Synthesis and separation of the enantiomers of the neuropeptide S receptor antagonist (9 R/S)-3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4- a ]pyrazine-7-carboxylic Acid 4-fluoro-benzylamide (SHA 68)

Article abstract of DOI:10.1021/jm200138r

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of the neuropeptide S receptor (NPSR) antagonist (9R/S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine- 7-carboxylic acid 4-fluoro-be

Full text of DOI:10.1021/jm200138r

ARTICLE
pubs.acs.org/jmc
Synthesis and Separation of the Enantiomers of the Neuropeptide
S Receptor Antagonist (9R/S)-3-Oxo-1,1-diphenyl-tetrahydro-
oxazolo[3,4-a]pyrazine-7-carboxylic Acid 4-Fluoro-benzylamide
(SHA 68)
Claudio Trapella,†,# Michela Pela,†,# Luisa Del Zoppo,† Girolamo Calo,‡ Valeria Camarda,‡ Chiara Ruzza,‡
||
Alberto Cavazzini,§ Valentina Costa,§ Valerio Bertolasi,§, Rainer K. Reinscheid,^ Severo Salvadori,† and
Remo Guerrini*,†
†Department of Pharmaceutical Sciences and Biotechnology Center and ‡Department of Experimental and Clinical Medicine, Section of
Pharmacology and Neuroscience Center, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 19, 44100
Ferrara, Italy
§Department of Chemistry and Centre for Structural Diffractometry, University of Ferrara, via L. Borsari 46, 44100 Ferrara, Italy
^Department of Pharmaceutical Sciences, University of California Irvine, 2214 Natural Sciences I, Irvine, California 92697, United States
S Supporting Information
b
ABSTRACT: This study reports the synthesis, chromatographic
separation, and pharmacological evaluation of the two enantio-
mers of the neuropeptide S receptor (NPSR) antagonist (9R/S)-
3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carbox-
ylic acid 4-fluoro-benzylamide (SHA 68). The (9R)-3-oxo-1,
1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid
4-fluoro-benzylamide (compound 10) and (9S)-3-oxo-1,1-di-
phenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid
4-fluoro-benzylamide (compound 10a) were synthesized and
their purity assessed by chiral chromatography. The absolute
configuration of the enantiomer 10 has been assigned from the crystal structure of the corresponding (S)-phenyl ethyl amine
derivative 8. Calcium mobilization studies performed on cells expressing the recombinant NPSR demonstrated that compound 10 is
the active enantiomer while the contribution of 10a to the NPSR antagonist properties of the racemic mixture is negligible.
’ INTRODUCTION
molecules able to interact with the NPSR was reported in the
patent literature by Takeda researchers.7 Among the different
moleculesdescribed in the patent, the compound (9R/S)-3-oxo-1,
1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic
acid 4-fluoro-benzylamide (SHA 68; compound 1) has been
pharmacologically characterized. In vitro, compound 1 behaves
as a selective, potent (pA2 = 8), and competitive antagonist at
human8 and murine9 NPSR. In vivo, compound 1 has been
reported to prevent the arousal promoting and anxiolytic-like
effects elicited by NPS in mice and rats.8,9 In addition, compound
1 reversed the protective effect of NPS on the NMDA receptor
antagonist MK-801-induced neurotoxicity in rats.10 Finally,
recent findings indicate that in the rat intracerebroventricular
injection of NPS increased conditioned reinstatement of cocaine
seeking, whereas peripheral administration of compound 1
reduced it.11 These pharmacological investigations were per-
formed using the racemic compound 1. Molecular modeling
Neuropeptide S (NPS) is the last neuropeptide identified via
the reverse pharmacology approach.1 NPS selectively binds and
activates a previously orphan GPCR receptor now referred to as
NPSR.1 NPSR is widely distributed in the brain, while the
expression of the NPS peptide precursor is limited to few discrete
brain areas.1,2 The supraspinal administration of NPS in rodents
produces a rather unique pattern of actions: stimulation of
wakefulness associated with anxiolytic-like effects.1 In addition,
NPS has been reported to inhibit food intake, facilitate memory,
elicit antinociceptive effects, and recent evidence suggests an
involvement of the NPS/NPSR system in drug addiction (see for
a review3).
Potent and NPSR selective antagonists are now required
for understanding the biological functions controlled by the
NPS/NPSR system. As far as peptide antagonists are concerned,
these molecules were recently discovered by replacing Gly5 in the
natural peptide sequence with a D-amino acid. Examples of such
compounds are [D-Cys(tBu)5]NPS,4 [D-Val5]NPS,5 and more
recently [tBu-D-Gly5]NPS.6 The first example of nonpeptide
Received: December 7, 2010
Published: April 05, 2011
r
2011 American Chemical Society
2738
dx.doi.org/10.1021/jm200138r J. Med. Chem. 2011, 54, 2738–2744
|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthesis of Compounds 10 and 10aa
a Reagents and conditions: (a) CH2Cl2, WSC, Boc-Gly-OH, room temp, 12 h; (b) LiAlH4, THF, 0 ꢀC, 1 h; (c) chloroacetyl-chloride, EtOAc, NaHCO3,
0 ꢀC to room temp, 24 h; (d) THF/DMF 1/1, NaH, 0 ꢀC to room temp, 24 h; (e) THF, LiAlH4, room temp, 4 h; (f) THF, benzophenone, sec-BuLi,
TMEDA, ꢀ78 ꢀC to ꢀ30 ꢀC to room temp, 24 h; (g) CH3CN, Fmoc-Cl, reflux, 12 h; (h) THF, DBU, p-fluoro-benzylisocyanate, room temp, 12 h.
studies investigated nonpeptide ligand binding to NPSR.12 In the
frame of these studies, docking analyses were performed and a
defined NPSR binding pocked was proposed; of note, only the
(S) enantiomer of compound 1 was used in such simulations.
The importance of ligand chirality for NPSR interaction has been
recently supported by the identification of two novel classes of
nonpeptide NPSR antagonists: the quinoline13 and the tricyclic
imidazole14 based compounds. In both cases, a single bioactive
enantiomer was obtained by chiral chromatography separation
from the corresponding racemic mixture.
procedure reported by Okamura et al.8 The purity grade and
enantiomeric excess of 10 and 10a were determined by chiral
HPLC analysis. The top panel of Figure 1 shows the chromato-
gram for the single enantiomer 10a (first eluted component), and
the middle panel that of 10 (second eluted species) and the
bottom panel of the figure displays the chromatogram for the
racemate. As is evident from this figure, there is no trace of 10 in
the chromatogram corresponding to the elution of 10a, nor of
10a in that of 10. To define the absolute configuration of the C9
chiral center, we explored different crystallization conditions for
compounds 8, 8a, 9, 9a, and for the final products. Only with
compound 8 were we able to obtain crystals suitable for further
X-ray investigation. In particular, compound 8 was crystallized
from ethanol/ethyl acetate, and its X-ray analysis demonstrated
the absolute configuration R at the chiral center C9. The absolute
C9 configuration of compound 8 has been assigned by reference
to the unchanged chiral center C10 in configuration S (Figure 2).
On the basis of the absolute configuration of 8, we were able to
assign the absolute C9 configuration to compounds 8a, 9, 9a, and
to the final products 10 and 10a.
In the present study, we report the synthesis, chiral HPLC
analysis, X-ray crystallographic assignment, and in vitro pharma-
cological evaluation of the two compound 1 enantiomers.
’ RESULTS AND DISCUSSION
The reference compound 1 was synthesized following the
procedures reported by Okamura et al.8 In Scheme 1 is described
the synthetic approach adopted for the synthesis of 10 and 10a
starting from (S)-phenyl ethyl amine. As reported in lit-
erature,15,16 the use of phenyl ethyl amine was expected to
induce the stereochemistry of C9 of the tetrahydro-oxazolo-
[3,4-a]pyrazine nucleus. Unfortunately, we obtained only a slight
chiral induction corresponding approximately to a 60/40% ratio
determined by NMR spectroscopy. Similar results were obtained
using (R)-phenyl ethyl amine as chiral auxiliary. Nevertheless,
diastereomers 8 and 8a were successfully separated in good yield
by flash chromatography. The removal of the chiral auxiliary to
obtain 9 and 9a and the acylation of N7 with p-fluoro-benzyli-
socyanate to obtain final compounds were achieved using the
In parallel, we performed a series of NMR experiments. In
Figure 3, the enlarged [1H]NMR spectra of the C9 proton region
of the 10a and 10 isomers are depicted. The coupling constant
analysis between Hx and Ha/Hb C8 protons are very similar
(11.3/3.7 Hz for 10a, Figure 3A and 11.2/3.7 Hz for 10,
Figure 3B) in both compounds. This result, together with
X-ray data of 8 (see Figure 2), confirms the axial position of
C9 proton in both enantiomers.
Next, we evaluated and compared the in vitro NPSR antago-
nist properties of compounds 1, 10, and . The three samples were
2739
dx.doi.org/10.1021/jm200138r |J. Med. Chem. 2011, 54, 2738–2744

Journal of Medicinal Chemistry
ARTICLE
Figure 2. ORTEP view of compound 8. The thermal ellipsoids are
drawn at 30% probability level.
challenged against the stimulatory effect of 10 nM NPS in
inhibition response curves (Figure 4). Compounds 1, 10, and
10a did not stimulate per se calcium mobilization up to 10 μM.
Compound 1 inhibited in a concentration dependent manner
the stimulatory effect of NPS showing similar high values of
potency (pKB = 8). These values of potency are superimpos-
able to those previously published.8,9 Compound 10 was also able
to antagonize in a concentration dependent manner the stimulatory
effect of NPS displaying values of potency similar or slightly
higher than the racemic mixture. By contrast, compound 10a
showed a slight inhibitory effect only at micromolar concentra-
tions. The values of potency of the three compounds in the three
cells lines are summarized in Table 1. Collectively, these results
demonstrated that compound 10 is the active enantiomer while
the contribution of compound 10a to the biological activity of the
racemic mixture is negligible. This information can be extremely
useful for the refinement of the recently proposed molecular
models of NPSR and its binding pocket.12 As already mentioned
in the Introduction, the relevance of ligand chirality for NPSR
binding is also corroborated by the fact that the biological activity
of chemically different molecules, such as the quinoline13 and the
tricyclic imidazole14 compounds, could be attributed to a single
bioactive enantiomer.
Figure 1. Chromatograms of compound 10a (top panel) and 10
(middle panel) in comparison with the racemate (bottom panel).
’ CONCLUSION
In conclusion, the present study described the synthesis and
separation of the two compound 1 enantiomers. The synthetic
scheme we used can be easily scaled up to multigrams. Com-
pound 10 was demonstrated to be the bioactive enantiomer.
Nowadays, this molecule represents the standard nonpeptide
NPSR antagonist that has been, and surely will be, used to
investigate the biological functions controlled by the NPS/NPSR
system and to evaluate the therapeutic potential of innovative
drugs acting as NPSR selective ligands.
tested in calcium mobilization studies performed on HEK293
cells expressing the murine NPSR or the two isoforms of the
human receptor (hNPSRAsn107 and hNPSRIle107).17
The natural peptide NPS was able to induce calcium mobi-
lization in a concentration dependent manner in HEK 293
mNPSR (pEC50 8.97 ( 0.11; Emax 250 ( 11%), hNPSRAsn107
(pEC50: 9.07 ( 0.11; Emax 316 ( 13%), and hNPSRIle107
(pEC50: 9.17 ( 0.15; Emax 333 ( 17%). The three samples were
2740
dx.doi.org/10.1021/jm200138r |J. Med. Chem. 2011, 54, 2738–2744

Journal of Medicinal Chemistry
ARTICLE
Figure 3. Enlarged [1H]NMR spectra of the C9 proton region of the 10a (A) and 10 (B) isomers.
’ EXPERIMENTAL SECTION
anhydrous THF, a solution of 3 (3.11 g, 11.19 mmol) was added
dropwise. The reaction was monitored by TLC (EtOAc/light petro-
leum 3:1), and after 24 h, the excess of hydride was quenched with water
and the salts were filtered trough a Celite pad. The solvent was
evaporated in vacuo to yield 4 (2.66 g, 10.07 mmol) in 90% yield. 1H
NMR (400 MHz, CDCl3): δ 7.32ꢀ7.27 (m, 5H, Ar); 4.96 (bs, 1H, NH-
Boc), 3.77ꢀ3.73 (q, 1H, CH3-CH-Ar, J = 6.6 Hz), 3.16ꢀ3.13 (m,
2H, NH-CH2-CH2), 2.59ꢀ2.51 (m, 2H, NH-CH2-CH2), 1.75 (bs,
Materials. HPLC grade solvent were purchased from Sigma Aldrich
(Steinheim, Germany). The purity of the tested compounds 1, 10, and
10a has been assessed by RP-HPLC. All compounds showed >95%
purity. One-dimensional and two-dimensional NMR spectra were
recorded on a VARIAN 400 MHz instrument. Chemical shifts are given
in ppm (δ) relative to TMS, and coupling constants are in Hz. MS
analyses were performed on a ESI-Micromass ZMD 2000. Optical
rotation data were recorded on a Perkin-Elmer polarimeter 241. Flash
chromatography was carried out on a silica gel (Merck, 230ꢀ400 Mesh).
Silica gel (Polygram SIL G/UV254) was used for thin layer chroma-
tography.
t
1H, ꢀNH), 1.42 (s, 9H, Buꢀ), 1.36ꢀ1.33 (d, 3H, CH3-CH-Ar,
20
J = 6.6 Hz). MS (ESI): [M þ H]þ = 265; [R]D = ꢀ29ꢀ (c =
0.11 g/100 mL, chloroform).
{2-[(2-Chloro-acetyl)-(1-phenyl-ethyl)-amino]-ethyl}-carbamic Acid
tert-Butyl Ester (5). To a stirred solution of 4 (1.94 g, 7.34 mmol) in
EtOAc (50 mL) at 0 ꢀC, saturated solution of NaHCO3 (5 mL) was
added. After 10 min, chloroacetyl chloride (1.17 mL, 14.68 mmol) was
added dropwise. The reaction was monitored by TLC (EtOAc/light
petroleum 3:1), and after 24 h at room temperature, NaHCO3 (2 mL)
was added to the organic phase. The organic layer was separated, and the
aqueous phase was extracted twice with EtOAc (50 mL). The combined
organic phases were concentrate to dryness to obtain 5 in quantitative
yield. 1H NMR (400 MHz, CDCl3): δ 7.22ꢀ6.98 (m, 5H, Ar),
5.59ꢀ5.43 (q, 1H, CH3-CH-Ar, J = 8 Hz), 4.85 (bs, 1H, NH-Boc),
4.10ꢀ3.97 (m, 2H, NH-CH2-CH2), 3.76 (s, 2H, CdO-CH2-Cl),
3.18ꢀ3.15 (m, 2H, NH-CH2-CH2), 1.40ꢀ1.25 (d, 3H, CH3-CH-Ar,
J = 8 Hz), 1.06 (s, 9H, tBu). 13C NMR (100 MHz, CDCl3):
δ 170.12,155.85, 139.47, 128.45, 128.20, 128.03, 80.69, 59.44, 42.86,
41.14, 38.24, 27.48, 19.98. MS (ESI): [M þ H]þ = 341.
Typical Procedures for the Synthesis of 10 and 10a.
[(1-Phenyl-ethylcarbamoyl)-methyl]-carbamic Acid tert-Butyl Ester
(3). To a stirred solution of Boc-Gly-OH (5 g, 28.5 mmol) in CH2Cl2
(50 mL), WSC (3.64 g, 19 mmol) and (S)-phenylethyl amine (3.45 g,
28.5 mmol) were added. After 24 h at room temperature, the reaction
was monitored by TLC (EtOAc/light petroleum 2:1). The organic layer
was washed with 10% citric acid (20 mL), 5% NaHCO3 (20 mL), and
brine (20 mL). The organic phase was dried, concentrated in vacuo, and
purified by flash chromatography (EtOAc/light petroleum 2:1) to
1
obtain 3 in 60% yield. H NMR (400 MHz, CDCl3): δ 7.31ꢀ7.24
(m, 5H, Ar), 6.62 (bs, 1H, NH-CO), 5.31 (bs, 1H, NH-Boc), 5.11
(m, 1H, CH-CH3), 3.75 (m, 2H, CH2-NH-Boc), 1.47 (d, 3H, CH3-CH-
t
Ar, J = 6.8 Hz), 1.42 (s, 9H, Buꢀ). 13C NMR (100 MHz, CDCl3):
δ 168.6, 156.2, 143.0, 128.7, 127.4, 126.1, 80.2, 48.7, 44.6, 28.3, 21.9.
20
MS (ESI): [M þ H]þ =279; [R]D = ꢀ41 (c = 0.121 g/100 mL,
3-Oxo-4-(1-phenyl-ethyl)-piperazine-1-carboxylic Acid tert-Butyl
ester (6). To a stirred suspension of 60% NaH (1.14 g, 28.61 mmol)
in a mixture of THF/DMF 1/1 (20 mL) at 0 ꢀC, a solution of 5 (3.25 g,
9.54 mmol) in THF/DMF 1/1 (10 mL) was added. After 24 h, the
chloroform).
[2-(1-Phenyl-ethylamino)-ethyl]-carbamic Acid tert-Butyl Ester (4).
To a stirred suspension of LiAlH4 (0.85 g, 22.38 mmol) at 0 ꢀC in
2741
dx.doi.org/10.1021/jm200138r |J. Med. Chem. 2011, 54, 2738–2744

Journal of Medicinal Chemistry
ARTICLE
δ 7.36ꢀ7.28 (m, 5H, Ar), 6.08 (q, 1H, CH3-CH-Ar, J = 8 Hz),
4.23ꢀ4.18 (d, 1H, NꢀCHeHa-CdO, J = 20 Hz), 4.10 (d, 1H,
N-CHeHa-CdO, J = 20 Hz), 3.62 (bs, 1H, CH2 piperazine), 3.27
(bs, 1H, CH2 piperazine), 3.19 (bs, 1H, CH2 piperazine), 2.83 (bs, 1H,
CH2 piperazine), 1.53ꢀ1.51 (d, 3H, CH3-CH-Ar, J = 8 Hz), 1.45 (s, 9H,
tBuꢀ). 13C NMR (100 MHz, CDCl3): δ 165.44, 153.78, 139.41, 128.63,
127.68, 127.36, 80.69, 50.08, 47.98, 40.24, 28.32, 15.34. MS (ESI):
[M þ H]þ = 305; [R]D20 = ꢀ116.0ꢀ (c = 0.318 g/100 mL, chloroform).
4-(1-Phenyl-ethyl)-piperazine-carboxylic Acid tert-Butyl Ester (7).
To a stirred suspension of LiAlH4 (453 mg, 18.9 mmol) in anhydrous
THF (20 mL) at room temperature, a solution of 6 (1.15 g, 3.78 mmol)
in THF (10 mL) was added. After 30 min, the reaction was completed as
showed by TLC analysis (EtOAc/light petroleum 1:2). The reaction was
quenched by adding 15% NaOH (1 mL) and Et2O (20 mL). The
resulting precipitate was filtered through a Celite pad, and the solvent
was concentrate to dryness. The crude product was purified by flash
chromatography (eluent: EtOAc/light petroleum 1:2) to give 7 (920 mg,
3.17 mmol) in 84% yield. 1H NMR (400 MHz, CDCl3): δ 7.31ꢀ7.25
(m, 5H, Ar), 3.41ꢀ3.35 (m, 5H, CH2-N-Boc, CH-CH3), 2.41ꢀ2.33
t
(m, 4H, CH2-N), 1.43 (s, 9H, Buꢀ), 1.36 (d, 3H, CH3-CH-Ar).
13C NMR (100 MHz, CDCl3): δ 146.80, 134.13, 128.38, 127.74,
127.10, 85.27, 50.36, 29.78, 28.49, 27.48. MS (ESI): [M þ H]þ
=
291; [R]D20 = ꢀ32ꢀ (c = 0.0104 g/100 mL, chloroform).
1,1-Diphenyl-7-(1-phenyl-ethyl)-hexahydro-oxazolo[3,4-a]pyrazin-3-
one (8 and 8a). To a stirred solution of 7 (380 mg, 1.31 mmol) in
anhydrous THF (5 mL), TMEDA (0.53 mL, 3.54 mmol) was added.
The reaction was cooled at ꢀ78 ꢀC, and sec-BuLi 1.4 M in hexane
(2.53 mL, 3.54 mmol) was added. The reaction was heated at ꢀ35 ꢀC,
and after 2 h, a solution of benzophenone (480 mg, 2.62 mmol) in
anhydrous THF (7 mL) was added dropwise. The reaction became
green and was stirred at room temperature for 24 h. After this time, the
reaction was monitored by TLC (EtOAc/light petroleum 1:2) and
quenched by adding NH4Cl saturated solution (20 mL). The solvent
was removed in vacuo and the aqueous phase extracted three times with
EtOAc (30 mL). The combined organic layer was dried and evaporated
to dryness. The crude diastereomers mixture was purified by flash
chromatography using EtOAc/light petroleum 1:2 as eluent to obtain
the fast running diastereomer 8a in 40% yield and the low running
distereomer 8 in 45% yield.
8a: 1H NMR (400 MHz, CDCl3): δ 7.55ꢀ7.49 (m, 2H), 7.41ꢀ7.21
(m, 11H), 7.19ꢀ7.14 (m, 2H), 4.51 (dd, 1H, J = 10.9, 3.6 Hz), 3.74
(ddd, 1H, J = 13.2, 3.5, 1.3 Hz), 3.34 (q, 1H, J = 6.7 Hz), 3.04 (ddd, 1H,
J = 13.0, 12.1, 3.6 Hz), 2.70ꢀ2.61 (m, 2H), 1.86 (td, 1H, J = 11.9, 3.6
Hz), 1.50ꢀ1.41 (m, 1H), 1.22 (d, 3H, J = 6.7 Hz). 13C NMR (100 MHz,
CDCl3): δ 156.17, 142.85, 142.52, 138.91, 128.69, 128.58, 128.50,
128.35, 128.01, 127.51, 127.37, 126.19, 125.95, 85.50, 64.52, 61.56,
52.66, 49.30, 42.07, 19.34. MS ESI [M þ Hþ] = 399; [R]D20= þ216
(c = 0.108 g/100 mL, chloroform).
Figure 4. Inhibition response curves to compounds 1 (SHA 68), 10,
and 10a in HEK293 cells expressing the murine NPSR and the human
NPSR isoforms. Data are mean ( SEM of four separate experiments
made in duplicate.
Table 1. Potencies (pKB) of Compounds 1 (SHA 68), 10, and
10a in HEK293 Cells Expressing the Murine NPSR and the
Human NPSR Isoforms
8: 1H NMR (400 MHz, CDCl3): δ 7.50ꢀ7.45 (m, 2H), 7.39ꢀ7.20
(m, 11H), 7.18ꢀ7.14 (m, 2H), 4.44 (dd, 1H, J = 3.56, 10.93 Hz),
3.86ꢀ3.79 (m, 1H), 3.48 (q, 1H, J = 6.8 Hz), 3.11 (ddd, 1H, J = 13.0,
12.0, 3.81 Hz), 2.80ꢀ2.73 (m, 1H), 2.44 (ddd, 1H, J = 11.5, 3.5, 1.6 Hz),
2.07ꢀ1.97 (m, 1H), 1.50 (m, 1H), 1.27 (d, 3H, J = 6.8 Hz). 13C NMR
(100 MHz, CDCl3): δ 156.20, 142.48, 142.28, 138.81, 128.65, 128.51,
128.32, 127.98, 127.59, 127.28, 126.11, 125.92, 125.84, 85.39, 63.86,
61.67, 53.24, 47.58, 42.11, 17.16; MS ESI [M þ Hþ] = 399; [R]D20 =ꢀ132ꢀ
(c = 0.11 g/100 mL, chloroform).
mNPSR
hNPSR Ile107
hNPSR Asn107
compd
pKB
pKB
pKB
1
8.16 (7.79ꢀ8.53)
8.29 (7.93ꢀ8.65)
<6
8.03 (7.77ꢀ8.37)
8.18 (7.90ꢀ8.46)
<6
7.99 (7.73ꢀ8.25)
8.28 (7.72ꢀ8.84)
<6
10
10a
3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic
Acid 9H-fluoren-9-ylmethyl Ester (9 and 9a). To a stirred solution of 8
or 8a (200 mg, 0.52 mmol) in acetonitrile (10 mL) at reflux, Fmoc-Cl
(148 mg, 0.57 mmol) dissolved in acetonitrile (7 mL) was added. The
reaction, monitored by TLC (EtOAc/light petroleum 1:2), was com-
pleted in 12 h. The desired precipitate was filtered off to obtain 9 or 9a in
about 67% yield and pure enough to be used in the next reaction.
reaction was quenched by adding NH4Cl saturated solution (15 mL),
and the solvent was removed in vacuo. The residue was dissolved in
EtOAc (50 mL) and washed twice with water (20 mL). The organic
layer was dried, evaporated under reduced pressure, and the crude
product purified by flash chromatography (eluent: EtOAc/light petro-
1
leum 1:1) to obtain 6 in 45% yield. H NMR (400 MHz, CDCl3):
2742
dx.doi.org/10.1021/jm200138r |J. Med. Chem. 2011, 54, 2738–2744

Journal of Medicinal Chemistry
ARTICLE
3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic
Acid 4-fluoro-benzylamide (10 and 10a). To a stirred solution of 9
(59 mg, 0.11 mmol) in anhydrous THF (15 mL), p-fluoro-benzyliso-
cyanate (34.4 mg, 0.228 mmol) and DBU (19.2 mg, 0.126 mmol) were
added. The reaction was monitored by TLC (EtOAc/light petroleum
1:2) and by mass spectrometry. After 24 h, the reaction was treated as for
8 and 8a. The organic phase was dried and evaporate to dryness to give
10 in 76% yield after column chromatography using EtOAc/light
hygromycin B (100 mg/L). HEK293hNPSRAsn107 cells were maintain-
ed in DMEM medium supplemented with 10% fetal bovine serum,
2 mM glutamine, and zeocin (100 mg/L). Cells were cultured at 37 ꢀC in
5% CO2 humidified air. Cells were seeded at a density of 50000 cells/
well into poly-D-lysine coated 96-well black, clear-bottom plates. The
following day, the cells were incubated with medium supplemented with
2.5 mM probenecid, 3 μM of the calcium sensitive fluorescent dye Fluo-
4 AM, and 0.01% pluronic acid for 30 min at 37 ꢀC. After that time, the
loading solution was aspirated and 100 μL/well of assay buffer (Hank’s
Balanced Salt Solution; HBSS) supplemented with 20 mM 4-(2-hydro-
xyethyl)-1-piperazineethanesulfonic acid (HEPES), 2.5 mM probene-
cid, and 500 μM Brilliant Black (Aldrich) was added. Concentrated
solutions (1 mM) of NPS were made in bidistilled water and kept at ꢀ20 ꢀC.
Compounds 1, 10, and 10a were dissolved DMSO at a final concentra-
tion of 10 mM, and stock solutions were kept at ꢀ20 ꢀC until use. The
successive dilutions were carried out in HBSS/HEPES (20 mM) buffer
(containing 0.02% bovine serum albumin fraction V). After placing both
plates (cell culture and master plate) into the fluorometric imaging plate
reader FlexStation II (Molecular Devices, Sunnyvale, CA), fluorescence
changes were measured. Online additions were carried out in a volume
of 50 μL/well. To facilitate drug diffusion into the wells in antagonist
type experiments, the present studies were performed at 37 ꢀC and three
cycles of mixing (25 μL from each well moved up and down 3 times)
were performed immediately after antagonist injection to the wells.
Inhibition response curves were determined against the stimulatory
effect of 10 nM NPS. Compounds 1, 10, and 10a were injected into the
wells 24 min before adding NPS.
1
petroleum 1/1 as eluent. H NMR (400 MHz, CDCl3): δ 7.51ꢀ7.47
(m, 2H), 7.41ꢀ7.18 (m, 10H), 7.03ꢀ6.94 (m, 2H), 4.95 (t, 1H, J = 5.5
Hz), 4.45ꢀ4.27 (m, 3H), 4.03 (ddd, 1H, J = 13.5, 3.5, 1.2 Hz), 3.81
(dd, 1H, J = 13.1, 2.7 Hz), 3.69ꢀ3.60 (m, 1H), 3.05 (td, 1H, J = 12.7, 3.7
Hz), 2.93ꢀ2.82 (m, 1H), 2.14 (dd, 1H, J = 13.3, 11.3 Hz). 13C NMR
(100 MHz, CDCl3): δ 157.20, 156.11, 141.81, 138.30, 134.91, 129.48,
129.41, 129.17, 129.09, 128.82, 128.72, 128.37, 125.99, 125.85, 115.70,
115.48, 85.90, 60.55, 46.58, 44.47, 43.76, 41.37. MS ESI [M þ Hþ] =
445.9; [R]D20 = þ92 (c = 0.1 g/100 mL, MeOH).
Compound 10a was obtained in the same manner, starting from 9a.
Analytical data: yield 83%. 1H NMR (400 MHz, CDCl3): δ 7.51ꢀ7.47
(m, 2H), 7.41ꢀ7.18 (m, 10H), 7.03ꢀ6.94 (m, 2H), 4.95 (t, 1H, J = 5.5
Hz), 4.45ꢀ4.27 (m, 3H), 4.03 (ddd, 1H, J = 13.3, 3.6, 1.3 Hz), 3.81
(dd, 1H, J = 13.1, 2.7 Hz), 3.69ꢀ3.60 (m, 1H), 3.05 (td, 1H, J = 12.7, 3.7
Hz), 2.93ꢀ2.82 (m, 1H), 2.14 (dd, 1H, J = 13.3, 11.3 Hz). 13C NMR
(100 MHz, CDCl3): δ 157.20, 156.11, 141.81, 138.30, 134.91, 129.48,
129.41, 129.17, 129.09, 128.82, 128.72, 128.37, 125.99, 125.85, 115.70,
115.48, 85.90, 60.55, 46.58, 44.47, 43.76, 41.37. MS ESI [M þ Hþ] =
445.9; [R]D20 = ꢀ91 (c = 0.12 g/100 mL, MeOH).
Data Analysis and Terminology. The pharmacological termi-
nology adopted in this paper is consistent with IUPHAR recommenda-
tions. Data were expressed as mean ( SEM of at least four independent
experiments made in duplicate. Maximum change in fluorescence,
expressed in percent of baseline fluorescence, was used to determine
agonist response. Nonlinear regression analysis using GraphPad Prism
software (version 4.0) allowed logistic iterative fitting of the resultant
responses and the calculation of agonist potencies and maximal effects.
Agonist potencies are given as pEC50 (the negative logarithm to base 10
of the molar concentration of an agonist that produces 50% of the
maximal possible effect). Compounds 1, 10, and compound 10a
antagonist properties were evaluated in inhibition response curve
experiments; the antagonist potency, expressed as pKB, was derived
from the following equation:
Chiral Chromatography Analysis. A micro HPLC (Agilent
1100 micro series, Agilent Technologies) equipped with a micro diode
array detector was employed. A 150 mm ꢁ 2 mm stainless steel column
packed with Lux Cellulose-1 (cellulose tris 3,5-dimethylphenylcarba-
mate from Phenomenex) was used for all the measurements. The
average size of the packing material was 3 μm. The mobile phase was
a binary mixture of hexane/isopropyl alcohol (80/20 v/v). Flow rate was
200 μL/min. Injection volume was 3 μL. Analyte solutions were filtered
with PFTE filters (0.45 μm, Supelco, Bellefonte, PA, USA) before
injection. All chromatograms were recorded at 230 nm. The retention
times for the first (10a) and second (10) eluted enantiomers were 6.5
and 7.9 min, respectively.
Crystal Structure Determination of Compound 8. The
crystal data of compound 8 were collected at room temperature using
a Nonius Kappa CCD diffractometer with graphite monochromated Mo
KR radiation. The data sets were corrected for Lorentz and polarization
effects. The structure was solved by direct methods18 and refined using
full-matrix least-squares with all non-hydrogen atoms anisotropically
and hydrogens included on calculated positions riding on their carrier
atoms. All calculations were performed using SHELXL-9719 and
PARST20 implemented in WINGX21 system of programs.
n 1=n
KB ¼ IC50=ð½2 þ ð½Aꢂ=EC50Þ ꢂ ꢀ 1Þ
where IC50 is the concentration of antagonist that produces 50%
inhibition of the agonist response, [A] is the concentration of agonist,
EC50 is the concentration of agonist producing a 50% maximal response,
and n is the Hill coefficient of the concentration response curve to the
agonist.
Crystal Data: C26H26N2O2, orthorhombic, space group P212121,
a = 11.2339(2), b = 11.6808(3), c = 16.4783(5) Å, V = 2162.30(9)
Å3, Z = 4, Dc = 1.224 g cmꢀ3, intensity data collected with θ e 26ꢀ, 4215
independent reflections measured, 3460 observed reflections [I >
2σ(I)], final R index = 0.0365 (observed reflections), Rw = 0.0904 (all
reflections), S = 1.048. The absolute configuration has not been
established by anomalous dispersion effects in diffraction measurements
on the crystal. The enantiomer has been assigned by reference to an
unchanging chiral center in the synthetic procedure. ORTEP22 view of
compound 8 is shown in Figure 2.
’ ASSOCIATED CONTENT
S
Supporting Information. Monodimensional and bidi-
b
mensional NMR spectra of compounds 8, 8a, and final products
and crystal data of compound 8 (CIF). This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
CCDC deposition number: 810351.
Corresponding Author
*Phone: þ39-0532-455-988. Fax: þ39-0532-455953. E-mail: r.
Calcium Mobilization Experiments. HEK293 cells stably expres-
sing the murine NPSR or the human receptor isoforms NPSRIle107 and
NPSRAsn107 were generated as previously described.17 HEK293mNPSR
and HEK293hNPSRIle107 cells were maintained in DMEM medium
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and
guerrini@unife.it.
Author Contributions
#These authors contributed equally to this work.
2743
dx.doi.org/10.1021/jm200138r |J. Med. Chem. 2011, 54, 2738–2744

Journal of Medicinal Chemistry
ARTICLE
’ ACKNOWLEDGMENT
(10) Okamura, N.; Reinscheid, R. K.; Ohgake, S.; Iyo, M.; Hashimoto,
K. Neuropeptide S attenuates neuropathological, neurochemical and
behavioral changes induced by the NMDA receptor antagonist MK-801.
Neuropharmacology 2010, 58, 166–172.
(11) Kallupi, M.; Cannella, N.; Economidou, D.; Ubaldi, M.; Ruggeri,
B.; Weiss, F.; Massi, M.; Marugan, J.; Heilig, M.; Bonnavion, P.; de Lecea, L.;
Ciccocioppo, R. Neuropeptide S facilitates cue-induced relapse to cocaine
seeking through activation of the hypothalamic hypocretin system. Proc.
Natl. Acad. Sci. U.S.A 2010, 107, 19567–19572.
(12) Dal Ben, D.; Antonini, I.; Buccioni, M.; Lambertucci, C.;
Marucci, G.; Vittori, S.; Volpini, R.; Cristalli, G. Molecular modeling
studies on the human neuropeptide S receptor and its antagonists.
ChemMedChem 2010, 5, 371–383.
(13) Melamed, J. Y.; Zartman, A. E.; Kett, N. R.; Gotter, A. L.;
Uebele, V. N.; Reiss, D. R.; Condra, C. L.; Fandozzi, C.; Lubbers, L. S.;
Rowe, B. A.; McGaughey, G. B.; Henault, M.; Stocco, R.; Renger, J. J.;
Hartman, G. D.; Bilodeau, M. T.; Trotter, B. W. Synthesis and evaluation
of a new series of Neuropeptide S receptor antagonists. Bioorg. Med.
Chem. Lett. 2010, 20, 4700–4703.
(14) Trotter, B. W.; Nanda, K. K.; Manley, P. J.; Uebele, V. N.;
Condra, C. L.; Gotter, A. L.; Menzel, K.; Henault, M.; Stocco, R.;
Renger, J. J.; Hartman, G. D.; Bilodeau, M. T. Tricyclic imidazole
antagonists of the Neuropeptide S Receptor. Bioorg. Med. Chem. Lett.
2010, 20, 4704–4708.
(15) Juaristi, E.; Leon-Romo, J. L.; Reyes, A.; Escalante, J. Recent
applications of alpha-phenylethylamine (alpha-PEA) in the preparation
of enantiopure compounds. Part 3: alpha-PEA as chiral auxiliary. Part 4:
alpha-PEA as chiral reagent in the stereodifferentiation of prochiral
substrates. Tetrahedron: Asymmetry 1999, 10, 2441–2495.
(16) Guizzetti, S.; Benaglia, M.; Rossi, S. Highly stereoselective
metal-free catalytic reduction of imines: an easy entry to enantiomeri-
cally pure amines and natural and unnatural alpha-amino esters. Org.
Lett. 2009, 11, 2928–2931.
(17) Reinscheid, R. K.; Xu, Y. L.; Okamura, N.; Zeng, J.; Chung, S.;
Pai, R.; Wang, Z.; Civelli, O. Pharmacological Characterization of
Human and Murine Neuropeptide S Receptor Variants. J. Pharmacol.
Exp. Ther. 2005, 315, 1338–1345.
(18) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.;
Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G.; Polidori, G.; Spagna, R.
SIR97: a new tool for crystal structure determination and refinement.
J. Appl. Crystallogr. 1999, 32, 115–119.
We are grateful to Dr. Alberto Casolari and Dr. Elisa Durini for
the NMR analysis and Professor Vinicio Zanirato for the helpful
discussion about NMR spectra of compound 8. This work was
supported by funds from the University of Ferrara (FAR grants to
G.C. and S.S.), the Italian Ministry of the University (PRIN grant
to G.C. and S.S. and CHEM-PROFARMA-NET grant to A.C.),
the Compagnia di S. Paolo Foundation (NPSNP grant to G.C.),
and the National Institute of Mental Health (MH-71313 grant to
R.K.R.).
’ ABBREVIATIONS USED
DBU, 1,8-Diazabicyclo[5.4.0]undec-7-ene; DMEM, Dulbecco’s
Modified Eagle’s Medium; DMF, N,N-dimethylformamide;
Fmoc-Cl, 9-fluorenylmethyl chloroformate; HBSS, Hank’s
Balanced Salt Solution; HEK, human embryonic kidney;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
MS-ESI, electron spray ionization mass spectrometry; NMDA,
N-methyl-D-aspartic acid; PFTE, polytetrafluoroethylene; RP-
HPLC, reversed-phase high-performance liquid chromatogra-
phy; THF, tetrahydrofuran; TMEDA, tetramethylethylenedia-
mine; WSC, 1-ethyl-3-(30-dimethylaminopropyl)carbodiimide
’ REFERENCES
(1) Xu, Y. L.; Reinscheid, R. K.; Huitron-Resendiz, S.; Clark, S. D.;
Wang, Z.; Lin, S. H.; Brucher, F. A.; Zeng, J.; Ly, N. K.; Henriksen, S. J.;
de Lecea, L.; Civelli, O. Neuropeptide S: a neuropeptide promoting
arousal and anxiolytic-like effects. Neuron 2004, 43, 487–497.
(2) Xu, Y. L.; Gall, C. M.; Jackson, V. R.; Civelli, O.; Reinscheid, R. K.
Distribution of neuropeptide S receptor mRNA and neurochemical
characteristics of neuropeptide S-expressing neurons in the rat brain.
J. Comp. Neurol. 2007, 500, 84–102.
(3) Guerrini, R.; Salvadori, S.; Rizzi, A.; Regoli, D.; Calo, G.
Neurobiology, pharmacology, and medicinal chemistry of neuropeptide
S and its receptor. Med. Res. Rev. 2010, 30, 751–777.
(4) Camarda, V.; Rizzi, A.; Ruzza, C.; Zucchini, S.; Marzola, G.;
Marzola, E.; Guerrini, R.; Salvadori, S.; Reinscheid, R. K.; Regoli, D.;
Calo, G. In vitro and in vivo pharmacological characterization of the
neuropeptide s receptor antagonist [D-Cys(tBu)5]neuropeptide S.
J. Pharmacol. Exp. Ther. 2009, 328, 549–555.
(19) Sheldrich, G. M. Program for the crystal structure refinement.
University of Gottingen: Gottingen, Germany, 1997; http://shelx.uni-
ac.gwdg.de/SHELX/.
(20) Nardelli, M. PARST95—an update to PARST: a system of
Fortran routines for calculating molecular structure parameters from the
results of crystal structure analyses. J. Appl. Crystallogr. 1995, 28, 659.
(21) Farrugia, L. J. WinGX suite for small-molecule single crystal
crystallography. J. Appl. Crystallogr. 1999, 32, 837–838.
(22) Farrugia, L. J. ORTEP-3 for Windows—a version of ORTEP-
III with a Graphical User Interface (GUI). J. Appl. Crystallogr. 1997,
30, 565.
(5) Guerrini, R.; Camarda, V.; Trapella, C.; Calo, G.; Rizzi, A.;
Ruzza, C.; Fiorini, S.; Marzola, E.; Reinscheid, R. K.; Regoli, D.;
Salvadori, S. Synthesis and biological activity of human neuropeptide
S analogues modified in position 5: identification of potent and pure
neuropeptide S receptor antagonists. J. Med. Chem. 2009, 52, 524–529.
(6) Guerrini, R.; Camarda, V.; Trapella, C.; Calo, G.; Rizzi, A.;
Ruzza, C.; Fiorini, S.; Marzola, E.; Reinscheid, R. K.; Regoli, D.;
Salvadori, S. Further studies at neuropeptide s position 5: discovery of
novel neuropeptide S receptor antagonists. J. Med. Chem. 2009,
52, 4068–4071.
(7) Fukatsu, K.; Nakayama, Y.; Tarui, N.; Mori, M.; Matsumoto, H.;
Kurasawa, O.; Banno, H. Bicyclic piperazine compound and use thereof.
PCT Int. Appl. WO2005021555, 2005.
(8) Okamura, N.; Habay, S. A.; Zeng, J.; Chamberlin, A. R.;
Reinscheid, R. K. Synthesis and pharmacological in vitro and in vivo
profile of 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-car-
boxylic acid 4-fluoro-benzylamide (SHA 68), a selective antagonist of
the neuropeptide S receptor. J. Pharmacol. Exp. Ther. 2008, 325, 893–
901.
(9) Ruzza, C.; Rizzi, A.; Trapella, C.; Pela, M.; Camarda, V.; Ruggieri,
V.; Filaferro, M.; Cifani, C.; Reinscheid, R. K.; Vitale, G.; Ciccocioppo,
R.; Salvadori, S.; Guerrini, R.; Calo, G. Further studies on the pharma-
cological profile of the neuropeptide S receptor antagonist SHA 68.
Peptides 2010, 31, 915–925.
2744
dx.doi.org/10.1021/jm200138r |J. Med. Chem. 2011, 54, 2738–2744