Synthesis and evaluation of a new series of Neuropeptide S receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.04.143

Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood-brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited ¹²⁵I-NPS binding in the CNS when administered to rats.

Full text of DOI:10.1016/j.bmcl.2010.04.143

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4700–4703
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a new series of Neuropeptide S receptor antagonists
a
a
b
b
Jeffrey Y. Melamed a, , Amy E. Zartman , Nathan R. Kett , Anthony L. Gotter , Victor N. Uebele ,
Duane R. Reiss b, Cindra L. Condra b, Christine Fandozzi c, Laura S. Lubbers b, Blake A. Rowe b,
Georgia B. McGaughey d, Martin Henault e, Rino Stocco e, John J. Renger b, George D. Hartman a,
Mark T. Bilodeau a, B. Wesley Trotter f
*
a Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA, USA
b Department of Neuroscience, Merck Research Laboratories, West Point, PA, USA
c Department of Drug Metabolism, Merck Research Laboratories, West Point, PA, USA
d Department of Chemistry Modeling and Informatics, Merck Research Laboratories, West Point, PA, USA
e In Vitro Sciences Group, Merck Canada, Montreal, Canada
f Department of Medicinal Chemistry, Merck Research Laboratories, Boston, MA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of nov-
elty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in
rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that read-
ily cross the blood–brain barrier. We detail here optimization efforts resulting in the identification of a
potent NPSR antagonist which dose-dependently and specifically inhibited 125I-NPS binding in the CNS
when administered to rats.
Received 8 March 2010
Revised 28 April 2010
Accepted 30 April 2010
Available online 5 May 2010
Keyword:
Neuropeptide S receptor
Ó 2010 Elsevier Ltd. All rights reserved.
The Neuropeptide S Receptor (NPSR or GPR154) is a G protein
coupled receptor, that is, expressed in regions of the central nervous
system that regulate sleep, wake and anxiety.1,2 NPS is a 20 amino
acid peptide, that is, a very potent agonist of NPSR (Kd = 0.33 nM).2
Central administration of NPS has been shown to produce arousal
and induce wakefulness by suppressing all stages of sleep, as dem-
onstrated by EEG recordings in rats.1 Studies have also shown that
the peptide shows anxiety reducing activity.1 These observations
suggest that an NPSR antagonist could represent a novel opportunity
for treatment of sleep-related and/or psychological disorders.
Though the de-orphanization of NPSR was reported relatively
recently, reports in the patent and medicinal chemistry literature
have identified a class of 1,1-diphenyl-hexahydro-oxazolo[3,4-
a]pyrazin-3-ones small molecule NPSR antagonists.3a,b A represen-
tative compound from this series was shown to prevent the effects
of ICV-administered NPS in mice, and recently the effects of the
compound in animal models of behavior were reported.3c
the interpretation of in vivo effects. In this Letter, we describe the
optimization of a lead series to achieve a potent, selective NPSR
antagonist, that is, well tolerated and demonstrates good occu-
pancy of NPSR in the brain.
An initial high-throughput screening effort led to quinolinone-
based lead 1 (Fig. 1). Compound 1 is a potent NPSR antagonist
(IC50 = 18 nM) but was shown to be a substrate for the P-glycopro-
tein transporter (P-gp) in vitro. In order to increase the likelihood
of compound exposure in the CNS, we set reduction of P-gp suscep-
tibility as a primary goal of initial optimization. Analogs with var-
ied cyclohexylamide substitution showed that the SAR in this
region was tight and not likely to afford the required improve-
ments in properties. The basic piperazine functionality of 1 was
NMe
N
The objective of this effort was to develop tool compounds that
are appropriate for assessing the effects of NPSR antagonism
in vivo. To achieve this, we required a potent antagonist with suf-
ficient free exposure in the brain to achieve high antagonism
in vivo and with minimal off-target activities that could confound
O
N
O
N
Me
1
NPSR IC50 = 18 nM
P-gp B:A/A:B = 10.6
* Corresponding author at present address: Synthonix, 3805 Old Easton Rd.,
Doylestown, PA 18902, USA. Tel.: +1 215 264 2570.
E-mail address: jmelamed@synthonix.com (J.Y. Melamed).
Figure 1. Quinolinone NPSR antagonist.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.143

J. Y. Melamed et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4700–4703
4701
postulated to be a potential contributor to P-gp transport of the
compound.4 However, we found no conservative alterations of this
moiety that both preserved potency and substantially reduced
P-gp susceptibility.
R
HN
O
CHO
O
a
b
R
O
N
O
N
N
Among the approaches that were attempted to replace the piper-
azinylcyclohexyl moiety, one ultimately fruitful effort involved
computationally evaluating commercial and proprietary amines to
select a set of potential replacements for the synthesis of a library.
This involved the use of a shape-matching computational algorithm
to identify replacements for the entire piperazinylcyclohexylmethyl
amine portion of the molecule. This was accomplished by applying a
proprietary computational approach called SQW.5 SQW seeks to
maximize volume overlap between a probe molecule or a set of
so-called sphere points with a target database using knowledge of
chemical typing and shape.
Me
Me
2
N
4
Me
5
Scheme 1. Reagents: (a) NaBH(OAc)3, amine, DCE; (b) cyclohexanecarbonyl
chloride, TEA, DCM.
Table 1
SAR studies of propyl diamine NPSR antagonists
More than 100 amines identified by the shape-matching
algorithm were incorporated into the lead series according to
Scheme 1. The synthesis begins with the reductive amination of
commercially available 1-methyl-2-oxo-1,2-dihydroquinoline-3-
carbaldehyde (2, Scheme 1) with an amine using sodium triacet-
oxyborohydride to provide 4. Unpurified 4 is then treated with
cyclohexanecarbonyl chloride and triethylamine to provide 5 in
moderate to 28–37% yields over two steps after automated re-
verse-phase purification.
R1
Me
N
R2
N
R3
O
N
Me
O
Evaluation of this set of compounds6 indicated that the 1,3-pro-
panediamine substructure represented a viable scaffold for non-
piperazinecyclohexyl NPSR antagonists (Table 1). Branched
analogs (6a–b, R = H) were moderately potent. Addition of a
methyl group to the propyl linker provided a significant boost in
potency (see 6a, 6b vs 6c, 6e). Replacement of the pyrrolidine in
6c with a piperidine ring led to 6d, which provided a 10-fold boost
in potency to 31 nM.
However, 6d remained a P-gp substrate with a B:A/A:B ratio of
36, presumably due in part to the presence of the basic amine.
Accordingly, morpholine derivative 6e, though less potent then
6d, was not a P-gp substrate (B:A/A:B = 2.0). We therefore targeted
further variation of this amine functionality to recover potency
while avoiding P-gp susceptibility. To this end, we envisioned a
synthetic route that would allow for variation of the amine at
the last step of the synthesis and allow alteration of the gem
dimethyl moiety. Yokozawa and co-workers have described a
three-component coupling procedure for aldehydes, allylsilanes,
and an ammonia equivalent that provides compounds of type 10
where R1 = H and R2 is a simple aryl or alkyl moiety.7
6
Compound
R1
NR2R3
Human NPSR
IC50 (nM)
B:A/A:Bb
Pappc
a
N
N
6a
6b
6c
6d
H
1577
725
326
31
n.d.
n.d.
n.d.
36
n.d.
n.d.
n.d.
25
H
O
N
N
Me
Me
N
6e
Me
144
2.0
23
O
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IC50, nM).
b
MDR1 Directional Transport Ratio (B to A)/(A to B). Values represent the average
of three experiments and interassay variability was 20%.
We found that this protocol could be extended to ketones by
proper choice of conditions and use of TES–OTf as the Lewis acid
c
Passive permeability (10ꢀ6 cm/s).
R2
R1
R2
R1
HN
R2
R1
H2N
O
c
b
a
O
N
H
+
+
SiMe3
Me3Si
N
H
OMe
R1
R2
N
Me
12
O
26-100%
91-94%
67-80%
O
OMe
8
7
9
11
10
R1 R2
N
R1 R2
d
R1 R2
O
R3
f
e
O
N
O
O
N
N
54-68%
R4
7-95%
26-88%
N
Me
O
N
Me
O
N
Me
O
13
14
15
Scheme 2. Reagents and conditions: (a) cat. TES–OTf, DCM, 0 °C to rt, 18 h; (b) NH2NH2, KOH, ethylene glycol, 200 °C; (c) 2, NaBH(OAc)3; (d) cyclohexanecarbonyl chloride,
DIEA; (e) OsO4, NMO, NaIO4; (f) HNRR0, NaBH(OAc)3, HOAc, NaOAc.

4702
J. Y. Melamed et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4700–4703
promoter. This procedure provided a rapid synthesis of the appro-
priate R1, R2 substituted N-homoallylcarbamates 10 (Scheme 2),
which could be deprotected and then reductively alkylated with
2 to provide the desired amines 12. Further elaboration provided
the key aldehyde intermediates 14, which could then undergo
reductive amination with various primary and secondary amines.
This sequence provided a broad array of amines, of which piper-
idines and morpholines were preferred. We also found that subtle
modifications of substitution in the linker region were effective for
enhancing antagonist potency. Ethyl, methyl-substituted com-
pound 16a (Table 2) showed more than fourfold improvement in
potency compared to the dimethyl analog 6d. The addition of fluo-
rine substituents to the piperidine ring decreased P-gp susceptibil-
ity, presumably by lowering the basicity of the amine, but also led
to a significant increase in plasma protein binding. We found that
the morpholine derivative 16d possessed a unique combination of
good physical properties and low P-gp susceptibility on this scaf-
fold. Further increasing the size of the geminal alkyl substituents
(see di-ethyl analog 16e and cyclohexyl analog 16f) decreased
activity (Table 2).
The two enantiomers of 16d were separated using chiral chro-
matography to give the potent antipode NPSR-QA18 (NPSR-quin-
olinone amide 1), as well as its much less potent enantiomer
16g. Compound NPSR-QA1 exhibited potent NPSR antagonism,
was not a P-gp substrate, and had a high plasma free fraction
(18% in human plasma). Consistent with these data, the compound
achieved good brain and CSF levels when dosed to rats. A 30 mg/kg
IP dose provided the following tissue levels at 0.5 h: plasma/brain/
Table 2
SAR studies of propyl diamine NPSR antagonists
R1
R2
O
N
N
X
N
O
Me
16
Compound
R1
R2
Human NPSR
B:A/A:Bb
Pappc
Prot. Bnd.
a
X
IC50 (nM)
16a (racemic)
16b (racemic)
16c (racemic)
16d (racemic)
16e (racemic)
Me
Me
Me
Me
Et
Et
Et
Et
Et
Et
CH2
CHF
CF2
O
7
14
22
23
103
43
28
33
25
18
n.d.
89%
n.d.
97%
91%
n.d.
2.6
1.1
0.5
n.d.
O
16f (racemic)
O
398
n.d
n.d.
n.d.
NPSR-QA1 (16d enantiomer 1)
16g (16d enantiomer 2)
Me
Et
Et
Me
O
O
10
785
1.7
n.d.
36
n.d.
82%
n.d.
a
b
c
Values represent the numerical average of at least two experiments. Interassay variability was 30% (IC50, nM).
MDR1 Directional Transport Ratio (B to A)/(A to B). Values represent the average of three experiments and interassay variability was 20%.
Passive permeability (10ꢀ6 cm/s).
A
B
C
TB
NS
100
100
90
80
70
60
50
40
30
20
10
0
AON
75
50
25
0
NPSR-QA1
NPSR-Inact
DEN
SC
AON
DEN
SC
-1.0
-0.5
0.0
-8
-7
-6
Plasma, Log [NPSR-QA1 (µM)]
Log [NPSR Ligand (M)]
Figure 2. NPSR-QA1 dose-dependently occupied 125I-hNPS binding sites in discrete regions of the rat brain. (A) Total 125I-hNPS binding (TB) relative to non-specific binding
(NSB) in anterior olfactory nucleus (AON), dorsal endopiriform nucleus (DEN) and superior colliculus (SC). TB in tissue sections from vehicle-treated animals (n = 2; 25% HPB
cyclodextrin: 75% sterile water, ip) was determined following incubation with 0.5 nM 125I-hNPS and compared to NSB in adjacent sections incubated with 125I-hNPS and an
excess of 2 lM unlabeled hNPS. Areas of interest in the coronal plane of the rat brain are demarcated by dashed lines. (B) Dose-dependent ex vivo occupancy by NPSR-QA1 in
discrete brain regions 30 min after ip dosing (n = 4 rats; n = 2–3 brain sections/rat). Tissue sections from vehicle- or NPSR-QA1-treated animals were incubated with 125I-
hNPS (0.5 nM, 30 min) and mean percent occupancy of NPSR-QA1 versus plasma concentration of the compound was determined for AON (filled circles, dotted line;
IC50 = 0.57 l l l
M; R2 = 1.0), DEN (open circles, dashed line; IC50 = 0.38 M; R2 = 0.97) and SC (open squares, solid line; IC50 = 0.13 M; R2 = 0.99) using non-linear regression. (C)
NPSR-QA1, but not an inactive NPSR ligand, inhibited binding of 125I-hNPS in SC. In vitro incubation of NPSR-QA1 (circles) dose-dependently attenuated mean ( SEM) 125I-
hNPS binding in SC (IC50 = 1.44 nM) of vehicle-treated animals while a structurally similar inactive compound, NPSR-Inact (triangles) did not. Total binding was determined
by incubating tissue with 125I-hNPS (0.5 nM, 30 min) in the absence of a NPSR ligand. Non-specific binding of tissue was determined by co-incubation of 125I-hNPS with 2
lM
unlabeled hNPS (n = 2–3 brain sections/concentration).

J. Y. Melamed et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4700–4703
4703
CSF (nM) = 4344, 1690, 101 and at 2.0 h: plasma/brain/CSF
References and notes
(nM) = 2297, 665, 45. NPSR-QA1 was tested against a broad panel
of receptor and enzyme assays and had no off-target activities with
an IC50 <10 lM (170 targets tested at MDS Pharma Services).
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.; Lecea, L.; Civelli, O. Neuron 2004,
43, 487.
NPSR-QA1 showed potent binding to the rat NPSR receptor in
in vitro binding displacement studies with an EC50 = 1.3 nM.9
These data suggest that NPSR-QA1 is a potent and selective tool
for studying the effects of NPSR antagonism in vivo and were sup-
plemented with a study looking at the ability of NPSR-QA1 to oc-
cupy the target when the compound was dosed IP.
2. Xu, Y.-L.; Gall, C. M.; Jackson, V. R.; Civelli, O.; Reinscheid, R. K. J. Comp. Neurol.
2007, 500, 84.
3. (a) Zhang, Y.; Gilmour, B. P.; Navarro, H. A.; Runyon, S. P. Bioorg. Med. Chem. Lett.
2008, 18, 4064; (b) Okamura, N.; Habay, S. A.; Zeng, J.; Chamberlin, A. R.;
Reinscheid, R. K. J. Pharmacol. Exp. Ther. 2008, 325, 893; (c) 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.
Peptides, in press.
The ability of NPSR-QA1 to penetrate the blood–brain barrier
and occupy NPS binding sites was demonstrated in studies exam-
ining ex vivo competition for 125I-hNPS binding. Intraperitoneal
administration of NPSR-QA1 (30 mg/kg; 25% HPB cyclodextrin)
attenuated 125I-hNPS binding in three different areas of the rat
brain including the anterior olfactory nucleus, dorsal endopiri-
form nucleus and superior colliculus (Fig. 2A and B). Specificity
for this binding was substantiated in experiments in which
in vitro pre-incubation of brain sections with NPSR-QA1 blocked
125I-hNPS binding in a concentration-dependent manner, while a
structurally similar inactive compound (NPSR-Inact, EC50 for
binding displacement on the rat receptor = 1400 nM9) did not
(Fig. 2C).
In summary, a new series of NPSR antagonists has been devel-
oped. A potent, highly selective and CNS penetrant antagonist,
NPSR-QA1, has been identified. This compound has been shown
to provide high occupancy of NPSR after IP dosing. Thus, NPSR-
QA1 represents a useful pharmacologic agent for further study of
the effects of central NPSR antagonism in vivo.
4. Cox, C. D.; Breslin, M. J.; Whitman, D. B.; Coleman, P. J.; Garbaccio, R. M.; Fraley,
M. E.; Zrada, M. M.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Lobell, R. B.; Tao, W.;
Abrams, M. T.; South, V. J.; Huber, H. E.; Kohl, N. E.; Hartman, G. D. Bioorg. Med.
Chem. Lett. 2007, 17, 2697.
5. Miller, M. D.; Sheridan, R. P.; Kearsley, S. L. J. Med. Chem. 1999, 42, 1505.
6. A dual sequence fluorometric imaging plate reader (FLIPR) Ca2+ mobilization
assay was used to evaluate compounds for agonist and antagonist activity on the
Neuropeptide S receptor expressed on recombinant CHOK1 cells plated in 384-
well PDL coated plates 24 h prior to experiment (15Kcells/well/20 ll). Following
washing with buffer and loading with Fluo4AM dye for 1 h at 37 °C in 5% CO2,
cell plates were placed in the FLIPR and incubated with titrated compounds for
4 min during sequence 1 data collection. In sequence 2, an EC80 of human NPS
was added to the assay plates and data collected for another 4 min. Percent
stimulatory activity (sequence 1) or inhibition (sequence 2) was calculated
relative to Ca2+ mobilization in NPS stimulated control wells (EC100 or EC80
NPS). Compound potencies were determined from plots of concentration
response curves using 4p logistical fits and reported as means SD of multiple
determinations.
7. Niimi, L.; Serita, K.; Hiraoka, S.; Yokozawa, T. Tetrahedron Lett. 2000, 41, 7075.
8. Single enantiomer (faster eluting peak) was obtained via chiral HPLC. Absolute
stereochemistry has not been determined.
9. Binding experiments employed radiolabeled compound 1. Radiolabeling of 1
was accomplished by tri-tritiation of the N-Me quinolinone moiety by alkylation
of the un-methylated quinolinone.