Identifying structural features on 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones critical for Neuropeptide S antagonist activity

Article abstract of DOI:10.1016/j.bmcl.2008.05.098

A series of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones were synthesized and tested for Neuropeptide S (NPS) antagonist activity. A concise synthetic route was developed, which features a DMAP catalyzed carbamate formation. 4-Fluorob

Full text of DOI:10.1016/j.bmcl.2008.05.098

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4064–4067
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identifying structural features on 1,1-diphenyl-hexahydro-
oxazolo[3,4-a]pyrazin-3-ones critical for Neuropeptide S antagonist activity
*
Yanan Zhang, Brian P. Gilmour, Hernán A. Navarro, Scott P. Runyon
Organic and Medicinal Chemistry, Research Triangle Institute, PO Box 12194, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones were synthesized and
tested for Neuropeptide S (NPS) antagonist activity. A concise synthetic route was developed, which fea-
tures a DMAP catalyzed carbamate formation. 4-Fluorobenzyl urea (1c) and benzyl urea (1d) were iden-
tified as the most potent antagonists among the compounds examined. Structure-activity relationships
(SARs) demonstrate that a 7-position urea functionality is required for potent antagonist activity and
alkylation of the urea nitrogen (1e) or replacement with carbon or oxygen (5a) results in reduced
potency. In addition, compounds with alpha-methyl substitution (1b) or elongated alkyl chains (1h
and 1j) had reduced potency, indicating a limited tolerance for 7-position substituents.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 23 April 2008
Revised 22 May 2008
Accepted 27 May 2008
Available online 12 June 2008
Keywords:
Neuropeptide S
Antagonist
NPSR
The coupling of putative transmitters with orphan receptors has
led to the identification of several interesting ligand-receptor pair-
ings that are now beginning to show novel pharmacology.1 In par-
ticular, the neuropeptide S receptor (NPSR) system deorphanized
by Sato and co-workers2 has recently been shown to modulate a
variety of physiological states such as sleep,3 feeding,4,5 anxi-
ety,3,6,7 drug abuse,8 and inflammation.9
The unique pharmacological properties of NPS were first de-
scribed by Xu and colleagues who determined that NPS was in-
volved in both arousal and anxiety.3 Administration of NPS (i.c.v.)
increased locomotor activity in both habituated and na mice.
NPS-treated mice also demonstrated anxiolytic-like behaviors in
the elevated plus maze, light dark box, and marble burying para-
digm. In more recent studies, Rizzi and co-workers confirmed the
arousal and anxiolytic promoting properties of NPS using stress-in-
duced hypothermia, which is a behavioral model insensitive to
alterations in locomotor activity.6
Recognizing that small molecule probes would greatly aid the
pharmacological characterization of NPS receptors, our laboratory
undertook a program to identify small molecule antagonists. The
development of small molecule antagonists based on a computa-
tionally generated structure of the endogenous peptide was not
considered reliable due to the significant conformational flexibility
inherent in the peptide.11 Therefore, our laboratory chose to em-
ploy a previously patented scaffold as a lead structure.13 Although
no functional or receptor-binding data were disclosed in the pat-
ent, structural features important for functional activity appeared
to center around 7-position derivatives of 1,1-diphenyl-hexahy-
dro-oxazolo[3,4-a]pyrazin-3-ones. Therefore, our laboratory syn-
thesized and evaluated a diverse series of 7-substituted analogs
(Tables 1 and 2). During the course of preparing this manuscript,
two small molecules described in the Takeda patent were also syn-
thesized and tested by Okamura et al.14 These compounds (SHA-
66, SHA-68) were shown to be potent and selective antagonists
of both the Asn107 and Ile107 variants of the NPS receptor (Fig. 1).
Target compounds (1b–1s), designed to identify structural fea-
tures important for NPS antagonist activity, were prepared using
The NPS receptor is activated by an endogenous 20 amino acid
peptide (Fig. 1) resulting in elevated intracellular calcium via its
cognate proteins Gq and Gs.3 Amino acids important for NPS ago-
nist activity have been identified through Ala scanning mutagene-
sis. In particular, Phe 2, Arg 3, Asn 4, and Val 6 are critical for
agonist activity, whereas residues 5–13 are hypothesized to form
an
a
-helical recognition sequence.10 NMR and circular dichroism
studies on NPS have indicated a significant degree of flexibility
among the amino acids critical for receptor activation, thus making
identification of the bioactive NPS conformer difficult using cur-
rently available data.10,11
Several single nucleotide-polymorphisms (SNP) of the NPS
receptor have been shown to exist. In particular, NPSR Asn107Ile
and NPSR C-alt have been fully characterized for agonist sensitiv-
ity.12 Radioligand binding of [125I]Tyr10-NPS is unaltered among
receptor variants, however, a 5- to 10-fold enhancement in func-
tional sensitivity using calcium flux is observed for the Ile107 vari-
ant.12 Importantly, the SNP (Asn107 to Ile107) in the NPS receptor has
been implicated as a risk factor for asthma and has been shown to
modulate the functional properties of NPS.9
* Corresponding author. Tel.: +1 919 316 3842; fax: +1 919 541 8868.
E-mail address: srunyon@rti.org (S.P. Runyon).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.098

Y. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4064–4067
4065
O
O
N
N
N
H
SFRNGVGTGMKKTSFQRAKS
NPS SEQUENCE
O
R = H, SHA-66
R = F, SHA-68
R
Figure 1. Neuropeptide S and previously described NPS antagonists.
Table 1
Table 2
NPS antagonist activity for urea derivatives
NPS antagonist activity for alternate 7-substitution
O
O
O
N
N
N
R
N
R
O
O
Compounda
R
NPS Ile107 Ke NPS Asn107 Ke Asn/Ile
Compound
R
NPS Ile107 Ke
NPS Asn107 Ke
Asn/Ile
O
O
N
1b
214 42
13.7 2.8
9.9 1.6
588 150
2.8
3.8
4.8
0.7
5a
354 51
353 22
1.0
CH3
O
F
F
N
H
1cb
1dc
1e
52
2
1m
339 76
460 130
1.4
F
N
H
47 15
O
O
O
1n
1o
244 42
911 130
1110 30
267 41
1.1
5.1
CH3
317
5
229 75
F
F
N
H
4640 1400
1100 300
F
NH2
1f
321 120
567 190
326 140
690 390
1.0
1.2
N
H
O
1p
1q
1r
1.0
—
O
1g
ACH3
>4
l
M
>4 lM
N
H
S
O
N
H
3760 470
2560 70
0.7
1h
1i
309 52
417 71
1.3
0.8
N
N
N
H
S
537 190
437 150
N
H
1s
280 41
354 96
1.3
N
H
O
O
aAll target compounds were fully characterized using NMR and mass spectrometry.
1j
726 107
1260 250
1.7
—
N
H
OEt
with two equivalents of hydrochloric acid at 60 °C afforded 3 in
nearly quantitative yield. Reprotection of the resulting amino
groups as carbamates seemed ideal, since the desired cyclic carba-
mate 5 could be readily prepared upon subsequent heating of 4
with base. Based on this hypothesis, carboxybenzyl (Cbz) and 2-
(trimethylsilyl)ethoxycarbonyl (Teoc) groups were investigated.
Compounds 4a and 4b were obtained as planned and cyclization
with sodium hydride in dimethyl formamide proceeded smoothly
to provide mono-protected derivatives 5a and 5b. Unfortunately,
removal of either protecting group proved problematic. Under nor-
mal deprotection conditions both cyclic carbamates 5a and 5b
either collapsed to give 3 or decomposed. In an effort to circum-
vent these issues an alternate protecting group was explored. Knöl-
ker and colleagues reported that 1,2-aminoalcohols, when treated
with di-tert-butyl dicarbonate (Boc2O) and dimethylaminopyridine
(DMAP), readily cyclize to provide oxazolidin-2-ones.15 Using this
rationale, diamine 3 was treated with Boc2O, triethylamine, and
DMAP in tetrahydrofuran to afford compound 5c in excellent yield.
1k
>4
l
M
>4 lM
N
H
OH
N
1l
109 23
490 173
4.5
N
H
a
All target compounds were fully characterized using NMR and Mass
Spectrometry.
b
c
SHA-68.
SHA-66.
the methods described in Schemes 1 and 2. The key intermediate
1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-one (1a) was
synthesized following a concise route as depicted in Schemes 1.
Treatment of commercially available dibenzyl ester 2 with excess
phenyllithium at ꢀ78 °C afforded (1,4-dibenzyl-piperazin-2-yl)-di-
phenyl-methanol in moderate yield. Subsequent debenzylation
using palladium on barium sulfate under a hydrogen atmosphere

4066
Y. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4064–4067
chlorochromate in dichloromethane, or formaldehyde using so-
dium triacetoxyborohydride in dichloroethane.17 Target com-
pounds were fully characterized by NMR and mass spectroscopy.
Considering the potential pharmacological significance of small
molecule NPS receptor antagonists, we have defined key structural
features of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-
a]pyrazin-3-ones required for antagonist activity. In keeping with
earlier reports,12,14 NPS was more potent at the Ile107 variant
(EC50 of 2.5 nM) than at the Asn107 variant (EC50 16.7 nM) using a
calcium flux functional assay.18 The number of spare receptors in
an expression system can affect potency, however, taken together,
these data support there being a functional difference between the
two variants. Compound 1c and a closely related 1,2,3,6-tetrahy-
dro-pyridine analog of 1l were resolved into their individual enan-
tiomers in the Takeda Patent although no indication of biological
activity was provided (Table 1).13 Okamura and colleagues recently
reported Kb’s of 27.9 nM (Ile107) and 16.9 nM (Asn107) for racemic
1c and 32.6 nM (Ile107) and 21.7 nM (Asn107) for the desfluoro ana-
log 1d, respectively.14 In our hands, racemic 1c (Ke = 13.7 and
51 nM) and 1d (Ke = 9.9 and 47 nM) were the most potent NPS
antagonists among the compounds described herein at both the
Ile107 and Asn107 variants, respectively. In contrast to Okamura,
we found these compounds to be slightly more selective for the
Ile107 variant. Methylation of urea 1c (1b, Ke = 214 and 588 nM) re-
duced potency by 16-fold at Ile107 and 12-fold at Asn107. Similarly,
replacement of the urea nitrogen with carbon (1m, Ke = 339 and
460 nM) or oxygen (5a, Ke = 354 and 353 nM) significantly reduced
potency at both receptor variants. This suggests a hydrogen on the
7-position urea is critical for antagonist activity. In order to deter-
mine if a hydrogen bond donor placed in close proximity to the
urea hydrogen could serve as a suitable surrogate, phenylalanine
derivative 1o was prepared. A 100-fold reduction in potency was
observed for 1o (Ke = 911 and 4640 nM). Because the urea func-
tionality appeared critical for NPS antagonist activity, we began
to evaluate alternate substitution of the 7-position urea. Addition
of an alpha methyl group (1e, Ke = 317 and 229 nM) reduced po-
tency by 32- and 48-fold compared to 1d. Lengthening the alkyl
spacer between the terminal phenyl ring to two carbons (1f,
Ke = 321 nM) reduced potency by 25-fold compared to 1c. Further
elongation of the alkyl spacer to three (1h,Ke = 309 nM) and four
carbons (1i,Ke = 537 nM) significantly reduced potency compared
to the parent 1d. These results suggest that the hypothetical recep-
tor pocket has specific size requirements. In order to assess if
hydrophilic or hydrophobic character of the urea substituent was
Ph
O
OH
Ph
R
N
OH
Ph
H
N
N
N
OMe
c
a,b
Ph
Ph
N
H
N
R
2
3
4a,
R = Cbz
Ph
e
4b, R = Teoc
O
O
O
O
Ph
Ph
Ph
Ph
N
N
d
f
N
N
R
H
5a,
5b,
5c, R = Boc
1a
R = Cbz
R = Teoc
Scheme 1. Synthesis of key intermediate 1a. Reagents and conditions: Synthesis of
amine 1a: (a) PhLi, THF, ꢀ78 °C; (b) H2, 40 psi, Pd/BaSO4, EtOH, 2 equiv, HCl, 60 °C;
(c) 4a, CbzCl, K2CO3, H2O/THF, 4b, Teoc-Succinimide, Et3N, CH3CN; (d) NaH, DMF;
(e) Boc2O, DMAP, Et3N, THF; (f) TFA,CH2Cl2.
Key intermediate 1a was then obtained in quantitative yield by
treatment of 5c with trifluoroacetic acid (TFA) in dichloromethane.
This approach now allows for the preparation of key intermediate
1a from diamine 3 in two high yielding steps.
The four-step procedure described in Schemes 1 provides key
intermediate 1a in good yield. Although the yield of step a in
Schemes 1 occurs in moderate yield (ꢁ50%), steps b,e, and f are
accomplished in almost quantitative yield. Compared to the proce-
dure adopted by both Takeda Pharmaceuticals13 and Okamura
et al.,14 orthogonal protection of the two amino groups was not
needed in our approach. In addition, the DMAP mediated cycliza-
tion of 3 to 5c makes this route an attractive alternative.
Target ureas 1b–l and thioureas 1r and 1s were prepared by
reacting 1a with various commercially available isocyanates or iso-
thiocyanates (Schemes 2). For 1l, where the isocyanate was not
available, reaction of 1a with triphosgene and N-(2-amino-
ethyl)piperidine proceeded well to give 1l in moderate yield.
Alternatively, 1a was coupled to a number of acids with benzotri-
azole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluoro-
phosphate (BOP) in tetrahydrofuran to provide amides 1m–o.
The N-alkyl amines 1p and 1q were prepared by reductive amina-
tion of 1a with 4-(4-fluorophenyl)-butanal, which was obtained
through oxidation of 4-(4-fluorophenyl)butanol16 with pyridinium
O
O
O
O
O
O
c (for 1m,n)
c, d (for 1o)
Ph
Ph
Ph
Ph
Ph
Ph
f
N
N
N
N
N
H
N
N
1a
O
N
H
O
R
e
a
1m–o
1l
O
O
O
O
O
O
Ph
Ph
Ph
Ph
Ph
Ph
N
N
b
N
N
N
N
R
R
O
N
X
N
H
CH3
F
1p,q
1c–k
1r,s
1b
X = O
X = S
Scheme 2. Synthesis of target compounds 1b–1s. Reagents: (a) Appropriate isocyanate or isothiocyanate, CH2Cl2; (b) Mel, NaHMDS, THF; (c) Appropriate acid, BOP, Et3H,
THF; (d) TFA, CH2Cl2; (e) Appropriate aldehyde, NaB(OAc)3H, dichloroethane; (f) N-(2-aminoethyl)piperidine, triphosgene, Et3N, CH2Cl2.

Y. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4064–4067
4067
4. Smith, K. L.; Patterson, M.; Dhillo, W. S.; Patel, S. R.; Semjonous, N. M.; Gardiner,
J. V.; Ghatei, M. A.; Bloom, S. R. Endocrinology 2006, 147, 3510.
5. Beck, B.; Fernette, B.; Stricker-Krongrad, A. Biochem. Biophys. Res. Commun.
2005, 332, 859.
essential for antagonist potency, carboxylic acid 1k and piperidine
1l were prepared. Intermediate ester 1j (Ke = 726 nM) and target
acid 1k (Ke > 4 lM) were both inactive as antagonists. Interest-
6. Rizzi, A.; Vergura, R.; Marzola, G.; Ruzza, C.; Guerrini, R.; Salvadori, S.; Regoli,
D.; Calo, G. Br. J. Pharmacol. 2008.
7. Leonard, S. K.; Dwyer, J. M.; Sukoff Rizzo, S. J.; Platt, B.; Logue, S. F.; Neal, S. J.;
Malberg, J. E.; Beyer, C. E.; Schechter, L. E.; Rosenzweig-Lipson, S.; Ring, R. H.
Psychopharmacology (Berl.) 2008, 197, 601.
8. Cioccioppo, R.; Economidou, D.; Cannella, N.; Braconi, S.; Stopponi, S. In Society
for Neuroscience 2007 2007. 271.18/Z1 San Diego.
9. Laitinen, T.; Polvi, A.; Rydman, P.; Vendelin, J.; Pulkkinen, V.; Salmikangas, P.;
Makela, S.; Rehn, M.; Pirskanen, A.; Rautanen, A.; Zucchelli, M.; Gullsten, H.;
Leino, M.; Alenius, H.; Petays, T.; Haahtela, T.; Laitinen, A.; Laprise, C.; Hudson,
T. J.; Laitinen, L. A.; Kere, J. Science 2004, 304, 300.
10. Bernier, V.; Stocco, R.; Bogusky, M. J.; Joyce, J. G.; Parachoniak, C.; Grenier, K.;
Arget, M.; Mathieu, M. C.; O’Neill, G. P.; Slipetz, D.; Crackower, M. A.; Tan, C. M.;
Therien, A. G. J. Biol. Chem. 2006, 281, 24704.
11. Tancredi, T.; Guerrini, R.; Marzola, E.; Trapella, C.; Calo, G.; Regoli, D.;
Reinscheid, R. K.; Camarda, V.; Salvadori, S.; Temussi, P. A. J. Med. Chem.
2007, 50, 4501.
12. Reinscheid, R. K.; Xu, Y. L.; Okamura, N.; Zeng, J.; Chung, S.; Pai, R.; Wang, Z.;
Civelli, O. J. Pharmacol. Exp. Ther. 2005, 315, 1338.
13. Fukatsu, K.; Nakayama, Y.; Tarui, N.; Mori, M.; Matsumoto, H.; Kurasawa, O.;
Banno, H. Bicyclic Piperazine Compound And Use Thereof, Patent Application
No.: PCT/JP2004/012683 (EPO 4772639.3), June 7, 2006.
14. Okamura, N.; Habay, S. A.; Zeng, J.; Chamberlin, A. R.; Reinscheid, R. K.
J. Pharmacol. Exp. Ther. 2008. doi:10.1124/jpet.107.135103.
15. Knölker, H. J.; Braxmeier, T. Tetrahedron Lett. 1998, 39, 9407.
16. Wan, A. S.; Ngiam, T. L.; Leung, S. L.; Go, M. L.; Heng, P. W.; Natarajan, P. N.;
Shafiee, A.; Vossoghi, M.; Savabi, F.; Francisco, C. G., et al Steroids 1983, 41,
309.
17. Matthews, J. M.; Dyatkin, A. B.; Evangelisto, M.; Gauthier, D. A.; Hecker, L. R.;
Hoekstra, W. J.; Liu, F.; Poulter, B. L.; Sorgi, K. L.; Maryanoff, B. E. Tetrahedron
2004, 15, 1259.
18. Functional determinations: identification of functional agonists as well as
antagonists at the NPS receptor utilized RD-HGA16 cells (Molecular Devices), a
ingly, 2-piperidinoethyl analog 1l had intermediate potency at
the Ile107 receptor variant (Ke = 109 nM) indicating bulky amine
substituents are tolerated, albeit at slightly reduced potency. Con-
verting urea 1l to thiourea 1s moderately reduced potency
(Ke = 280 nM), whereas replacement of the piperidine with a more
water soluble morpholino group (1r, Ke = 3760 nM) dramatically
reduced potency. Finally, evaluation of 4-(4-fluorophenyl)butyl
analog 1p (Ke = 1100) and methyl analog 1q (Ke > 4 lM) were inac-
tive as antagonists. In general, it appears that tolerance to 7-substi-
tution is limited.
This study demonstrates that antagonists were generally more
potent at the Ile107 variant (1- to 5-fold) with limited exceptions
(Tables 1 and 2). This suggests that future development of antago-
nists selective for one receptor variant may be possible. In addition,
none of the compounds presented in this study possessed measur-
able intrinsic activity at 10 l
M in the NPS Ile107 cell line.
In conclusion, we have provided a novel synthetic route to 7-
substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones
and have begun to identify the key structural features required for
NPS antagonist activity. In particular, we have demonstrated the
importance of the urea functionality possessing a free hydrogen
and that ethylpiperidine (1l) can serve as a substitute for the ben-
zyl group of 1d. The combination of these results provides a basis
for the design and testing of novel scaffolds with enhanced potency
and drug-like properties for the NPS receptor.
CHO cell line stably over-expressing the promiscuous Gq-protein Ga16. Two
individual cell lines were created that stably express each NPS receptor variant
(NPS Ile107 and Asn107). Cells are loaded with the calcium sensitive dye
calcium3 (Molecular Devices) for 1 h and compounds are assayed in separate
experiments for intrinsic activity and for the ability to inhibit NPS activity as
measured by calcium mobilization in the FlexStation assay. Test compound Ke
values were determined by running an 8-point half-log NPS concentration
response curve in the presence and absence of a single concentration of test
compound. EC50 values were calculated for NPS (A) and NPS + test compound
(A0), and these used to calculate the test compound Ke. A three-parameter
logistic equation was fit to the concentration response data with Prism (v5 for
Windows, GraphPad Software; San Diego, CA) to calculate the EC50 values. At
least two different concentrations of test compound were used for these
experiments, and these were chosen such that they at least caused a 4-fold
rightward shift in the NPS EC50. The Ke was calculated from the formula:
Ke = [L]/(DR-1), where [L] equals the concentration of test compound in the
assay and DR equals the dose ratio (A0/A). The data represent the mean SE
from at least three independent experiments.
Acknowledgments
This work was supported by a United States National Institute
of Mental Health research Grant (MH081247-01). We also thank
Mr. Keith Warner, Ms. Tiffany Langston, and Ms. Kathleen Kitsopo-
ulos for their valuable technical assistance.
References and notes
1. Civelli, O. Trends Pharmacol. Sci. 2005, 26, 15.
2. Sato, S. S. Y.; Miyajima, N.; Yoshimura, K. Novel G-protein coupled receptor
protein and DNA thereof. World Patent Application WO 02/31145 A1. 2002.
3. 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. Neuron
2004, 43, 487.