (ESI) calculated for C26H24FN3O3Na (MϩNa) 468.1700,
added FmocCl (2.58 g, 9.96 mmol) in one portion, and the mixture found 468.1700.
was brought to reflux at 90°C. After approximately 10 min, a white
precipitate formed in the reaction flask. The suspension was allowed
to stir at reflux for an additional 5 h, after which the mixture was
Functional Characterization in Vitro. Generation of stable
cell lines expressing human NPSR Asn107, NPSR Ile107, and mouse
NPSR has been described before (Xu et al., 2004; Reinscheid et al.,
cooled and vacuum filtered. The precipitate was washed with an 2005). Agonist-induced mobilization of intracellular Ca2ϩ was mea-
additional portion of cold acetonitrile (20 ml). The crude white solid sured using the fluorometric imaging plate reader technology as
7 (3.76 g, 80%) was then used without further purification. IR (neat): described previously (Reinscheid et al., 2005). Antagonist compounds
1762, 1702, 1449, 1426, 1229, 986, 757, 702 cmϪ1 1H NMR (400 were dissolved as 10 mM stock solutions in dimethyl sulfoxide
;
MHz, CDCl3) (spectrum acquired at room temperature): ␦ 7.74–7.80 (DMSO) and then diluted into assay buffer containing 0.1% bovine
(m, 2H), 7.25–7.55 (m, 15H), 7.14–7.16 (m, 1H), 4.50–4.88 (m, 2H), serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO) with final
4.21 (br s, 1.4H), 4.16 (br s, 0.6H), 3.98 (br s, 1H), 3.75 (br s, 1.6H), DMSO levels at Ͻ1% (v/v). Half-maximal inhibitory concentrations
3.45 (br s, 0.4H), 2.70–2.92 (m, 2H), 2.13 (t, 1H, J ϭ 12.4 Hz); HRMS (IC50) of antagonists were determined in triplicate by preincubating
(ESI) calculated for C33H28N2O4Na (MϩNa) 539.1947, found cells stably expressing NPSR for 10 min with the compounds before
539.1951.
challenging with various concentrations of NPS peptide. IC50 values
SHA 66. To a mixture of 7 (2.00 g, 3.87 mmol) and benzyl isocya- were calculated by nonlinear regression analysis using GraphPad
nate (962 ␮l, 7.74 mmol) in THF (20 ml) was added 1,8- Prism (GraphPad Software Inc., San Diego, CA). Dose ratios for
diazabicyclo[5.4.0]undec-7-ene (637 ␮l, 4.26 mmol) drop-wise. The agonist dose responses in the presence of different antagonist con-
reaction was stirred for 15 min at room temperature, quenched by centrations were determined for Schild analysis to obtain Kb values
addition of saturated aqueous NH4Cl (10 ml), extracted with ethyl for antagonist binding. IC50 values at functional agonist EC50 were
acetate (3 ϫ 40 ml), dried (Na2SO4), filtered, and concentrated. The obtained by first running an agonist dose response and then using
residue was purified by column chromatography (40% EtOAc/hex- the calculated agonist EC50 on the same batch of cells for antagonist
anes then 10% MeOH/CHCl3) to give SHA 66 (1.61 g, 98%) as a dose responses.
yellow oil that foamed under vacuum. IR (neat): 3354, 1755, 1633,
Radioligand binding in cells stably expressing NPSR Ile107 was
1538, 1450, 1263, 1082, 1030, 982, 752, 699; 1H NMR (400 MHz, performed as described before (Xu et al., 2004). SHA 68 was tested in
CDCl3): ␦ 7.46–7.48 (m, 2H), 7.15–7.40 (m, 13H), 5.90 (t, 1H, J ϭ 5.5 triplicate for displacement of [125I]Tyr10-NPS. Nonspecific binding
Hz), 4.25–4.41 (m, 3H), 4.05 (dd, 1H, J ϭ 2.7, 13.3 Hz), 3.77 (br d, 1H, was determined in the presence of 1 ␮M NPS. Ki values were calcu-
J ϭ 11.4 Hz), 3.64 (dd, 1H, J ϭ 2.7, 13.0 Hz), 2.92 (dt, 1H, J ϭ 3.5, lated with GraphPad Prism.
12.7 Hz), 2.67 (dt, 1H, J ϭ 3.4, 13.3 Hz), 2.06 (dd, 1H, J ϭ 11.4, 13.2
SHA 68 was tested for selectivity against a number of G protein-
Hz); 13C NMR (100 MHz, CDCl3): ␦ 157.4, 156.0, 141.7, 139.4, 138.2, coupled receptors (GPCRs), expressed stably, transiently, or endog-
128.7, 128.6, 128.5, 128.4, 128.2, 127.4, 127.2, 125.9, 125.7, 85.8, enously in various cell lines as listed in Table 1. Receptors were
TABLE 1
Selectivity profile of SHA 68
Receptor
Cell Line
Expression
Agonist
Vasopressin 1a
HEK 293
HEK 293
U 373 MG
CHO
Transient
Transient
Endogenous
Stable
Arg8-vasopressin
Oxytocin
Oxytocin
Tachykinin 1
␮-Opioid
␬-Opioid
Substance P
Dermorphin
Dynorphin A
Orphanin FQ/nociceptin
Somatostatin-28
MCH
Neuropeptide Y
Dopamine
Isoproterenol
Tyramine
CHO
Stable
Nociceptin/orphanin FQ
Somatostatin 2
Melanin-concentrating hormone 1
Neuropeptide Y 2
D2 dopamine
␤2-adrenergic
Trace amine 1
P2Y1
CHO
Stable
Transient
Stable
Transient
Transient
Endogenous
Stable
HEK 293
HEK 293
HEK 293
HEK 293
HEK 293
HEK 293
1321 N1
CHO
Stable
Stable
ADP
ADP
P2Y12
CHO, Chinese hamster ovary.

896
Okamura et al.
preincubated with 10 ␮M SHA 68 for 10 min and then activated with plasma or brain sample) using peak area ratio of analyte to internal
100 nM of their corresponding agonists (Table 1) to test for both standard. Finally, concentrations for analyte in plasma and brain
agonistic and antagonistic activity. Receptor activation was moni- samples were interpolated. Plasma and brain concentrations (nano-
tored as mobilization of intracellular Ca2ϩ using the fluorometric grams per milliliter and nanograms per gram, respectively) were
imaging plate reader technology. In cases of Gi-linked receptors (e.g., back-calculated from the interpolated concentrations and the dilu-
D2 dopamine receptor or opioid receptors), the corresponding cell tion factors.
lines were cotransfected with a chimeric G protein that allows for
coupling of Gi-linked receptors to the phospholipase C pathway, as
described before (Saito et al., 1999).
NPS-Induced Hyperlocomotion. Locomotion of mice was mon-
itored in an automated activity system equipped with IR sensors
for both horizontal and vertical activity measurements (Versamax;
AccuScan Instruments, Inc., Columbus, OH). Male C57BL/6 mice
were allowed to habituate to the recording chamber for 2 h. Vehicle
or SHA 68 (in PBS, 10% Cremophor EL) were injected i.p. 10 min
before central administration of either NPS (1 nmol in 2 ␮l of PBS,
0.1% BSA) or vehicle (PBS, 0.1% BSA). Recording of locomotor ac-
tivity began 5 min after the i.c.v. injections and continued for 90 min.
Horizontal activity represents IR beam breaks in the x and y dimen-
sions, whereas vertical activity was recorded by IR sensors located 10
cm above the chamber floor (z dimension). Stereotypic behavior is
defined as repetitive breaks of a single beam that is not followed by
a consecutive beam break of an adjacent sensor. The automated
system does not allow for differentiation of individual stereotypic
behaviors, such as grooming or sniffing.
Statistical Analysis. PK data for SHA 68 (plasma concentration
versus time) were obtained at 1 mg/kg i.v. and 2.5 mg/kg i.p. PK
curves were analyzed using a noncompartmental approach (noncom-
partmental analysis, model 200, extravascular input, for i.p. injec-
tion, and NCA model 201, i.v. bolus input, for i.v. dosing) with linear
trapezoidal interpolation and uniform weighting in WinNonlin ver-
sion 5.1.1 (Pharsight, Mountain View, CA).
In Vivo Studies. Male C57BL/6 mice (National Cancer Institute,
Bethesda, MD; or Charles River Laboratories, Wilmington, MA), 8 to
12 weeks old, were group-housed (four animals per cage) under
controlled conditions (temperature, 21 Ϯ 2°C; relative humidity,
50–60%; 12-h light/dark cycle; lights on 6:00 AM) with free access to
food and water. For i.c.v. drug injections, mice were briefly anesthe-
tized with halothane. NPS was dissolved in phosphate-buffered sa-
line (PBS), pH 7.4, containing 0.1% BSA and injected i.c.v. (total
volume, 2 ␮l) as described before (Xu et al., 2004). SHA 68 was
dissolved in PBS containing 10% Cremophor EL (Sigma-Aldrich),
and 100 ␮l was injected i.p.
For pharmacokinetic (PK) studies, mice were weighed and then
received i.p. or i.v. injections with the drug dissolved in a vehicle
suitable for injection (5% dimethylacetamide, 5% Cremophor EL,
and 90% PBS at 0.2 mg/ml for i.v. and 2.5% dimethylacetamide, 2.5%
Cremophor EL, and 95% PBS at 0.5 mg/ml for i.p.). A total of 24
animals were used for a total of six time points with four animals per
time point. Animals were euthanized, and blood was collected in
heparinized tubes. Plasma was isolated by centrifugation, and drug
levels were determined by liquid chromatography-coupled tandem
mass spectrometry (LC/MS/MS) according to the analytical method
described below. Likewise, 12 animals were used for blood-brain
barrier (BBB) penetration studies for a total of three time points
(0.25, 1, and 2 h) and four animals per time point. At the indicated
time intervals, animals were euthanized. Their blood was collected,
and plasma was isolated for plasma drug level determination by
LC/MS/MS. Brains were harvested, blotted on absorbent paper, and
homogenized in a 50:50 (v/v) mixture of saline and ethanol (3 ml/g
brain). Drug concentration in brain tissue was determined using the
same LC/MS/MS method. All experiments were carried out in accor-
dance with the Guidelines for the Care and Use of Mammals in
Neuroscience and Behavioral Research (National Research Council,
2003) and approved by the local Institutional Animal Care and Use
Committees.
Cumulative horizontal, vertical, or stereotypic activities for the
complete 90-min session were analyzed by one-way analysis of vari-
ance (ANOVA) followed by Bonferroni’s post hoc test, where appro-
priate. Selected data sets of cumulative activity data were also com-
pared by unpaired Student’s t test. The time course of horizontal,
vertical, or stereotypic activity over the 90-min session was divided
in 5-min intervals and analyzed by two-way ANOVA with time and
drug treatment as variables. Values of p Ͻ 0.05 were considered
significant.
Results
Synthesis and Structure Verification. The piperazine
derivatives SHA 66 and SHA 68 were both synthesized (Fig.
1) in five steps from cheap, commercially available piperazine
hexahydrate 1 in 54 and 55% overall yields, respectively.
Monobenzylation of 1 was accomplished in the presence of
one equivalent of piperazinium dihydrochloride monohydrate
2 (synthesized from 1) to yield 3 in 90% yield after recrystal-
lization (two crops) (Cymerman-Craig, 1959; Cymerman-
Craig and Young, 1962, 1973). Standard Boc protection of 3
gave the differentially protected 4 in quantitative yield
(Berkheij et al., 2005). Intermediate 4 was then treated with
sec-butyllithium/N,N,NЈ,NЈ-tetramethylethylene diamine, at
Ϫ78°C, followed by trapping of the intermediate anion with
Analytical Method. Plasma proteins were precipitated by mix-
ing plasma samples with a stock solution of reserpine in acetonitrile
(1 volume of plasma for 3 volumes of acetonitrile). After centrifuga-
tion, the supernatant was isolated and analyzed by LC/MS/MS.
Homogenized brain samples were treated in a similar fashion with 3
volumes of the acetonitrile solution containing internal standard
(reserpine). Precipitated material was removed by centrifugation,
and the supernatant was analyzed by LC/MS/MS. Standard curves
were prepared by spiking plasma or brain homogenate with a stock
solution of test compound dissolved in DMSO. The spiked samples
were treated as described above.
Samples were analyzed on a Finnigan TSQ Quantum Ultra
(Thermo Electron Corporation, Waltham, MA) mass spectrometer,
equipped with a Turbo-Ionspray interface, in positive-ion mode benzophenone. The formation of 6 occurred smoothly, in good
(source temperature, 400°C) and with an Agilent 1100 Series LC
system comprising autosampler, pump, and column oven (Agilent
Technologies, Santa Clara, CA). A Kromasil 100-5 C4 (30 ϫ 4.6
mm) column was used for the separation (3-min gradient using
0.1% formic acid in water as solvent A and 0.1% formic acid
in acetonitrile as solvent B). Transitions m/z 446.2/167.0 and
609.7/195.0 were monitored for SHA-68 and the internal standard
(reserpine), respectively.
yield (76%), with only a small amount of the dialkylated side
product (Ͻ10%) observed. After several unsuccessful at-
tempts to selectively remove the benzyl-protecting group of 6
via hydrogenolysis, an alternate strategy was employed. For-
tunately, amine debenzylation could be accomplished by ad-
dition of FmocCl to an acetonitrile solution of 6 at room
temperature. After approximately 10 min, 7 precipitated
from the solution and could be filtered and used without
further purification. The yield of the reaction could be in-
Chromatograms were automatically integrated using Finnigan
Xcalibur Software. A 1/x2-weighted least-squares linear regression
was applied to calibration standards (nanograms per milliliter in creased to 80% by heating to 90°C for 5 h, after which time,

Characterization of NPSR Antagonist
897
no further conversion was observed. Compounds SHA 66 and activation by the endogenous agonist NPS in HEK cells sta-
SHA 68 were then obtained in almost quantitative yield from bly expressing human NPSR Asn107 or NPSR Ile107, respec-
a mixture of 7 and the requisite isocyanate (8 or 9) by treat- tively. Schild analysis revealed that SHA 66 inhibited NPS-
ment with 1,8-diazabicyclo[5.4.0]undec-7-ene, to remove the induced Ca2ϩ mobilization with pA2 values of 7.662 at NPSR
Fmoc-protecting group, followed by addition of the secondary Asn107 and 7.486 at NPSR Ile107, respectively (Fig. 2E;
amine to the isocyanate. The structures of SHA 66 and SHA Table 2). The fluorinated analog SHA 68 displayed pA2 val-
68 were fully characterized by IR, 1H and 13C NMR, and ues of 7.771 at NPSR Asn107 and 7.554 at NPSR Ile107,
mass spectrometry. Both compounds were insoluble in water respectively. Because the slope of the Schild plot for both
or aqueous buffers, and calculated logP values of 4.21 for compounds at both receptor isoforms was close to 1.0 (Table
SHA 66 and 4.35 for SHA 68, respectively, supported this 2) and therefore indicated that they are competitive antago-
observation. Therefore, stock solutions of the compounds nists, Kb values were extrapolated from the linear regression
were prepared in 100% DMSO and diluted appropriately curves (Table 2). Schild analysis also indicated that both
thereafter.
compounds have approximately 1.5-fold higher affinity at
In Vitro Pharmacological Activity. As shown in Fig. 2, NPSR Asn107 than NPSR Ile107. At functional agonist EC50
A to D, SHA 66 and SHA 68 potently antagonized NPSR concentrations, SHA 66 displayed IC50 values of 26.1 and
Fig. 2. Pharmacological activity of SHA 66 (A and B) and SHA 68 (C and D) at NPSR Asn107 (NPSR-N107) and NPSR Ile107 (NPSR-I107). Both
compounds were tested in the presence of increasing concentrations of NPS to generate dose ratios for Schild analysis by measuring mobilization of
intracellular Ca2ϩ in HEK 293 cells stably expressing the different NPSR isoforms. Concentrations of antagonist compounds are given in the figure
legend of B. Data are shown as means Ϯ S.E.M. E, Schild analysis of dose ratios followed by linear regression analysis. F, displacement of radioligand
binding by SHA 68 in cells stably expressing NPSR Ile107. [125I]Tyr10-NPS (40 pmol) was used as a tracer, and incubations were done in triplicate.
Data are shown as means Ϯ S.E.M.

898
Okamura et al.
TABLE 2
Pharmacological characterization of SHA 66 and SHA 68
SHA 66
SHA 68
NPSR Asn107
NPSR Ile107
NPSR Asn107
NPSR Ile107
pA2 (95% CI)
7.662 (7.997–7.426)
21.7 nM
1.007 Ϯ 0.06363
26.1 Ϯ 8.1 nM
7.486 (7.886–7.225)
32.6 nM
1.041 Ϯ 0.07110
28.8 Ϯ 12.2 nM
7.771 (8.167–7.502)
16.9 nM
1.066 Ϯ 0.08553
22.0 Ϯ 4.4 nM
7.554 (7.823–7.354)
27.9 nM
0.9817 Ϯ 0.05494
23.8 Ϯ 9.4 nM
47.7 nM
Kb
Schild slope
IC50 (at functional agonist EC50
Ki (binding)
)
pA2, negative logarithm of antagonist concentration shifting the agonist EC50 by a factor of 2; Kb, dissociation constant of antagonist binding extrapolated from Schild
analysis; Schild slope, slope from linear regression curve of dose ratios; IC50, half maximal concentration of antagonist required to block agonist response at functional agonist
EC50 (EC50 in this experiment: NPSR Asn107 ϭ 15.6 nM; NPSR Ile107 ϭ 4.3 nM); Ki, equilibrium dissociation constant determined by radioligand binding; CI, confidence
interval.
28.8 nM at NPSR Asn107 and NPSR Ile107, respectively, steady state was calculated to be 2.5 l/kg, showing extensive
whereas SHA 68 yielded IC50 values of 22.0 and 23.8 nM at distribution of the compound outside the vasculature and the
the two NPSR isoforms, respectively (Table 2). Cells stably extracellular space.
expressing mouse NPSR showed similar inhibition of NPS-
BBB penetration of SHA 68 was tested by i.p. administra-
induced Ca2ϩ mobilization by SHA 66 and SHA 68 with IC50 tion of 5 and 50 mg/kg in male C57BL/6 mice. Animals (four
values of 60.8 Ϯ 13.8 and 48.7 Ϯ 14.7 nM against a single per group) were euthanized at 0.25, 1, and 2 h postdose.
agonist concentration of 12.5 nM NPS, respectively (data not Graphs of plasma and brain concentrations of SHA 68, in
shown). In radioligand binding experiments, SHA 68 com- nanograms per milliliter and nanograms per gram, respec-
peted for labeled NPS binding at NPSR Ile107 with a Ki value tively, as well as their ratio, are presented in Fig. 3B. Al-
of 47.7 nM (95% confidence interval, 39.01–58.31 nM), which though the brain/plasma ratio increases with progressing
is reasonably close to the calculated Kb of 27.9 nM for SHA 68 time at the 5 mg/kg dose, both brain and plasma concentra-
at NPSR Ile107 (Fig. 2F; Table 2). Nonspecific binding was tions of SHA 68 drop dramatically over the same period of
approximately 14% of total binding under these conditions.
time, as expected from PK data (decrease factor ϳ50 for
Selectivity Profile of SHA 68. Fourteen different GPCRs plasma and ϳ8 for brain concentration ratio between 0.25
were tested to establish a selectivity profile for SHA 68 (Ta- and 2 h). On the contrary, at the 50 mg/kg dose, plasma and
ble 1). Each receptor was expressed in a suitable cellular brain concentrations of SHA 68 are more stable over time
environment, endogenously, transiently, or as a stable clone, (decrease factor ϳ5 for plasma and ϳ3 for brain concentra-
and activated with its endogenous ligand at 100 nM. SHA 68 tion ratio between 0.25 and 2 h). This is probably due to a
was tested at a concentration of 10 ␮M. No agonistic or slowdown in elimination of the compound as the dose in-
antagonistic activity of SHA 68 was observed at any of the creases. Conversion to molar concentrations indicated that at
GPCRs tested, indicating that the compound appears to be a dose of 50 mg/kg i.p., pharmacologically relevant concen-
selective for NPSR. In particular, no activity of SHA 68 was trations of SHA 68 at or above its Kb value can be reached for
detected at vasopressin or oxytocin receptors, which are se- at least 1 h in both plasma and brain (plasma, 87.99 Ϯ 21.99
quentially closest to NPSR, albeit at only 25 to 29% amino ␮M at 15 min, 49.63 Ϯ 10.98 ␮M at 60 min; brain, 6.33 Ϯ 0.33
acid identity.
␮M at 15 min, 6.06 Ϯ 0.23 ␮M at 60 min; data are averages Ϯ
Pharmacokinetic Profile of SHA 68. SHA 66 and SHA S.E.M., n ϭ 5).
68 display very similar pharmacological profiles in vitro, but
In Vivo Activity of SHA 68. NPS is known to induce hy-
SHA 68 appears to have slightly higher potency at mouse perlocomotion in mice after central administration. To test
NPSR than SHA 66. Therefore, we selected SHA 68 for fur- whether this central effect of NPS can be antagonized by SHA
ther in vivo experiments in mice. Due to the lipophilic nature 68, we recorded motor activity in mice that had received injec-
of SHA 68 (calculated logP ϭ 4.35), we first analyzed the tions with vehicle or SHA 68 (5 and 50 mg/kg i.p.) and subse-
pharmacokinetic profile of the compound in mice. SHA 68 quently received central administrations of either vehicle or 1
was injected into male C57BL/6 mice at 1 and 2.5 mg/kg for nmol NPS i.c.v. As shown in Fig. 4A, injection of NPS ϩ vehicle
i.v. bolus and i.p. administration, respectively. Plasma sam- increased horizontal activity that lasted for at least 90 min.
ples were collected from four mice at six different time points, Injection of NPS ϩ SHA 68 (50 mg/kg) reduced horizontal
and concentration of drug in plasma was determined by a activity to approximately 50% of the activity recorded from mice
standard LC/MS/MS protocol. Dose-normalized PK curves receiving NPS ϩ vehicle injections, demonstrating that this
(plasma concentration, Cp, as a function of time) are pre- dose of SHA 68 was effective in at least partially blocking
sented in Fig. 3A. Noncompartmental analysis of the data NPS-induced horizontal activity. Statistical analysis revealed
(WinNonlin; Pharsight) was used to generate PK parameters significant main effects of drug treatment (F4,68 ϭ 211.52, p Ͻ
that are summarized in Table 3.
0.0001) and time (F17,68 ϭ 19.12; p Ͻ 0.0001) with interaction
Bioavailability of the compound administered i.p., calcu- (F ϭ 1.47; p ϭ 0.0087). Analysis of cumulative activity data
lated as the ratio of dose-normalized area under the curve revealed that SHA 68 alone (Veh ϩ SHA 68, 50 mg/kg) was able
between i.p. and i.v. dosing, is almost quantitative. Elimina- to reduce basal horizontal activity compared with mice receiv-
tion t1/2 is shorter for i.p. than for i.v. route of administration. ing vehicle injections (p ϭ 0.022, unpaired Student’s t test; Fig.
However, this observation is driven by the last time points of 4D). However, this effect is not statistically significant across
the PK curve, which are the most susceptible to experimental the five treatment groups of the experiment by one-way
errors. Mean residence times, on the other hand, are similar ANOVA. At the highest dose (50 mg/kg), SHA 68 was able to
between the two injection routes. Volume of distribution at block approximately 50% of 1 nmol NPS-induced motor activa-

Characterization of NPSR Antagonist
899
Fig. 3. Pharmacokinetic analysis of SHA 68 in mice. A, dose-normalized plasma concentrations [in (nanograms per milliliter)/(milligrams per
kilogram)] as a function of time (hours) for i.v. administration of SHA 68 at 1 mg/kg dose (open squares) and i.p. administration at 2.5 mg/kg (triangles)
dose. Plasma and brain concentrations of SHA 68 administered i.p. at 5 (B) and 50 mg/kg (C), 0.25, 1, and 2 h postdose. D, brain/plasma ratio (milliliter
per gram) calculated from B and C as a function of dose and time. Animal numbers were n ϭ 5 at all time points. Data are shown as means Ϯ S.E.M.
TABLE 3
0.0114), although this effect was not significant across the
five treatment groups (Fig. 4E). Furthermore, central NPS
administration was found to significantly increase stereo-
typic behavior (defined as repetitive movements recorded by
breaks of a single IR beam), as shown in Fig. 4C. Statistical
analysis by two-way ANOVA revealed significant main ef-
fects of drug treatment (F4,68 ϭ 135.65, p Ͻ 0.0001) and time
(F17,68 ϭ 12.75; p Ͻ 0.0001) without interaction. NPS in-
creased the cumulative duration of stereotypic behavior ap-
proximately 3-fold compared with Veh ϩ Veh-treated ani-
mals, and SHA 68 (50 mg/kg) blocked this effect by
approximately 50% (Fig. 4F).
Pharmacokinetic parameters of SHA 68
PK parameters obtained by noncompartmental analysis of PK curves (see text) for
i.v. administration of SHA 68 at 1 mg/kg and i.p. dosing at 2.5 mg/kg. Data were
calculated as described under Materials and Methods.
i.v.
i.p.
AUC_last (h/ng/ml)
AUC_inf (h/ng/ml)
CL (l/h/kg)
231
233
4.29
2.53
0.54
0.74
548
549
4.56
Vss (l/kg)
MRT (h)
0.52
0.43
t1/2 (h)
Bioavailability
94%
AUC_last, area under the curve between time zero and the last measured time
Together, these data demonstrate that peripheral admin-
istration of SHA 68 is able to, at least partially, antagonize
behavioral effects produced by central administration of
NPS. Thus, SHA 68 behaves as an NPSR antagonist in vivo.
point; AUC_inf, area under the curve extrapolated to infinity; CL, clearance; Vss
volume of distribution at steady state; MRT, mean residence time.
,
tion. In contrast, a 10-fold lower dose of SHA 68 (5 mg/kg) did
not produce significant effects in blocking NPS-induced hori-
zontal activity.
SHA 68 displayed similar effects on vertical activity, i.e.,
rearing and climbing, in mice. As shown in Fig. 4B, central
Discussion
Analysis of physiological functions of any neurotransmitter
NPS administration increased vertical activity, and this ef- system depends on the availability of selective, high-affinity
fect was at least partially blocked by coadministration of 50 agonists and antagonists. So far, the recently discovered NPS
mg/kg SHA 68, whereas the lower dose of the antagonist had system has only been studied by using the endogenous pep-
no effect. Statistical analysis by two-way ANOVA revealed tide agonist NPS. To our knowledge, the present study is
significant main effects of drug treatment (F4,68 ϭ 53.24, p Ͻ therefore the first report of compounds with antagonistic
0.0001) and time (F17,68 ϭ 1.98; p ϭ 0.0102) without inter- activity at the NPS receptor.
action. Analysis of cumulative vertical activity showed that
The data presented in this study demonstrate that SHA 66
SHA 68 also reduced basal vertical activity compared with and SHA 68 are potent antagonists at NPSR in vitro, and the
Veh ϩ Veh-treated animals (unpaired Student’s t test; p ϭ selectivity profile of SHA 68 indicates that this compound

900
Okamura et al.
Fig. 4. Effect of SHA 68 on motor activity in mice. Mice had been habituated for 2 h before injection. SHA 68 was dissolved in 10% Cremophor EL
(vehicle) and injected (i.p.) 10 min before NPS or vehicle (PBS, 0.1% BSA) were injected centrally (i.c.v.). Group sizes: Veh ϩ Veh, n ϭ 12; Veh ϩ SHA
68 (50 mg/kg), n ϭ 12; NPS ϩ Veh, n ϭ 18; NPS ϩ SHA 68 (50 mg/kg), n ϭ 15; NPS ϩ SHA 68 (5 mg/kg), n ϭ 22. Time course of horizontal activity
(A), vertical activity (B), and stereotypic behavior (C) over 90 min. Cumulative horizontal activity (D), cumulative vertical activity (E), and cumulative
stereotypy time (F) during the 90-min observation period. ءءء, p Ͻ 0.001, Veh ϩ Veh versus NPS ϩ Veh; #, p Ͻ 0.05; ###, p Ͻ 0.001 NPS ϩ Veh versus
NPS ϩ SHA 68 (50 mg/kg), one-way ANOVA with Bonferroni post hoc test.
might be selective for NPSR, although the panel of 14 GPCRs tional role of NPSR in these pathologies remains unclear
used in the screening is certainly not exhaustive. The two (Laitinen et al., 2004; Feng et al., 2006; D’Amato et al., 2007;
closely related bicyclic piperazines SHA 66 and SHA 68 dis- Malerba et al., 2007).
play almost identical affinities, indicating that the fluorina-
The present study also provides evidence that SHA 68 is
tion does not affect receptor binding. The pharmacological able to enter the brain and block NPS-induced behavioral
profiles of the two compounds do however reveal a slightly responses in vivo. However, the pharmacokinetic profile of
higher affinity for the NPSR Asn107 variant over NPSR SHA 68 indicates that the compound has only limited BBB
Ile107. These observations indicate that the amino acid in penetration. This might explain our observation that even at
position 107 of NPSR might interfere with binding of the two a relatively high dose of 50 mg/kg, SHA 68 was able to block
antagonist compounds. This is of particular interest because only approximately 50% of the motor-activating effect pro-
we have previously shown that the polymorphism at position duced by NPS. It is obvious that compounds with improved
107 of NPSR (Asn versus Ile) does not affect agonist binding pharmacokinetic profiles are needed to extend these studies,
affinity but has in turn profound effects on agonist efficacy by and the chemical scaffold of SHA 66 and SHA 68 may be
shifting the EC50 5 to 10-fold (Reinscheid et al., 2005). There- useful as a lead structure to design new compounds with
fore, the Ile107 side chain might stabilize a receptor confor- improved potency and pharmacokinetic properties. It should
mation that is promoting agonist activation, whereas the also be mentioned that the automated behavioral observation
Asn107 side chain might facilitate antagonist binding. Using system used in this study does not allow for detection of more
the bicyclic piperazine scaffold, it should therefore be possi- complex behaviors that are often associated with arousal,
ble to develop compounds that display enhanced selectivity such as increased grooming or sniffing. These behaviors and
toward one of the two isoforms of the human NPS receptor. other small repetitive movements are detected and summa-
Such compounds could not only be useful in understanding rized as stereotypic activity. Differentiation of locomotion
the structural role of this critical amino acid within the into horizontal, vertical, and stereotypic activity has been
receptor protein but might lead to individualized drugs that chosen to illustrate the arousing properties of NPS and the
target specific genotypes of NPSR. We have recently shown attenuation of these effects by SHA 68 coadministration.
that the NPSR Asn107 isoform is underrepresented in male
In summary, we present evidence that two closely related
panic disorder patients (Okamura et al., 2007); thus, specif- bicyclic piperazines are potent and selective antagonists at
ically targeting NPSR Ile107 with selective NPSR antago- NPSR in vitro and are able to antagonize NPS-induced ef-
nists might be a viable concept for developing novel thera- fects in vivo. These molecules should therefore be useful in
peutics to treat panic disorder. In addition, specific NPSR the characterization of physiological functions of the NPS
genotypes were repeatedly found to be associated with system and might lead to the development of novel therapeu-
asthma or other immunological disorders, although the func- tic compounds.

Characterization of NPSR Antagonist
901
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Acknowledgments
We thank S. D. Clark and D. M. Duangdao for helpful discussion
and O. Civelli for generous access to equipment resources.
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Address correspondence to: Dr. Rainer K. Reinscheid, Department of Phar-
maceutical Sciences, University of California Irvine, 360 Med Surge II, Irvine,
CA 92697-4625. E-mail: rreinsch@uci.edu