SAR analysis of novel non-peptidic NPBWR1 (GPR7) antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.12.030

In this Letter we report on the advances in our NPBWR1 antagonist program aimed at optimizing the 5-chloro-2-(3,5-dimethylphenyl)-4-(4-methoxyphenoxy) pyridazin-3(2H)-one lead molecule previously obtained from a high-throughput screening (HTS)-derived hit. Synthesis and structure-activity relationships (SAR) studies around the 3,5-dimethylphenyl and 4-methoxyphenyl regions resulted in the identification of a novel series of non-peptidic submicromolar NPBWR1 antagonists based on a 5-chloro-4-(4-alkoxyphenoxy)-2-(benzyl)pyridazin-3(2H)- one chemotype. Amongst them, 5-chloro-2-(9H-fluoren-9-yl)-4-(4-methoxyphenoxy) pyridazin-3(2H)-one 9h (CYM50769) inhibited NPW activation of NPBWR1 with a submicromolar IC₅₀, and displayed high selectivity against a broad array of off-targets with pharmaceutical relevance. Our medicinal chemistry study provides innovative non-peptidic selective NPBWR1 antagonists that may enable to clarify the biological role and therapeutic utility of the target receptor in the regulation of feeding behavior, pain, stress, and neuroendocrine function.

Full text of DOI:10.1016/j.bmcl.2012.12.030

Bioorganic & Medicinal Chemistry Letters 23 (2013) 614–619
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
SAR analysis of novel non-peptidic NPBWR1 (GPR7) antagonists
Miguel Guerrero a, Mariangela Urbano a, Marie-Therese Schaeffer b,d, Steven Brown b,d, Hugh Rosen b,c,d
,
Edward Roberts a,b,
⇑
a Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
b Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
c Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
d The Scripps Research Institute Molecular Screening Center, 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:
In this Letter we report on the advances in our NPBWR1 antagonist program aimed at optimizing the
5-chloro-2-(3,5-dimethylphenyl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one lead molecule previously
obtained from a high-throughput screening (HTS)-derived hit. Synthesis and structure–activity relation-
ships (SAR) studies around the 3,5-dimethylphenyl and 4-methoxyphenyl regions resulted in the
Received 23 October 2012
Accepted 10 December 2012
Available online 20 December 2012
identification of
a novel series of non-peptidic submicromolar NPBWR1 antagonists based on a
Keywords:
NPBWR1 (GPR7) antagonists
Anorexia
Analgesia
Stress
5-chloro-4-(4-alkoxyphenoxy)-2-(benzyl)pyridazin-3(2H)-one chemotype. Amongst them, 5-chloro-
2-(9H-fluoren-9-yl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one 9h (CYM50769) inhibited NPW
activation of NPBWR1 with a submicromolar IC50, and displayed high selectivity against a broad array
of off-targets with pharmaceutical relevance. Our medicinal chemistry study provides innovative non-
peptidic selective NPBWR1 antagonists that may enable to clarify the biological role and therapeutic util-
ity of the target receptor in the regulation of feeding behavior, pain, stress, and neuroendocrine function.
Ó 2012 Elsevier Ltd. All rights reserved.
Neuropeptide W (NPW) and neuropeptide B (NPB) bind and
activate two G-protein coupled receptors (GPCRs), namely
NPBWR1 (GPR7) and NPBWR2 (GPR8).1 NPB mRNA is widely dis-
tributed throughout the mouse brain and is present in the hippo-
campus, hypothalamin nucleus, Edinger–Westphal nucleus (EW),
locus coerulius, inferior olive and lateral parabrancheal nucleus.1,2
The expression of NPW is more confined to EW, ventral tegmental
area (VTA), periaqueductal gray and dorsal raphe nucleus.3
Human NPBWR1 was mapped to chromosome 10q11.2-121.1
and NPBWR2 to chromosome 20q13.3. NPBWR1 and NPBWR2
share 64% sequence homology and decrease intracellular cAMP
by coupling to the Gi-class of GPCRs.1,4
Intrathecal (i.t.) administration of NPW produced analgesic ef-
fects in inflammatory pain in the mouse formalin test, but not
mechanical or thermal pain.9 Interestingly, samples taken from pa-
tients with inflammatory/immunomediated neuropathies showed
a higher expression of NPBWR1 restricted to myelin-forming
Schwan cells. Similarly, animal models of acute inflammatory
and trauma induced neuropathic pain exhibited an increased mye-
lin-associated expression of NPBWR1, suggesting a central role of
this receptor in analgesia.10
Remarkably, i.c.v. administration of NPW in the brain reduced
seizure activity in mice, suggesting that NPWR1 may be a suitable
target for epilepsy.11
In situ hybridization and tissue binding studies showed that
NPBWR1 mRNA is localized in the extended amygdala (central nu-
cleus of the amygdala, bed nucleus of the stria terminalis) and the
hypothalamus (suprachiasmatic nucleus, VTA).1,5
This neuropeptide system has been hypothesized to play an
important role in modulating feeding behavior.6,1 Intracerebroven-
tricular (i.c.v.) administration of NPW to fasting rats or free-feeding
rats before dark phase suppressed food intake and increased both
heat production and body temperature.7 Furthermore, NPBWR1
knockout mice developed adult-onset obesity.8
I.c.v. injection of NPB in male rats elevated prolactin and corti-
costerone, and decreased growth hormone levels in circulation,
suggesting that this peptide may play a physiological role in the
neuroendocrine response to stress via the activation of the hypo-
thalamus-pituitary-adrenal axis.12 NPBWR1 knockout mice
showed abnormality in the contextual fear condition test in addi-
tion to an increased autonomic and neuroendocrine response to
physical stress, suggesting that the impairment of NPBWR1 leads
to stress vulnerability.13 A recent study provided evidence that hu-
man genetic differences in NPBWR1 modulate emotional responses
to facial expressions, particularly towards angry facial expression;
it is speculated that this behavior may be linked to the role of
NPBWR1 in modulating fear.14
⇑
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.2012.12.030

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 23 (2013) 614–619
615
tribromide (BBr3) yielded the key intermediate 5. Alkylation of 5
with a series of electronically and structurally diverse alkyl halides
6a-6ag and 6aj–6al furnished the final compounds 7a–7ag and
7aj–7al (Scheme 1). Compound 7ag was reduced with lithium
borohydride (LiBH4) to give 7ah which was transformed to 7ai
using acetyl chloride (Scheme 2).
The cyclopentyl analog 7a was ꢀ14-fold less potent than 1.
Interestingly, the benzyl derivative 7b was only ꢀ3-fold less potent
than 1 indicating that the insertion of a methylene between the
aromatic nucleus and the pyridazinone is tolerated in region a of
the chemotype. The attachment of a methyl group in the ortho po-
sition of the phenyl ring (7c) led to twofold increase in potency
compared to the unsubstituted 7b, whereas a ꢀ2-fold decrease of
potency was observed for the meta- and para-methyl substituted
analogs (7d, 7e). Similarly, the 2-trifluoromethyl analog 7f was
twofold more potent than 7b, whereas a mild decrease of potency
was observed for the 3-trifluoromethyl 7g and 4-trifluoromethyl
7h analogs. Interestingly, an increase of potency versus the
mono-substituted counterparts was observed when a second
methyl group was added in the ortho, meta or para position of 7c,
7d, 7e. Indeed, the 2,6-dimethyl 7i and 3,5-dimethyl 7j analogs
were ꢀ1.5- and ꢀ3.4-fold more potent than 7c and 7d, respec-
tively, while the 3,4-dimethyl analog 7k was ꢀ2- and ꢀ3-fold more
potent than 7d and 7e, respectively. Furthermore, the 4-methoxy
7l, 4-phenyl 7m and 4-isopropyl 7n analogs were less potent than
7b by ꢀ5-, ꢀ12- and ꢀ13-fold, respectively, thus showing that
installing electron-donating, aromatic or alkylic groups in para po-
sition is detrimental for the potency. The potency was impacted to
a lesser extent when a chlorine was inserted in para position, with
7o being slightly more potent than 7b. Further exploring the ortho
position showed that increasing the size from a methyl (7c) to an
ethyl group (7p) led to a small increase in potency, while the iso-
propyl derivative 7q was slightly less potent than 7p. The 2-meth-
Figure 1. NPBWR1 lead molecule.
Recently, we reported the identification, design and structure–
activity relationships (SAR) of a novel non-peptidic NPBWR1
antagonist chemotype obtained from a high-throughput screening
(HTS) of the Molecular Libraries-Small Molecule Repository
(MLSMR) library. The 5-chloro-2-(3,5-dimethylphenyl)-4-(4-
methoxyphenoxy)pyridazin-3(2H)-one 1 (CYM50557) was identi-
fied as a lead molecule in our NPBWR1 antagonist program
(Fig. 1).15 Its sub-micromolar potency and excellent selectivity
against a panel of pharmacologically relevant off-target proteins
prompted us to further optimize 1 in an effort to develop an
in vivo pharmacological tool to better elucidate the biological role
of NPBWR1. In this letter we report on the expansion of the SAR
studies around the coil region a and the pendant 4-methoxyphenyl
moiety b (Fig. 1).
Our SAR studies on 1 started by exploring the region a. The syn-
thesis of 7a–7al is depicted in Schemes 1 and 2 and their biological
results are listed in Table 1.16 MOM-protection of N-2 followed by
substitution on position 4 of the pyridazinone using the 4-metoxy-
phenoxide furnished 4. MOM-deprotection of
4 using boron
Scheme 1. Synthesis of 7a–7ag, 7aj–7al.
Scheme 2. Synthesis of 7ah–7ai.

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M. Guerrero et al. / Bioorg. Med. Chem. Lett. 23 (2013) 614–619
Table 1
7w was ꢀ2-fold more potent than 7b and slightly less potent than
the pyrazole 7v. Remarkably, the naphtalen-2-yl analog 7x was
ꢀ4-fold less potent than 7c, while the naphtalen-1-yl 7y
(CYM50719) was ꢀ3-fold more potent than 7c. Based on these re-
sults we explored the SAR around the naphthalen-1-yl moiety.
Introducing an additional methylene spacer between the pyridaz-
inone and the naphthalenyl ring (7z) led to 30-fold loss of potency.
Installing a methyl in position 2 (7ab) led to a small decrease in po-
tency compared to 7y, and a 2- to 3-fold loss of potency was ob-
NPBWR1 antagonist activity of compounds 7a–7al (IC50
l
M)
R2
Compound
R1
IC50
3.70
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
7w
7x
Cyclopentyl
Phenyl
2-Methylphenyl
3-Methylphenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.78
0.37
1.40
1.70
0.37
1.80
1.00
0.24
0.41
0.61
3.80
9.40
4-Methylphenyl
2-Trifluoromethylphenyl
3-Trifluoromethylphenyl
4-Trifluoromethylphenyl
2,6-Dimethylphenyl
3,5-Dimethylphenyl
3,4-Dimethylphenyl
4-Methoxyphenyl
4-Phenylphenyl
4-Isopropylphenyl
4-Chlorophenyl
2-Ethylphenyl
2-Isopropylphenyl
2-Methoxyphenyl
2-(Methoxycarbonyl)phenyl
2-Cyanophenyl
(1,10-Biphenyl)-2-yl
2-(Pyrazol-1-yl)phenyl
Anthracen-9-yl
served for the 2-methoxy analog 7ac. The installation of
a
fluorine (7aa), methyl (7ae) and bromine (7ad) at position 4 led
to ꢀ3-, ꢀ3- and ꢀ14-fold loss in potency, respectively, confirming
that substitutions in this position are not tolerated. Interestingly,
the quinoline 7af was ꢀ24-fold less potent than the naphthalene
7y indicating that the basic atom in this position is detrimental
for the potency. Next, a series of analogs with disubstituted ben-
zylic position was explored keeping, first, the naphtalen-1-yl as
the constant moiety. Interestingly, the ethyl ester 7ag was ꢀ3-fold
less potent than the non-substituted 7y. Surprisingly, the acetate
7ai and the primary alcohol 7ah were ꢀ83- and ꢀ18-fold less po-
tent than 7y, respectively. Interestingly, the methyl 7aj and phenyl
7ak substituted analogs were ꢀ13- and ꢀ128-fold less potent than
7y. Additionally, the phenyl ketone 7al was slightly more potent
than the non-substituted 7b. This data showed that steric interac-
tions in this portion of the molecule are important for the potency,
and indicated only a minor decrease of the potency when the sec-
ond substituent contains a carbonyl group immediately attached to
the benzylic carbon (7ag, 7al). We speculated that the partial keto-
enol tautomerization could positively impact the potency by forc-
ing the benzylic substituents into a quasi-planar conformation.
Based on this working hypothesis, we synthesized planar or pla-
nar-like tricyclic structures (9a–9h). The synthesis of these deriva-
tives is depicted in Schemes 3 and 4. Furthermore, the biphenyl
system was opened and a carbonyl group was inserted to obtain
the quasi-planar ketone 9j and the amide 9k. Additionally, the
bicyclic amide 9i was studied. The synthesis of 9i–9k is depicted
in Scheme 5. Coupling of pyridazinone 5 with a series of tricyclic
systems 8a–8e using Ullman conditions led to the products 9a–
9e (Scheme 3). Alkylation of intermediate 5 with benzylchlorides
10a–10c using sodium hydride as the base led to the formation
10.0
0.59
0.26
0.64
0.67
1.20
1.40
0.54
0.22
0.32
1.50
0.14
4.20
0.36
0.22
0.35
2.00
0.37
3.40
0.45
2.50
11.60
1.90
18.00
0.58
Naphtalen-2-yl
Naphtalen-1-yl
7y
7z
Naphthalen-1-ylmethyl
4-Fluoronaphtalen-1-yl
2-Methylnaphtalen-1-yl
2-Methoxynaphtalen-1-yl
4-Bromonaphtalen-1-yl
4-Methylnaphtalen-1-yl
Quinolin-5-yl
Naphtalen-1-yl
Naphtalen-1-yl
Naphtalen-1-yl
Naphtalen-1-yl
7aa
7ab
7ac
7ad
7ae
7af
7ag
7ah
7ai
7aj
7ak
7al
CO2Et
CH2OH
CH2OAc
Me
Phenyl
COPh
Naphtalen-1-yl
Phenyl
aData are reported as mean of n = 3 determinations.
oxy analog 7r was nearly equipotent to the isopropyl analog 7q.
Interestingly, the methyl ester 7s and cyano 7t derivatives were
3- to 4-fold less potent than 7c. Notably, the biphenyl derivative
7u was only 1.5-fold less potent than 7c, while the pyrazole 7v
was slightly more potent than 7c and 7p. Taken together this data
suggests that both steric and electronic factors in the ortho position
modulate the potency. Next, we explored the installation of bicy-
clic and tricyclic aromatic systems in the region a. The anthracene
of 9f–9h. Alkylation of 5 with the
a-halo carbonyl 11, 12a and
12b using potassium carbonate as the base furnished 9i–9k. The
biological data of 9a–9k is reported in Table 2.16
Remarkably, the dibenzoxazepines 9a and 9b were slightly
more potent than the ketone 7at. Surprisingly, the dibenzothiaze-
pine 9c was >50-fold less potent than 9b. Interestingly the
Scheme 3. Synthesis of 9a–9e.

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 23 (2013) 614–619
617
Scheme 4. Synthesis of 9f–9h.
Scheme 5. Synthesis of 9i–9k.
nyl ketone 9j was nearly equipotent to the tricyclic system 9b and
slightly more potent than 7at. Interestingly, the biphenylamide 9k
was slightly more potent than the carba-analog 9j.
Table 2
NPBWR1 antagonist activity of compounds
9a–9k (IC50
lM)
Compound
IC50 (l
M)a
Next, we explored the region b while selecting three moieties
from the region a. The synthesis of 15a–15w is depicted in Scheme
9a
9b
9c
9d
9e
9f
9g
9h
9i
0.37
0.49
>25
0.53
2.20
3.10
18.00
0.12
0.78
0.43
0.28
16
6. The results are listed in Table 3. Alkylation of pyridazinone 2
with chlorides 6c, 6ac and 10c using potassium carbonate yielded
the intermediates 13a–13c. Substitution with a series of phenols in
position 4 of pyridazinones 13a–13c using sodium hydride as the
base yielded the final products 15c–15w and the intermediates
16a, 16b which were deprotected to the primary alcohols 15a,
15b using tetrabutyl ammonium fluoride (TBAF).
9j
9k
First, we attached solubility-enhancing groups in this portion of
the molecule. Interestingly the 2-methylbenzyl primary alcohol
15a was only ꢀ2-fold less potent than the 4-methoxy counterpart
7c, while the naphthalenyl alcohol 15b was ꢀ31-fold less potent
than 7y indicating that the 2-methylbenzyl and naphthalenyl
series differ in their SAR. We further explored the region b while
keeping the naphthalene in the region a. Adding a fluorine in posi-
tion 2 (15c) or 3 (15d) of the phenyl ring, decreased the potency by
ꢀ4- and ꢀ2-fold, respectively compared to 7y, indicating that
changing the dipolar moment in the phenyl ring influences nega-
tively the antagonist activity. Next, the SAR at position 4 was ex-
plored. The ethoxy analog 15e (CYM50775) was slightly more
potent than 7y. Conversely, the n-propoxy 15f and isopropoxy
15i were ꢀ8- and ꢀ4-fold less potent than 7y, respectively, sug-
gesting that steric factors modulate the potency. Remarkably, the
ethyl 15g was less than twofold less potent than 7y indicating that
the hydrogen bond acceptor capability in this position is not funda-
mental for the functional activity. Furthermore, the trifluorometh-
oxy 15h was ꢀ39-fold less potent than 7y, and a dramatic loss of
a
Data are reported as mean of n = 3
determinations.
benzopyridoxazepine 9d was less than twofold less potent than the
carba-analog 9a, showing that the insertion of a basic center in this
series was tolerated. When the oxygen from 9b was exchanged for
a carbonyl group (9e) the potency decreased by ꢀ4-fold. Removing
the imine from the tricyclic system led to the tricyclic compounds
9f–9h. Remarkably, the fluorene 9h (CYM50769) was slightly more
potent than the naphtalen-1-yl derivative 7y. Increasing the mid-
dle-ring size from 5 to 7 members (9g) led to 150-fold loss in po-
tency. The xanthene 9f was ꢀ26-fold less potent than 9h.
Interestingly, the elongated bicyclic analog 9i was only ꢀ6-fold less
potent than the fluorene 9h indicating that is possible to increase
the distance between the pyridazinone and the aromatic rings
without major decrement in the potency. Furthermore, the biphe-

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M. Guerrero et al. / Bioorg. Med. Chem. Lett. 23 (2013) 614–619
Scheme 6. Synthesis of 15a–15w.
Table 3
NPBWR1 antagonist activity of compounds 15a–15w
R3
Ar
IC50 (lM)
a
Compound
15a
15b
15c
15d
15e
15f
15g
15h
15i
2-Methylbenzyl
4-(Hydroxymethyl)phenyl
4-(Hydroxymethyl)phenyl
2-Flouro-4-methoxyphenyl
3-Fluoro-4-methoxyphenyl
4-Ethoxyphenyl
0.73
4.40
0.64
0.36
0.10
1.10
0.25
5.50
0.53
>20.00
0.43
0.61
0.98
8.50
0.29
1.80
0.82
0.43
0.83
0.25
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
Naphthalen-1-ylmethyl
9H-Fluoren-9-yl
4-Propoxyphenyl
4-Ethylphenyl
4-Trifluoromethoxyphenyl
4-Isopropoxyphenyl
4-(Methoxycarbonyl)phenyl
4-(Methylthio)phenyl
3-Methoxyphenyl
15j
15k
15l
15m
15n
15o
15p
15q
15r
15s
15t
3-Ethoxyphenyl
3-Ethylphenyl
3,4-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
Benzofuran-5-yl
2,3-Dihydrobenzofuran-5-yl
2,3-Dihydro-1H-inden-5-yl
4-Ethoxyphenyl
15u
15v
15w
9H-Fluoren-9-yl
9H-Fluoren-9-yl
9H-Fluoren-9-yl
4-Ethylphenyl
4-Trifluoromethoxyphenyl
4-Propoxyphenyl
0.24
>20.00
>20.00
a
Data are reported as mean of n = 3 determinations.
potency was observed for the methyl ester 15j, while changing the
oxygen of 7y for sulfur (15k) decreased the potency only by three-
fold. Next, we explored the position 3 of the phenyl ring. The meth-
oxy 15l, ethoxy 15m and ethyl 15n analogs were ꢀ4-, 10- and
34-fold less potent than 7y, 15e and 15g respectively. Remarkably,
the 3,4-dimethoxy 15o and 3,4,5-trimethoxy 15p were 2- and
ꢀ13-fold less potent than 7y suggesting that the negative effect
of polisubtitution on the aromatic ring is additive. Since the meth-
oxy on position 3 of 15o was tolerated, we speculated that bicyclic
systems may improve the potency. However, the benzofuran 15q
was ꢀ3-fold less potent than 15o, while the dihydrobenzofurane
15r was less than ꢀ2-fold less potent than 15o and ꢀ2-fold more

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 23 (2013) 614–619
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potent than the indene 15s. Different SAR was observed within the
fluoren-9-yl series. The ethoxy 15t and ethyl 15u analogs were
equipotent and twofold less potent than 9h, while a dramatic de-
crease in potency was observed for the 4-trifluoromethoxy 15v
and 4-propoxy 15w.
Amongst the synthesized compounds, 9h (CYM50769) was se-
lected for further characterization.17 The solubility of 9h in a phos-
phate buffered saline (PBS) at pH 7.4 is 0.17
lM. The compound is
non-cytotoxic to U2OS cells at 20 M and chemically stable in PBS
l
at pH 7.4 with half-life higher than 48 h. The selectivity profile was
investigated against the Ricerca panel of off-target proteins includ-
ing GPCRs, enzymes, transporters and ion channels at a concentra-
tion of 30 lM. Remarkably, out of 35 tested targets only CYP450
1A2, 5-HT2B and CYP450 2C19 showed 67%, 63% and 51% inhibi-
tion, respectively.
In summary, we have reported the synthesis and SAR studies
around the coil and 4-methoxyphenyl regions (a, b) of novel
non-peptidic NPBWR1 antagonists based on a 5-chloro-4-(4-alk-
oxyphenoxy)-2-(benzyl)pyridazin-3(2H)-one chemotype. Small
changes in region b had a negative impact on the potency, while
the region a was found to interact with a liphophilic pocket that
can accommodate a great variety of bulky quasi-planar substitu-
ents. Our studies resulted in the identification of a novel series of
submicromolar NPBWR1 antagonists including 7y (CYM50719),
9h (CYM50769) and 15e (CYM50775) endowed with greater po-
tency than our previously reported lead 1. Amongst them, 9h
was further profiled and found to be highly selective against a
broad array of off-targets with pharmaceutical relevance, making
this compound suitable for further development. Our medicinal
chemistry advances around this chemotype will be communicated
in due course.
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16. 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 Gqi3 (hGPR7
HEK293T/Gqi3 cell line) were used for this study. Cells were plated at 10,000
cells/well of a 384 well plate in 25
lL media and incubated overnight. Next,
25 L of Fluo8 NW (ABD Bioquest) was added to all wells and the assay plate
l
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
Acknowledgments
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
This work was supported by the National Institute of Health
Molecular Library Probe Production Center Grant U54 MH084512
(Peter Hodder, Hugh Rosen).
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–
response curve was then fitted using GraphPad Prism (GraphPad Software Inc)
normalized from to 100 for each assay. In cases where the highest
concentration tested (i.e. 20 M) did not result in greater than 50%
lM. For each test
A
a
References and notes
0
l
1. Tanaka, H.; Yoshida, T.; Miyamoto, N.; Motoike, T.; Kurosu, H.; Shibata, K.;
Yamanaka, A.; Williams, S. C.; Richardson, J. A.; Tsujino, N.; Garry, M. G.; Lerner,
activation, the IC50 was determined manually as greater than 20
17. http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1880.
lM.