Design, synthesis and SAR analysis of novel potent and selective small molecule antagonists of NPBWR1 (GPR7)

Article abstract of DOI:10.1016/j.bmcl.2012.09.074

Novel small molecule antagonists of NPBWR1 (GPR7) are herein reported. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 5-chloro-4-(4-methoxyphenoxy)-2-(p-tolyl) pyridazin-3(2H)-one as a NPBWR1 hit antagonist with micromolar activity. Design, synthesis and structure-activity relationships study of the HTS-derived hit led to the identification of 5-chloro-2-(3,5-dimethylphenyl)-4-(4-methoxyphenoxy) pyridazin-3(2H)-one lead molecule with submicromolar antagonist activity at the target receptor and high selectivity against a panel of therapeutically relevant off-target proteins. This lead molecule may provide a pharmacological tool to clarify the molecular basis of the in vivo physiological function and therapeutic utility of NPBWR1 in diverse disease areas including inflammatory pain and eating disorders.

Full text of DOI:10.1016/j.bmcl.2012.09.074

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7135–7141
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and SAR analysis of novel potent and selective small
molecule antagonists of NPBWR1 (GPR7)
Mariangela Urbano a, Miguel Guerrero a, Jian Zhao a, Subash Velaparthi a, S. Adrian Saldanha b,
Peter Chase b, Zhiwei Wang c, Olivier Civelli c, Peter Hodder b,d, Marie-Therese Schaeffer e,f
,
Steven Brown e,f, Hugh Rosen e,f,g, Edward Roberts a,
⇑
a Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
b Scripps Research Institute Molecular Screening Center, Lead Identification Division, Translational Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States
c Department of Pharmacology, University of California Irvine, Irvine, CA 92697, United States
d Department of Molecular Therapeutics, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
e Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
f The Scripps Research Institute Molecular Screening Center, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
g Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2012
Revised 17 September 2012
Accepted 19 September 2012
Available online 2 October 2012
Novel small molecule antagonists of NPBWR1 (GPR7) are herein reported. A high-throughput screening (HTS)
of the Molecular Libraries-Small Molecule Repository library identified 5-chloro-4-(4-methoxyphenoxy)-
2-(p-tolyl)pyridazin-3(2H)-one as a NPBWR1 hit antagonist with micromolar activity. Design, synthesis
and structure–activity relationships study of the HTS-derived hit led to the identification of
5-chloro-2-(3,5-dimethylphenyl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one lead molecule with sub-
micromolar antagonist activity at the target receptor and high selectivity against a panel of therapeuti-
cally relevant off-target proteins. This lead molecule may provide a pharmacological tool to clarify the
molecular basis of the in vivo physiological function and therapeutic utility of NPBWR1 in diverse disease
areas including inflammatory pain and eating disorders.
Keywords:
NPBWR1 (GPR7)
Selective small molecule antagonists
Feeding behavior and energy homeostasis
Inflammatory pain
Ó 2012 Elsevier Ltd. All rights reserved.
In 1995 O’Dowd et al. reported the existence of two structurally
related orphan G-protein coupled receptors termed GPR7 and
GPR8 which were identified by the cloning of opioid-somato-
statin-like receptor genes from human genomic DNA.1 GPR7 and
GPR8 share a 70% nucleotide and a 64% amino acid identity with
each other. Although orthologs of GPR7 and GPR8 have been iso-
lated by PCR from many species, in rodents GPR8 has not been
found.2 Neuropeptide W (NPW) and neuropeptide B (NPB) were
successively identified as the endogenous ligands for these recep-
tors.3,4 NPB mRNA is widely distributed throughout the mouse
brain and is present in the hippocampus, hypothalamic nucleus,
Edinger-Westphal nucleus (EW), locus coerulius, inferior olive
and lateral parabrancheal nucleus.3,5 NPW expression is more con-
fined to EW, ventral tegmental area (VTA), periaqueductal gray and
dorsal raphe nucleus.6
with the two NPW isoforms NPW23 and NPW30, whereas
NPBWR2 shows
a potency rank order of NPW23 > NPW30 >
NPB.3,4,8 Both NPBWR1 and NPBWR2 couple to the Gi-class of
GPCRs and decrease intracellular cAMP.3
In situ hybridization in rodents and tissue binding studies
showed that NPBWR1 mRNA is localized in the suprachiasmatic
nucleus, hippocampus, arcuate nucleus, bed nucleus of the stria
terminalis, extended amygdala, VTA.2,3
Based on the distribution of peptides (NPW and NPB) and
receptors (NPBWR1, NPBWR2) it was hypothesized that this neu-
ropeptide system plays an important role in modulating feeding
behavior and energy homeostasis,3,4 although its effects are incom-
pletely understood and some discrepancies are found in the litera-
ture. Indeed, intracerebroventricular (i.c.v.) administration of NPW
to fasting rats or free-feeding rats before dark phase suppressed
food intake and increased energy expenditure as a result of aug-
mented heat production, oxygen consumption and body tempera-
ture.9 However, two groups demonstrated an orexigenic effect in
rats followed by acute administration of NPW.4,10 I.c.v. injection
of NPB into rats induced a biphasic feeding effect at low doses with
early and mild orexigenic action followed by marked anorexia,
whereas higher doses caused anorexia at all time points.3 Although
further studies are needed to resolve the molecular mechanisms of
Following the receptor deorphanizing, GPR7 and GPR8 were
reclassified by IUPHAR as NPBWR1 (GPR7) and NPBWR2 (GPR8).7
NPW and NPB activate both receptors with varying degree of affin-
ity. NPBWR1 has a slightly higher affinity for NPB as compared
⇑
Corresponding author. Tel.: +1 858 784 7770; fax: +1 858 784 7745.
E-mail address: eroberts@scripps.edu (E. Roberts).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.074

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M. Urbano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7135–7141
administration of NPW in the brain reduced seizure activity in mice,
suggesting that NPBWR1 may be a novel target for epilepsy.19
Despite the accumulating evidence proving the implication of
NPBWR1 in the regulation of feeding behavior, neuroendocrine
function and pain, aspects of the receptor biological role remain
unclear, partly due to the lack of in vivo pharmacological tools. Re-
cently, a series of novel small molecule antagonists of NPBWR1
have been disclosed; however, these molecules were found to be
P-glycoprotein substrates and suffered from poor metabolic stabil-
ity having less than 10% parent remaining at 30 min in mouse liver
microsomes.20
Figure 1. In vitro potency of the NPBWR1 antagonist hit.
Herein we report on the discovery, synthesis and structure–
activity relationships (SAR) of novel NPBWR1 antagonists which
may allow experiments aimed to elucidate the biological profile
and the therapeutic application of the target receptor. The 5-
chloro-4-(4-methoxyphenoxy)-2-(p-tolyl)pyridazin-3(2H)-one 1a
( Fig. 1) was identified by in house high-throughput screening
(HTS) of the Molecular Libraries-Small Molecule Repository
(MLSMR) library as a NPBWR1 antagonist hit with an IC50 value
its anorectic properties, NPBWR1 has been recently hypothesized
to play an important role in the regulation of feeding and energy
metabolism by stimulating lipolysis and inhibiting leptin secre-
tion.11,12 Remarkably, NPBWR1 knockout mice developed adult-
onset obesity and had elevated plasma leptin concentration.13
Additionally, rats injected with NPW showed decreased plasma
insulin and leptin concentration.14 Compatible with these studies,
NPBWR1 has been demonstrated to be expressed in rat adipocytes,
while NPW and NPB are detectable in preadipocytes and
macrophages.11
Intrathecal administration of either NPW23 or NPB produced
analgesic effect in inflammatory pain models in rats (plantar injec-
tion of formalin or carrageenan) but had no effects in thermal and
mechanical nociceptive tests.15 Although the precise mechanisms
responsible for the analgesic effect elicited by NPW23 or NPB in
the rat formalin and carrageenan tests are not known, these data
suggested that both spinally-applied NPW23 and NPB suppressed
the input of nociceptive information to the spinal dorsal horn
and indicated a new potential therapeutic approach to treating
inflammatory pain.15 Interestingly, samples taken from patients
of 2.2 l
M at the target receptor.21 The structural integrity of the
hit was confirmed by its re-synthesis via condensation of p-tolyl
hydrazine 2a with mucochloric acid 3. The 4,5-dichloro pyrida-
zin-3(2H)-one 4a thus obtained underwent nucleophilic addition
at position 4 with phenol 5a using anhydrous 1,4-dioxane (Scheme
1).22
Our SAR studies on 1a involved the preparation and testing of
analogs 1b–1ae varying the p-tolyl region (A) while maintaining
the 4-methoxyphenoxy group (C) and the pyridazin-3(2H)-one
central core (B) as constant moieties (Scheme 1, Table 1).23 The tar-
get compounds were obtained analogously to 1a starting from
commercially available or readily synthesized aryl hydrazines
2b–2ae.24
Interestingly, the unsubstituted phenyl analog 1b was ꢀsixfold
less potent than the hit. When the 4-methyl group of the hit was
exchanged by fluorine (1c), chlorine (1g) or bromine (1f) the po-
tency varied inversely to the electronegativity of the halogen. In-
deed, the bromide 1f was threefold more potent than the hit,
whereas the chloride 1g and fluoride 1c were slightly more and
less potent than the hit respectively. The 4-ethylcarboxylate analog
1h was slightly more potent than the hit. Moderately polar substit-
uents such as trifluoromethyl (1d) and isopropoxy (1e) groups in
the para position were also tolerable with a ꢀthreefold increase
in potency. A 3- to 4-fold increase in potency over the hit was ob-
served when in the same position were inserted bulkier and/or
longer substituents such as ethyl (1i) sec-butyl (1l), tert-butyl
(1n). A greater increase of activity (eightfold) was observed for
the isopropyl derivative 1j, whereas the n-pentyl analog 1m was
with inflammatory/immunomediated neuropathies showed
a
higher expression of NPBWR1 restricted to myelin-forming
Schwan cells where the receptor is constitutively present at low
levels. Consistently, animal models of acute inflammatory and
trauma-induced neuropathic pain exhibited an increased myelin-
associated expression of NPBWR1, suggesting a therapeutic role
of this receptor in analgesia and as a marker of inflammatory neur-
opathies.16 I.c.v. injection of NPB in male rats elevated prolactin
and corticosterone, and decreased growth hormone (GH) levels
in circulation, suggesting that this peptide may play a physiological
role in the neuroendocrine response to stress via the activation of
the hypothalamus-pituitary-adrenal axis.17 Additionally, i.c.v.
injection of NPB has been recently shown to induce slow wave
sleep (SWS) in wild-type mice in a dose-dependent manner when
administered during the dark period. Interestingly, the NPB did not
impact SWS in NPBWR1 knockout mice thus suggesting that
NPBWR1 may have a role in modulating the sleep/waking state.18
Remarkably, consistent with the dual analgesic and anti-
epileptic activity of several anticonvulsant neuropeptides, i.c.v.
slightly less potent than 1a. Interestingly, the presence of a
p- or
p-like system was tolerated but did not significantly improve the
potency as observed for the phenyl 1o and cyclopropyl 1k analogs.
Adding a methyl group in the ortho position led to a slight incre-
ment of potency compared to the hit as observed for the 2,4-,
2,5- and 2,3-dimethyl substituted derivatives 1p–1r. Remarkably,
Scheme 1. Synthesis of 1a–1ae.

M. Urbano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7135–7141
7137
Table 1
Table 2
NPBWR1 antagonist activity of 1a–1ae
NPBWR1 antagonist activity of 6a–6n
Compd.
Ar
IC50
(l
M)a
Compd.
Ar
Ar1
IC50 (l
M)a
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
4-Methylphenyl
Phenyl
4-Fluorophenyl
4-Trifluoromethylphenyl
4-Isopropoxyphenyl
4-Bromophenyl
4-Chlorophenyl
4-Ethylcarboxylatephenyl
4-Ethylphenyl
4-Isopropylphenyl
4-Cyclopropylphenyl
4-(sec-Butyl)phenyl
4-(n-Pentyl)phenyl
4-(tert-Butyl)phenyl
4-Phenylphenyl
2,4-Dimethylphenyl
2,5-Dimethylphenyl
2,3-Dimethylphenyl
3,4-Dimethylphenyl
3-Methylphenyl
3-Chlorophenyl
3-Bromophenyl
3-Methoxyphenyl
3-Ethylphenyl
3-Isopropylphenyl
3,5-Dimethylphenyl
3-Methyl-4-isopropylphenyl
5,6,7,8-Tetrahydronaphthalen-1-yl
5,6,7,8-Tetrahydronaphthalen-2-yl
Naphthalen-1-yl
2.20
13.00
3.60
0.75
0.77
0.69
1.77
1.20
0.58
0.27
1.10
0.65
2.57
0.68
1.71
0.98
1.40
1.30
0.59
0.57
0.69
0.71
0.41
0.23
0.28
0.27
0.30
0.65
0.65
0.36
0.16
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
3,5-Dimethylphenyl
3,5-Dimethylphenyl
Phenyl
4-Ethoxyphenyl
4-Hydroxyphenyl
4-Trifluoromethoxyphenyl
4-Trifluoromethyl
20.00
0.99
1.30
2.20
0.88
0.68
2.30
20.00
4.80
12.20
0.65
2.10
0.21
0.44
4-Ethylphenyl
4-Hydroxymethylphenyl
3,4-Dimethoxyphenyl
4-Hydroxyphenyl
4-Trifluoromethoxyphenyl
4-Hydroxymethylphenyl
4-Aminoacetylphenyl
4-Ethoxyphenyl
4-Hydroxymethylphenyl
a
Data are reported as mean of n = 3 determinations.
1s
1t
biological results are reported in Table 2.23 First, the para substituent
of C was varied while keeping the p-tolyl coil A (6a–6h). Removing
the methoxy group from 1a led to ninefold loss in potency (6a).
Interestingly, the hydroxyphenoxy analog 6c was slightly more
potent than 1a, while the potency remained unchanged by install-
ing a trifluoromethoxy (6d) or hydroxymethyl (6g) group. The
ethoxy (6b) as well as trifluoromethyl (6e) and ethyl (6f) substi-
tuted compounds were ꢀ2- to 3-fold more potent than the hit, thus
suggesting that a hydrogen bond interaction in this position is not
fundamental for the potency. The 3,4-dimethoxyphenoxy analog
6h was ninefold less potent than the hit. The results from the
molecules containing the 4-isopropylphenyl coil (6i–6l) did not
parallel those from the p-tolyl series as observed for the
4-hydroxyphenoxy 6i and 4-trifluoromethoxyphenoxy 6j deriva-
tives which were less potent than 1j by ꢀ18- and 45-fold, respec-
tively. Moreover, the 4-hydroxymethylphenoxy analog 6k was
only ꢀ2-fold less potent than 1j and ꢀ3-fold more potent than
1a. In the presence of 4-aminoacetyl substituent (6l) the potency
decreased by eightfold as compared to 1j.
1u
1v
1w
1x
1y
1z
1aa
1ab
1ac
1ad
1ae
Naphthalen-2-yl
a
Data are reported as mean of n = 3 determinations.
Within the 3,5-dimethyl series (6m and 6n), the 4-ethoxyphenoxy
6m was almost equipotent to the 4-methoxyphenoxy derivative 1z
while the 4-hydroxymethylphenoxy analog 6n was less than
twofold less potent than 1z. Interestingly, since some polar groups
were tolerated, the para position of C may be exploited for install-
ing solubility-enhancing features and improving physicochemical
properties of the hit class.
Scheme 2. Synthesis of 6a–6n.
Next, we modified the pyridazin-3(2H)-one region B while
maintaining constant the p-tolyl or 4-isopropyl phenyl in A and
the 4-methoxy or 4-ethoxy phenoxy in C. The synthesis and biolog-
ical results of compounds 9a–9u are reported in Schemes 3–6 and
Table 3.23 The 5-bromopyridazin-3(2H)-ones 9a–9c were synthe-
sized analogously to the hit (Scheme 3). Mucobromic acid 7 was
condensed with aryl hydrazine 2a, 2j or 2t under acidic conditions
to yield 4,5-dibromo pyridazin-3(2H)-ones 8a–8c which under-
went nucleophilic addition at position 4 with the phenoxyde of
5a or 5c. Suzuki coupling of boronic acids 10a–10c with 9a led to
derivatives 9d–9f (Scheme 3). Stille coupling of bromide 9b with
tetramethyltin 11 or tributyl(vinyl)tin 12 furnished 9g and 9h
(Scheme 3). Reduction of the olefin 9h using palladium/carbon
(Pd/C) under hydrogen atmosphere yielded the ethyl derivative
9i. Oxidative cleavage of 9h using sodium periodate and osmium
tetroxide led to the formation of the aldehyde 9j which was re-
duced to the alcohol 9k using sodium borohydride. Conversion of
9j into the corresponding oxime followed by dehydration using
mesylate chloride furnished the nitrile 9l. 9j was oxidized with so-
dium hypochlorite to the carboxylic acid 9m which was esterified
to 9n (Scheme 3).
3,4-dimethyl substituted analog 1s was ꢀfourfold more potent
than 1a and slightly more potent than the 2,4-dimethyl analog
1p. 3-Monosubstituted analogs bearing a small group such as
methyl (1t), chlorine (1u) or ethyl (1x) were 2- to 4-fold more
potent than their 4-subtituted counterparts. In the same trend,
the 3,5-dimethyl derivative 1z was twofold more potent than its
3,4-substituted analog 1s. However, similar activity was found
for 3- and 4-bromo substituted analogs (1v, 1f) as well as for
3- and 4-isopropyl derivatives (1y, 1j). The 3-methoxyphenyl
derivative 1w was slightly less potent than the 3-ethylphenyl ana-
log 1x indicating a minor negative impact of the electrondonating
properties of the methoxy group. The 3-methyl-4-isopropylphenyl
analog 1aa was almost equipotent to the 4-isopropylphenyl analog
1j. Surprisingly, tetrahydronaphtalen-1-yl 1ab was equipotent to
tetrahydronaphtalen-2-yl analog 1ac while naphtalen-2-yl 1ae
was twofold more potent than the naphatalen-1-yl analog 1ad.
We selected the aromatic coil A of representative examples (1a,
1j, 1z) to investigate the SAR at the 4-methoxyphenoxy head C. The
analogs 6a–6n were prepared similarly to the hit (Scheme 2). The

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M. Urbano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7135–7141
Scheme 3. Synthesis of 9a–9n.
1j was dechlorinated using Pd/C and hydrogen to give 9o
of 17 using bromine followed by chlorination at position 5 using
phosphoryl chloride and selective substitution of bromine with
5a under mild basic conditions provided the title compound
(Scheme 6). Hydrolysis of 9s gave the carboxylic derivative 9t
which was methylated to 9u. The p-tolyl bromide 9a was ꢀthreefold
more potent than 1a, while the 4-isopropylphenyl 9b was ꢀ2.5-fold
less potent than its chloride analog 1j and equipotent to the
4-ethoxyphenoxy analog 9c. Derivatization of 9a by installing an
aromatic ring such as phenyl (9d) or furanyl (9e) led to drastic loss
in potency. Installing a cyclopropyl (9f) led to ꢀeightfold loss in
potency compared to 9a. Within the 4-isopropylphenyl series, the
methyl 9g, vinyl 9h and ethyl 9j were ꢀ28-, 9-, and 40-fold less
potent than 1j. Interestingly, hydrogen bond acceptors such as
carbaldehyde (9j), nitrile (9l) and methyl ester (9n) were ꢀ23-
and ꢀ15- and 10-fold less potent than 1j, respectively. The primary
(Scheme 4)22b 1j reacted with sodium azide to furnish the corre-
sponding azide derivative which was reduced with Pd/C and
hydrogen to the corresponding primary amine 9p. Reaction of 9p
with acetyl chloride furnished 9q (Scheme 4).
Condensation of 3-methylphenyl hydrazine 2t with dichloro-
maleic anhydride 13 under acidic conditions followed by O-meth-
ylation using methyl sulfate afforded 15. Nucleophilic substitution
of 5a on 15 led to the formation of the final product 9r (Scheme
5).25
The synthesis of 9s involved the condensation of 4-isopropyl-
phenyl hydrazine 2j with diethyl ketomalonate 16 followed by
acetylation using acetyl chloride/zinc chloride and cyclization
using lithium bis(trimethylsilyl)amide at low temperature to fur-
nish the intermediate 17. Subsequent bromination at position 4

M. Urbano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7135–7141
7139
Scheme 4. Synthesis of 9o–9q.
Scheme 5. Synthesis of 9r.
Scheme 6. Synthesis of 9s–9u.

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M. Urbano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7135–7141
Table 3
NPBWR1 antagonist activity of 9a–9u
R1
R2
O
O
N
R
N
Ar
Compd.
Ar
R
R1
R2
IC50 (l
M)a
9a
9b
9c
9d
9e
9f
9g
9h
9i
4-Methylphenyl
Methoxy
Methoxy
ethoxy
Bromine
Bromine
Bromine
Phenyl
Furan-2-yl
Cyclopropyl
Methyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.78
0.72
0.72
>20
14.28
6.20
7.50
2.40
10.70
6.20
5.60
4.20
>20
2.7
>20
>20
>20
2.10
3.60
>20
4-Isopropylphenyl
4-Isopropylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
3-Methylphenyl
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Vinyl
Ethyl
9j
9k
9l
Carbaldehyde
Hydroxymethyl
Nitrile
Carboxylic acid
Methyl carboxylate
H
Amine
Acetamide
Chlorine
9m
9n
9o
9p
9q
9r
9s
9t
H
Methoxy
4-Isopropylphenyl
4-Isopropylphenyl
4-Isopropylphenyl
Chlorine
Chlorine
Chlorine
Ethyl Carboxylate
Carboxylate
Methyl carboxylate
9u
3.60
a
Data are reported as mean of n = 3 determinations.
alcohol 9k was ꢀ21-fold less potent than 1j while the carboxylic
acid 9m was inactive. Total loss of potency was found for the unsub-
stituted 9o, the amine 9p and acetamide 9q analogs. When a meth-
oxy group was introduced in position 6 (9r) the potency decreased
by ꢀ3.5-fold compared to 1t. Electron-withdrawing groups in the
same position were also detrimental for the activity with the ethyl
and methyl carboxylate analogs (9s, 9u) being 13-fold less potent
than 1j. Total loss of activity was observed for the carboxylic acid 9t.
In order to assess the potential of drug developability, the selec-
tivity profile of 1z was investigated against a Ricerca panel of 35
therapeutically relevant off-target proteins including GPCRs, en-
zymes and ion channels. Remarkably, the compound was found
to be highly selective against the pool of tested targets (stimula-
Acknowledgments
This work was supported by the National Institute of Health
Molecular Library Probe Production Center grant U54 MH084512
(Peter Hodder, Hugh Rosen).
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9. Mondal, M. S.; Yamaguchi, H.; Date, Y.; Shimbara, T.; Toshinai, K.; Shimomura,
Y.; Mori, M.; Nakazato, M. Endocrinology 2003, 144, 4729.
10. Baker, J. R.; Cardinal, K.; Bober, C.; Taylor, M. M.; Samson, W. K. Endocrinology
2003, 144, 2816.
phosphate buffered saline (PBS) at pH 7.4 with half-life higher than
48 h, thus indicating the lack of reactive sites.21 However, 1z dis-
plays poor water solubility (2 lM in PBS at pH 7.4, room tempera-
ture).21 Overall, 1z is as suitable lead molecule for optimization
studies addressed at enhancing its in vitro potency and physico-
chemical properties.
In summary, we have reported the design, synthesis and SAR
analysis of novel small molecule NPBWR1 antagonists based on
5-chloro-4-(4-alkoxyphenoxy)-2-(aryl)pyridazin-3(2H)-one chem-
otype. Systematic SAR studies of the MLSMR hit 1a led to the
identification of a lead molecule 1z (CYM50557) with submicrom-
olar antagonist activity at NPBWR1 and high selectivity against a
panel of off-targets. 1z may provide a novel valuable pharmacolog-
ical tool to explore the effects of NPBWR1 signaling cascade and
probe the molecular basis of the in vivo physiological function
and therapeutic utility of the target receptor. Details of further
research efforts will be communicated in due course.
´
´
11. Skrzypski, M.; Pruszynska-Oszmalek, E.; Rucinski, M.; Szczepankiewicz, D.;
Sassek, M.; Wojciechowicz, T.; Kaczmarek, P.; Kolodziejski, P. A.; Strowski, M.
Z.; Malendowicz, L. K.; Nowak, K. W. Regul. Pept. 2012, 176, 51.
12. Date, Y.; Mondal, M. S.; Kageyama, H.; Ghamari-Langroudi, M.; Takenoya, F.;
Yamaguchi, H.; Shimomura, Y.; Mori, M.; Murakami, N.; Shioda, S.; Cone, R. D.;
Nakazato, M. Endocrinology 2010, 151, 2200.
13. Ishii, M.; Fei, H.; Friedman, J. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10540.
´
14. Rucinski, M.; Nowak, K. W.; Chmielewska, J.; Ziolkowska, A.; Malendowicz, L. K.
J. Mol. Med. 2007, 19, 401.
15. Yamamoto, T.; Saito, O.; Shono, K.; Tanabe, S. Brain Res. 2005, 1045, 97.

M. Urbano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7135–7141
7141
16. Zaratin, P. F.; Quattrini, A.; Previtali, S. C.; Comi, G.; Hervieu, G.; Scheideler, M.
A. Mol. Cell. Neurosci. 2005, 28, 55.
17. Samson, W. K.; Baker, J. R.; Samson, C. K.; Samson, H. W.; Taylor, M. M. J.
Neuroendocrinol. 2004, 16, 842.
18. Hirashima, N.; Tsunematsu, T.; Ichiki, K.; Tanaka, H.; Kilduff, T. S.; Yamanaka, A.
Sleep 2011, 34, 31.
25 lL of Fluo8 NW (ABD Bioquest) was added to all wells and the assay plate
incubated for 50 min at 37 °C, 5% CO2 and 95% relative humidity. Test
compounds were added and the assay plate was incubated for 15 min at
room temperature. The assay was started by performing a basal read of
fluorescence (495 nm excitation and 515 nm emission) for 15 s on the FLIPR
Flexstation II 384 (Molecular Devices). Next, 5.5 ll of GPR7 agonist (20 nM
19. Green, B. R.; Smith, M.; White, K. L.; White, H. S.; Bulaj, G. ACS Chem. Neurosci.
2011, 2, 51.
20. Romero, F. A.; Hastings, N. B.; Moningka, R.; Guo, Z.; Wang, M.; Di Salvo, J.; Lei,
Y.; Trusca, D.; Deng, Q.; Tong, V.; Terebetski, J. L.; Ball, R. G.; Ujjainwalla, F.
Bioorg. Med. Chem. Lett. 2012, 1014, 22.
final concentration = EC80) in FLIPR Buffer (HBSS/20 mM Hepes/0.1% BSA) or
FLIPR Buffer alone were dispensed to the appropriate wells. Then a real time
fluorescence measurement was immediately performed for the remaining 45 s
of the assay. Tested compounds were assayed in triplicate in an 8-point 1:3
dilution series starting at a nominal concentration of 20
compound, percent inhibition was plotted against the log of the compound
concentration. three parameter equation describing sigmoidal dose-
lM. For each test
21. (a) http://www.ncbi.nlm.nih.gov/books/NBK98923/.; (b) http://pubchem.ncbi.
nlm.nih.gov/assay/assay.cgi?aid=1880.
A
a
22. (a) Suree, N.; Yi, S. W.; Thieu, W.; Marohn, M.; Damoiseaux, R.; Chan, A.; Jung,
M. E.; Clubb, R. T. Bioorg. Med. Chem. 2009, 17, 7174; (b) The regiochemistry of
the hit-class molecules was corroborated by 1H NMR of compound 9o: the
pyridazin-3(2H)-one protons at positions 5,6 (H5–H6) originated two doublets
at d 6.22 and 7.65 ppm with a coupling constant (J) of 4.6 Hz consistent with
the H5–H6 J of pyridazin-3(2H)-one rings. The 1H NMR spectrum of 9o was
acquired on spectrometer Varian Inova-400: 1H NMR (400 MHz, CDCl3):
d = 7.65 (d, J = 4.6 Hz, 1H), 7.48 (d, J = 7.5 Hz, 2H), 7.26 (d, J = 7.5 Hz, 2H),7.06
(d = 8.9 Hz, 2H), 6.93 (d = 8.9 Hz, 2H), 6.22 (d, J = 4.6 Hz, 1H), 3.81 (s, 3H), 2.39
(s, 3H).
response curve was then fitted using GraphPad Prism (GraphPad Software
Inc) normalized from 0 to 100 for each assay. In cases where the highest
concentration tested (i.e. 20
the IC50 was determined manually as greater than 20
l
M) did not result in greater than 50% activation,
M.
l
24. Konakanchi, D. P.; Subba R.P.; Ananthaneni, L.; Pilli, R.; Reddy, M. P.; Satya, B.;
Rao, A. K.; Chowdary, N.V. WO2009/098715 A2.
25. The regiochemistry of 9r was verified by 2D ROSY experiment recorded using
spectrometer Bruker DRX-600. NOE effects were observed between the protons
of the methoxy group on the 4-methoxyphenoxy moiety C (CH3-reg.C) and the
aromatic protons in ortho position to this group. The protons of the methoxy
group (CH3- reg.B) at position 6 of the pyridazin-3(2H)-one core B gave rise to
a single cross-peak with the aromatic proton (Ha) on carbon 2 of the 3-
methylphenyl group. A cross-peak was also observed between Ha and the
vicinal methyl group. No NOE effects were observed between CH3- reg.B and
the protons of the 4-methoxyphenoxy system.
23. The biological inhibition assay employed a chimeric cell line that forces the
receptor to use Gqi3; therefore the assay readout was calcium release. HEK
cells stably co-transfected with the human NPBWR1 and
HEK293T/Gqi3 cell line) were used for this study. Cells were plated at 10,000
cells/well of a 384 well plate in 25 L media and incubated overnight. Next,
Gaqi3 (hGPR7
l