Structure-activity relationship of imidazopyridinium analogues as antagonists of neuropeptide s receptor

Article abstract of DOI:10.1021/jm400904m

The discovery and characterization of a novel chemical series of phosphorothioyl-containing imidazopyridines as potent neuropeptide S receptor antagonists is presented. The synthesis of analogues and their structure-activity relationship with respect to the Gq, Gs, and ERK pathways is detailed. The pharmacokinetics and in vivo efficacy of a potent analogue in a food intake rodent model are also included, underscoring its potential therapeutic value for the treatment of sleep, anxiety, and addiction disorders. This article not subject to U.S. Copyright. Published 2013 by the American Chemical Society.

Full text of DOI:10.1021/jm400904m

Article
pubs.acs.org/jmc
Structure−Activity Relationship of Imidazopyridinium Analogues as
Antagonists of Neuropeptide S Receptor
Samarjit Patnaik,† Juan J. Marugan,*,† Ke Liu,† Wei Zheng,† Noel Southall,† Seameen J. Dehdashti,†
Annika Thorsell,‡ Markus Heilig,‡ Lauren Bell,‡ Michelle Zook,‡ Bob Eskay,‡ Kyle R. Brimacombe,†
and Christopher P. Austin†
†National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville,
Maryland 20850, United States.
‡National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: The discovery and characterization of a novel chemical series of
phosphorothioyl-containing imidazopyridines as potent neuropeptide S receptor
antagonists is presented. The synthesis of analogues and their structure−activity
relationship with respect to the Gq, Gs, and ERK pathways is detailed. The
pharmacokinetics and in vivo efficacy of a potent analogue in a food intake rodent
model are also included, underscoring its potential therapeutic value for the
treatment of sleep, anxiety, and addiction disorders.
Leading in vivo studies published by Xu et al.3 showed an
increase in locomotor activity and reduction of all stages of
INTRODUCTION
■
Neuropeptides are short chains of amino acids or small proteins
sleep upon supraspinal administration of NPS in mice.
that play a crucial role in controlling intracellular neuronal
Paradoxically, they also demonstrated a reduction in anxiety
signaling. They bind to cell-surface receptors and elicit a variety
of physiological responses.1 The 21-amino acid neuropeptide S
(NPS), named “S” because of a conserved N-terminus serine
residue found across vertebrate species, has received consid-
erable attention in the recent past. The cognate receptor for
in four different validated mouse models of anxiety, earning
NPS the unique distinction of being a novel activating
anxiolytic.7 Experiments with Long−Evans rats also showed
that administration of NPS in the lateral brain ventricle leads to
anorexia-like symptoms with suppression of food intake.8
NPS, neuropeptide S receptor (NPSR), was first described by
Ciccocioppo and co-workers reported that administration of
Sato in 2002, but the endogenous peptide was left
uncharacterized.2 The Reinscheid group deorphanized the
NPS in the lateral hypothalamus of the rat brain resulted in a
significant increase in cue-induced alcohol-seeking behavior in
receptor and also identified regions of the brain where the
an alcohol relapse rat model.9 This effect was reduced by the
selective oxerin receptor antagonist SB-334867,10 indicating the
presence of a strong link between the hypocretin1/orexinA and
NPS signaling pathways. Similar in vivo work has also
implicated this circuitry’s relevance toward addiction to cocaine
and morphine.11 The types of physiological changes observed
in various rodent models after central administration of NPS
have been summarized in several reviews.12
peptide and its receptor are expressed.3 They also detailed the
pharmacology of the NPS/NPSR system using Chinese
hamster ovary (CHO) and human embryonic kidney 293
(HEK 293) cells stably expressing human and murine variants
of NPSR. NPS acted as an agonist in these cell lines, causing a
dose-dependent increase in the levels of intracellular calcium,
cyclic adenosine monophosphate (cAMP), and p42/p44
mitogen-activated protein kinase (MAPK) phosphorylation.
This suggested that NPSR is coupled to the Gq and Gs proteins
and the MAPK pathway, respectively. Furthermore, they
synthesized the human [125I]Tyr10-hNPS radiolabeled peptide
and showed it to be an agonist that is equipotent to the natural
ligand NPS and can be used in radiolabeled ligand displacement
assays.4 The pharmacological characterization was extended to
an Asn107Ile mutant and a C-terminal splice variant that were
linked to asthma.5,6
Systematic exploration of the amino acids along the
SFRNGVGTGMKKTSFQRAKS peptide sequence of NPS
has not only identified key residues essential for agonist
activity but also led to the discovery of novel peptide
antagonists.13 Recently several small-molecule antagonists of
Received: June 19, 2013
This article not subject to U.S. Copyright.
Published XXXX by the American Chemical
Society
A
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a
b
Figure 1. Potent small-molecule antagonists of the NPSR pathway. Activity described in the literature. Activity determined in house.
a
Scheme 1. Synthetic Scheme for the Screening Hit and Analogues
a
Reagents and conditions. (a) EtOH, CHCCH2Br, reflux, 47% yield; (b) Pd(PPh3)4, CuI, DMF, rt, 72% yield; (c) TMSI, toluene; (d) py, Et3N,
PPh2I; (e) 30% H2O2, 30−50% yield over two steps; (f) sulfur, 30−50% yield over two steps; (g) Me2SO4, 1,4-dioxane, sealed tube, 100−120 °C,
30−50% yield.
the NPS/NPSR pathway have also been reported in the
literature. This has been a promising development, as these
molecules serve not only as vital tools for understanding
pharmacology but also hold potential for drug development
toward anxiety, food, and addiction disorders. In this regard,
two molecules disclosed by Takeda Pharmaceuticals,14 SHA66
B
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a
Scheme 2. Synthetic Scheme toward Tertiary Alcohols 19a and 19b
a
Reagents and conditions. (a) NBS; (b) iPrMgCl·LiCl, THF, Ph2CO, 18% yield for 18a and 31% yield for 18b; (c) Me2SO4, 1,4-dioxane, sealed
tube, 100−120 °C, 50−60% yield.
Table 1. SAR of Initial Analogues: The cAMP and Ca2+ Assay Activities Are Mean IC50 Values with Standard Deviations (SDs)
from Two Separate Experiments, Each Run in Duplicate; The ERK Assay Activities Are Mean IC50 Values with SDs from One
Experiment Run in Duplicate
a
b
“Inact” = inactive. This compound showed partial efficacy in its antagonistic behavior. This compound showed <50% inhibition at the highest
c
concentration (50 μM) tested. Both IC50 values are reported here, as we consider the standard deviation between the replicates to be significant.
(1a) and SHA68 (1b) (Figure 1), have been characterized
extensively as potent inhibitors of the NPS/NPSR signaling
pathway. A 50 mg per kilogram (mpk) intraperitoneal (IP)
dose of 1b was also able to reduce NPS-induced hyper-
locomotion by about 50% in a 90 min mouse model.15
Reported pharmacokinetic (PK) studies showed brain levels of
∼6 μM at 15 min and ∼2 μM at 2 h, which are well beyond its
in vitro half-maximal inhibitory concentration (IC50). More-
over, the bioactive enantiomer of 1b has recently been
identified.16 Merck has disclosed potent inhibitors from two
separate chemical series (2 and 3).17,18 The best compound
from a series of quinolinones, 2, demonstrated high ex vivo
occupancy of NPSR in discrete regions of the rat brain after a
30 mpk IP dose. GSK and Actelion Pharmaceutical Ltd. have
also reported novel NPSR antagonists around the scaffolds
represented by pyrroloimidazolone (4) and indanone (5),
respectively.19,20 Our efforts within the NIH have resulted in
the disclosure of a naphthapyranopyrimidine probe (6), where
C
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Figure 2. Overlap model of 1b and screening hit 7 (green).
structural changes in the same chemical series led to the
discovery of selective modulators of either the Gq or Gs
pathway.21 Dal Ben has summarized the structure−activity
relationship (SAR) around these small NPSR antagonists in a
recent review.22 Herein we disclose the discovery of another
structurally uncommon chemotype that after medicinal
chemistry optimization produced a compound with potent
antagonism of the NPS/NPSR signaling pathway.
addition, we also analyzed the inhibition of ERK activation, as
the NPSR is also coupled to the MAPK pathway. To that end,
the synthetic route shown in Scheme 1 was developed for our
SAR exploration. While the desmethyl compound 10a was
commercially available, 2-methylimidazo[1,2-a]pyridine (10b)
could be accessed via a known palladium-catalyzed internal
cyclization of the amino group on the pendant alkyne in 2-
amino-1-(propargyl)pyridinium bromide (9).25 The Tolmachev
protocol allowed for the electrophilic aromatic substitution at
the 3-position for the introduction of the diphenylphosphino
group.26 While 10a could be reacted with chlorodiphenylphos-
phine, the 2-methyl counterpart 10b required the use of the
more reactive iododiphenylphosphine, which was generated
from chlorodiphenylphosphine and trimethylsilyl iodide.27
Treatment of crude phosphines 11a and 11b with either
elemental sulfur or hydrogen peroxide provided the diphenyl-
phosphorothioyl species 12b and 12d or the diphenylphos-
phoryl species 12a and 12c, respectively. A final alkylation of
12b and 12d with dimethyl sulfate led to imidazopyridinium
salts 13a and 13b, respectively.28 These imidazopyridinium
salts could be purified by normal or reversed-phase chromato-
graphic techniques, and the nature of the counteranion proved
to be inconsequential toward the bioactivity. The generation of
the indole variants 15a and 15b were carried out in similar
fashion from commercially available 14a and 14b, respec-
tively.29
We were also interested in evaluating a tertiary diphenyl
alcohol group as a possible bioisosteric replacement for the
phosphorothioyl group. To that end, the 3-bromoimidazopyr-
idines 17a and 17b were generated from 16a and 16b,
respectively, with N-bromosuccinimide (NBS) (Scheme 2).
Subsequent halogen−metal exchange using the chemistry
described by Krasovskiy and Knochel30 was followed by
addition of benzophenone to generate the corresponding
tertiary alcohols 18a and 18b. These alcohols could be
chemoselectively alkylated at N1 in dioxane to yield analogues
19a and 19b, respectively.
RESULTS AND DISCUSSION
■
Our efforts to discover novel antagonists for the NPS pathway
started with the development of a high-throughput homoge-
neous time-resolved fluorescence (HTRF) assay that could
measure the formation of cAMP upon binding of NPS to NPSR
using CHO cells stably expressing NPSR. This cAMP assay was
used to screen 222 256 compounds in the quantitative high-
throughput screening (qHTS) format.23 This effort led to the
discovery of the inhibitor 3-(diphenylphosphorothioyl)-1,2-
dimethylimidazo[1,2-a]pyridin-1-ium iodide (7) (Figure 1).24
The antagonistic activity of this compound was further
confirmed in orthogonal assays measuring the reduction of
calcium mobilization in the same cell line and in a radiolabeled
NPS displacement assay. The unusual functionality within the
molecule prompted us to evaluate its stability in various
aqueous buffers. We found that 7 did not decompose into any
other species after exposure to Hank’s balanced buffer solution
(HBBS) or phosphate-buffered saline (PBS) over a period of
48 h. This confirmed its robust chemical stability and spurred a
medicinal chemistry effort aimed at examining the key
structural elements that are necessary for the antagonistic
activity. In the previous chemotype reported by us (6),20 we
observed a structure-related differential in the antagonist
activities toward the Gs and Gq pathways within the same
NPS antagonistic series.20 At the present moment, it is not clear
which signaling pathways are linked to different biological
responses observed by NPSR activation. Therefore, we decided
to examine and report here the inhibition of both the Gs and
Gq pathways with the cAMP and Ca2+ assays, respectively. In
D
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As an initial confirmation of our HTS data, we obtained
IC50’s similar to our previous values in both assays for our
resynthesized original hit compound 7. In addition, the activity
data in Table 1 indicate the sensitivity of the 2-methylimida-
zopyridine core and the phosphorothioyl moieties toward
structural change. Thus, elimination of the 2-methyl substituent
reduced the activity 3−25-fold (e.g., 12b vs 12d and 13a vs
13b) in the cAMP assay. Replacement of the sulfur atom in the
thiophosphoryl functional group by an oxygen (12c vs 12d) or
its elimination (11a) abolished the antagonist activity. Similarly,
diphenyl alcohols 18a and 18b and their corresponding N1-
methylated analogues 19a and 19b were inactive. The indole
Replacement of the imidazopyridinium core with indole in 15a
and 15b also proved deleterious toward the activity. In general,
the active compounds appeared to have enhanced potencies in
the Ca2+ and ERK assays.31
Table 2. SAR of Analogues at Position 1: The cAMP and
Ca2+ Assay Activities Are Mean IC50 Values with SDs from
Two Separate Experiments, Each Run in Duplicate; The
ERK Assay Activities Are Mean IC50 Values with SDs from
One Experiment Run in Duplicate
The activity results of the first set of synthetic compounds
(Table 1) convinced us to preserve the 2-methylimidazopyr-
idinium core and the thiophosphorus moiety in our subsequent
analogue design. The striking structural similarity between the
diphenylphospho group in our screening hit and the geminal
diphenyl group in 1a and 1b prompted us to consider simple
overlap models between these compounds (Figure 2).
Moreover, in-house [125I]Tyr10-NPS displacement experiments
had disclosed that both chemical series are able to compete
with the natural ligand, and therefore, we hypothesized that
both orthosteric antagonists would bind to the same region,
probably with similar three-dimensional geometry. When we
overlapped the geminal diphenyl substituents, we realized that
the oxazolidinone dipole aligned well with the phosphorothioyl
moiety. The piperidine in 1b also overlapped reasonably with
the imidazole of the imidazopyridine. However, it seemed that
we were probably missing a hydrophobic phenyl ring at the
lower portion of our compound which may have been the
critical element in the higher potency of 1a and 1b. Fortunately,
we were able to test this hypothesis by utilizing the chemistry
that had been developed to resynthesize screening hit 7. To
that end, 12d would be an ideal intermediate for alkylation at
N1 with various commercially available bromides that would
place a phenyl ring different numbers of spacer units away from
the core of the molecule (Scheme 3).
a
The two replicate IC50 values are reported here, one (<0.5 nM) being
b
less than could be detemined accurately by the assay. Both IC50
values are reported here, as we consider the standard deviation
between the replicates to be significant.
the more constrained propenylbenzene appendage, which
recorded a benchmark potency of 45 nM in the cAMP assay.
Spacing the phenyl ring by four (20f) or five (20g) methylene
units led to erosion of the potent activity achieved with three
spacer units. Each analogue in Table 2 showed at least a 25-fold
drop in its potency in the cAMP assay compared with its
individual potency in the calcium or ERK assay. At this
juncture, we also evaluated additional substitution in the phenyl
ring when it was optimally separated from N1 of the
imidazopyridine core by a propenyl group. Table 3 displays
the activities of such compounds in the context of the Gs and
Gq pathways. Although many types of substitutions were
tolerated, unsubstituted 20e remained the most potent
compound. It appeared that meta substitution was slightly
less tolerated than para substitution, as exemplified by
compounds 21g and 21c versus 21f and 21d, respectively.
The weakest inhibitor in this series was the 2-naphthyl-
substituted compound 21i, indicating a size restriction that
might be associated with the hydrophobic region that
accommodates the phenyl group in 20e.
Scheme 3. Synthetic Route to Analogues with Chemical
Diversity at Position 1
While we had discovered potent antagonists of the NPS/
NPSR signaling pathway, it was crucial to compare the activities
of this structural class with those of the best-in-class small-
molecule inhibitors in the literature. We did this in the context
of our functional assays as well the radiolabeled binding assay
(Table 4). We observed that 20e was 5-fold more potent than
1b in the calcium and displacement assays and 9-fold more
potent in the cAMP assay. On the other hand, the potencies in
these assays were comparable to those of the active enantiomer
of 2 reported by Merck. Comopound 20e appeared to be a
more potent inhibitor of ERK phosphorylation. We also
decided to compare the intrinsic stabilities of these key NPSR
Table 2 summarizes the activities of the analogues generated
to test our hypothesis. Compound 20a with a phenyl ring one
methylene unit away from N1 had similar potency as the
original hit 7. This also held true for 20b and 20c with ethylene
and ethanone spacer units, respectively, between the phenyl
ring and N1. Remarkably, compound 20d with the phenyl-
propyl chain recorded substantial improvements in potency
(IC50 = 110, 1, and 2 nM in the cAMP, calcium, and ERK
assays, respectively). The same trend was observed for 20e with
E
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(tBU)5]NPS was able to reverse the anorectic effect of NPS.32
While we predicted a reasonable exposure in plasma, we were
unsure of the blood−brain barrier penetration of 20e. Hence, at
the outset, we decided to dose 20e by the intracerebroven-
tricular (ICV) route as well. To that end, Wistar rats were
habituated to palatable food consumption and then treated with
ICV injections of NPS, which led to a marked reduction in food
intake measured at 15, 30, and 60 min. Gratifyingly, a single 10
μg dose of 20e was able to reverse the suppression of food
intake induced by NPS at all three time points (Figure 3).
The success in the food intake in vivo model experiment led
to further characterization of 20e. While ICV administration of
the drug was a robust way to check for proof of principle, the
evaluation of pharmacokinetics after IP administration of the
drug was a more suitable path forward toward more long-term
in vivo studies. Table 5 enumerates the exposure levels that
were achieved after a 10 mpk dose in mice. In plasma, a Cmax of
1.5 μM was reached 15 min post dose, and the concentrations
steadily declined with a half-life of 8.8 h to about 54 nM at 24 h.
More importantly, the drug crossed the blood−brain barrier,
and drug levels (52 nM) that were above the in vitro IC50
values in all three functional assays were observed even at 24 h.
No adverse reactions were observed in this single-dose study.
This bodes well for further in vivo characterization of this
chemical series in other disease models. While we are aware
that the total drug concentration in the brain may not be a
good indicator of a pharmacodynamic effect,33 detailed in vivo
studies of 20e in rat alcohol models showed efficacy with an IP
dose of 1.0 mpk.34
Table 3. SAR of the Substituted Phenyl Ring: The cAMP and
Ca2+ Assay Activities Are Mean IC50 Values with SDs from
Two Separate Experiments, Each Run in Duplicate
Compound 20e was also profiled at 10 μM against 55 targets.
We observed >90% inhibition of control in seven targets, which
were followed with IC50 determination studies (Table 1 in the
Supporting Information). We were particularly concerned with
the activity in the μ-agonist displacement assay. We decided to
compare this activity with those for compounds having potent
affinity toward the μ-opioid receptor (Figure 4). Thus, in an in-
house assay to monitor the displacement of radiolabeled
DAMGO, a peptide with potent affinity for the μ-opioid
receptor, we observed that 20e was more than 200-fold less
active than naloxone or morphine. With an IC50 of 1 nM in the
[
125I]NPS displacement assay, it appears that a reasonable
therapeutic window exists between the probe’s affinities toward
NPSR and the μ-opioid receptor.
CONCLUSIONS
■
The qHTS paradigm at NCGC was used to identify a
structurally novel small molecule as an antagonist to the
NPS−NPSR neurocircuitry. Further medicinal chemistry
revealed the uniqueness of this chemotype toward receptor
binding and showed that even slight modifications to the
structure resulted in dramatic loss of activity. Simple overlap
models with a previously disclosed inhibitor prompted an SAR
study that led to the synthesis of the potent analogues 20d and
20e. The latter was characterized as a potent antagonist in
functional and binding assays. Administration of this compound
by the ICV route completely reversed NPS-induced suppres-
sion of palatable food intake in rats. In vitro stability studies in
mouse liver microsomes showed that this compound has a
lower rate of metabolism compared with the most characterized
compounds in the literature. This translated to a 10 mpk IP
dose in mice, which proved to be safe and maintained high
levels of drug in the plasma with slow elimination. This
experiment also demonstrated that the drug was able to cross
a
Both IC50 values are reported here, as we consider the standard
deviation between the replicates to be significant.
antagonists, and to that end, we analyzed the percentages of
parent compound remaining at 15, 30, and 60 min after
incubation in mouse liver microsomes. We were pleased to
learn that 20e had the lowest rate of intrinsic clearance, which
boded well for prospective in vivo studies and was a clear
advantage over 1b and 2.
The discovery of the potent NPSR antagonist 20e warranted
an in vivo evaluation of its activity. As a preliminary study, we
chose to examine the efficacy of this compound in a rat food
intake model where the NPSR peptide antagonist [D-Cys-
F
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Table 4. Comparison of Key Potent NPSR Antagonists: The Reported cAMP and Ca2+ Assay Activities Are Mean IC50 Values
from Two Separate Experiments, Each Run in Duplicate; The Reported ERK, [125I]Tyr10-hNPS Displacement, and Microsome
Assay Results Are Mean Values from Respective Single Experiments Run in Duplicate
a
% remaining
IC50 (nM)
ERK
with NADPH
without NADPH
#
Ca2+
cAMP
[
125I]Tyr10-hNPS
0
15
30
60
0
15
30
60
1b
2
5.3
420
55
14
11
1.3
25
100
100
100
29
0
9
0
2
0
98
96
94
105
103
102
96
97
101
94
1.2
6.7
3.5
20e
0.96
45
72
46
26
102
102
a
Percentage of parent compound remaining after incubation in mouse liver microsomes for the indicated times in minutes.
Table 5. Mouse Pharmacokinetics of 20e35 (The
Concentration at Each Time Point Presented Here Is the
Mean Derived from N = 3)
10 mpk IP
plasma
SD
brain
SD
sampling
mean
mean
mean
mean
a
a
time (h) (ng/mL) (ng/mL) (nM) (ng/g)
(ng/g) (nmol/g)
0
BQL
N/A
306
BQL
1503
1325
1150
556
386
245
214
126
54
BQL
14
24
25
21
27
24
28
43
24
N/A
2.41
8.16
3.57
5.14
6.95
3.31
8.06
17.8
2.45
BQL
30
52
53
44
57
53
60
93
52
0.083
0.25
0.5
1
700
617
111
536
235
259
5.03
7.64
18.3
34.6
9.66
4.33
2
180
4
114
8
99
12
24
58
25
Figure 3. Reversal of NPS-induced suppression of palatable food
intake. Three groups of rats (each with N = 7) were treated with two
successive (10 min apart) 10 μL ICV injections of (a) vehicle (10 μL
of 2% DMSO in distilled water) followed by vehicle (veh/veh); (b)
vehicle followed by 10 μg of NPS in vehicle (veh/NPS); and (c) 10 μg
of 20e in vehicle followed by 10 μg of NPS in vehicle (antagonist/
NPS). Following the second injection, animals were placed back into
their cages. Palatable food (17% H2O, 33% sweetened condensed milk,
51% rat chow) was introduced 20 min thereafter, and intake was
measured at the indicated time points. #, p < 0.5 veh/NPS group vs
veh/veh group; *, p < 0.05 antagonist/NPS group vs veh/NPS group.
PK parameters
plasma
brain
tmax (h)
0.083
700
12
Cmax (ng/mL)
t1/2 (h)
43
8.8
N/A
746
N/A
AUClast (h ng/mL)
AUCinf (h ng/mL)
2240
2560
33
AUCbrain/AUCplasma (%)
a
Mean from N = 3
the blood−brain barrier and maintain concentrations higher
than the in vitro functional assay IC50 for 24 h in the brain.
Thus, in spite of its unusual structure, we have described the
progression of these phosphorothioyl-containing imidazopyr-
idinium compounds to physiologically relevant antagonists of
the NPS/NPSR pathway. Recently, we completed an evaluation
of 20e in animal models of alcoholism, where antagonism of
NPSR is predicted to have an effect.32 The results of these
studies showed a suppression of alcohol self-administration in
rats with a dose as low as 1 mpk. We also showed that at the
same dose, 20e is able to reduce alcohol-induced ERK
phosphorylation in the central amygdala of these rats.
500 μM Ro 20-1724, EC80 of NPS) was added to each well, cells were
incubated at 37 °C, 5% CO2 for 30 min. D2-conjugated cAMP (1.25
μL) and cryptate-conjugated anti-cAMP antibody (1 μf) were then
added. D2-conjugated cAMP and cryptate-conjugated anti-cAMP
antibody were both prepared in cell lysis buffer according to the
manufacturer’s instructions. After 30 min, the plates were then read
with a Viewlux plate reader (PerkinElmer) using the TRF detection
mode optimized for HTRF. This assay was done routinely as
compounds were generated. The reported data are mean IC50 values
with SDs from two independent experiments, each run in duplicate.
ERK Assay. CHO-NPSR cells were cultured using F12K medium
(ATCC) supplemented with 10% FBS and 250 μg/mL hygromycin b
EXPERIMENTAL SECTION
■
cAMP Assay. A Chinese hamster ovary cell line stably expressing
the NPSR was generated in the Heilig lab. The cells were maintained
in F12 medium containing 10% FBS, 100 units/mL penicillin, 100 μg/
mL streptomycin, and 200 μg/mL geneticin at 37 °C, 5% CO2.
Suspended CHO-NPSR cells were seeded into 1536-well tissue-
culture-treated white plates at a density of 1800 cells/well in 4 μL of
medium without geneticin and incubated at 37 °C, 5% CO2 overnight.
After 1 μL of stimulation buffer (1× PBS, 0.1% BSA, 0.05% Tween-20,
G
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compounds or in DMEM containing 0.1% BSA at 20 °C for 1.5 h.
Increasing concentrations of compounds or unlabeled human NPS
were used to compete with 0.15 nM [125I]Y10-NPS. Nonspecific
binding was determined in the presence of 1 μM unlabeled human
NPS. Cells were washed twice with cold PBS and lysed with 1 N
NaOH. Bound radioactivity was counted in a liquid scintillation
counter. The reported activities are mean IC50 values with SDs from
one experiment run in duplicate.
General Methods. Unless otherwise stated, reactions were carried
out under an atmosphere of dry argon or nitrogen in dried glassware.
The indicated reaction temperatures refer to the reaction bath, while
room temperature (rt) is noted as ∼25 °C. All anhydrous solvents,
commercially available starting materials, and reagents were purchased
from Aldrich Chemical Co. and used as received. Analytical thin-layer
chromatography (TLC) was performed with Sigma-Aldrich TLC
plates (5 cm × 20 cm, 60 Å, 250 μm). Visualization was accomplished
by irradiation under a 254 nm UV lamp. Chromatography on silica gel
was performed using forced flow (liquid) of the indicated solvent
system on Biotage KP-Sil prepacked cartridges and a Biotage SP-1
automated chromatography system. Reversed-phase preparative
purification was performed on a Waters semipreparative HPLC
instrument. The column used was a Phenomenex Luna C18 (5 μm, 30
mm × 75 mm) at a flow rate of 45 mL/min. The mobile phase
consisted of acetonitrile and water (each containing 0.1% trifluoro-
acetic acid). A gradient of 10% to 50% acetonitrile over 8 min was used
during the purification. Fraction collection was triggered by UV
detection (220 nM). 1H spectra were recorded on a Varian Inova 400
MHz spectrometer. Chemical shifts are reported in parts per million
with the solvent resonance as the internal standard (CDCl3 7.27 ppm
and DMSO-d6 2.50 ppm for 1H). Data are reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, br s = broad singlet, m = multiplet), coupling constant(s),
number of protons.
The reported analytical purity analyses and retention times were
obtained by the following two methods on an Agilent LC/MS (Agilent
Technologies, Santa Clara, CA): Method 1: A 7 min gradient of 4% to
100% acetonitrile (containing 0.025% trifluoroacetic acid) in water
(containing 0.05% trifluoroacetic acid) was used with an 8 min run
time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3
μm, 3 mm × 75 mm) was used at a temperature of 50 °C. Method 2:
A 3 min gradient of 4% to 100% acetonitrile (containing 0.025%
trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid)
was used with a 4.5 min run time at a flow rate of 1 mL/min. A
Phenomenex Luna C18 column (3 μm, 3 mm × 100 mm) was used at
a temperature of 50 °C. Unless otherwise stated, analogues were
determined to have >95% purity using the above methods.
Mass determinations were performed using an Agilent 6130 mass
spectrometer with electrospray ionization in the positive mode.
Molecular weights were confirmed using an Agilent time-of-flight
(TOF) mass spectrometer. A 3 min gradient from 4% to 100%
acetonitrile (0.1% formic acid) in water (0.1% formic acid) was used
with a 4 min run time at a flow rate of 1 mL/min. A Zorbax SB-C18
column (3.5 μm, 2.1 mm × 30 mm) was used at a temperature of 50
°C. Molecular formulas were confirmed using electrospray ionization
in the positive mode with the Agilent Masshunter software (version
B.02).
2-Amino-1-(prop-2-yn-1-yl)pyridin-1-ium Bromide (9). Pyr-
idin-2-amine (9.5 g, 0.10 mol) and 3-bromoprop-1-yne (10 mL, 0.10
mol) were added to ethanol (50 mL) and refluxed for 2 h. The
reaction was left to cool overnight. A light-yellow solid precipitated out
of the reaction mixture. This was filtered via a Buchner funnel, washed
with cold ethanol, and air-dried to obtain 2-amino-1-(prop-2-
ynyl)pyridinium bromide (10 g, 47 mmol, 47% yield). 1H NMR
(400 MHz, DMSO-d6): δ 3.82 (t, J = 2.5 Hz, 1H), 5.08 (d, J = 2.5 Hz,
2H), 6.95 (td, J = 6.9, 1.4 Hz, 1H), 7.11 (m, 1H), 7.91 (ddd, J = 8.9,
7.1, 1.7 Hz, 1H), 8.17 (m, 1H), 8.63 (br s, 2H).
Figure 4. μ-Opioid receptor affinity of 20e compared with those of
naloxone and morphine in the human recombinant μ-opioid receptor
OPRM1 stable cell line: (blue curve) morphine, IC50 = 2.5 0.05 nM;
(red curve) naloxone, IC50 = 1.87 0.07 nM; (green curve) 20e, IC50
= 580 0.11 nM.
(Life Technologies) selection antibiotic. Cells were harvested in Opti-
MEM (ATCC) supplemented with 1% FBS and plated in Greiner 96-
well white tissue-culture-treated clear-bottom plates at a seeding
density of 25 000 cells/well and grown overnight at 37 °C, 5% CO2.
The medium in the wells was replaced with 100 μL of Opti-MEM
only, and the cells were incubated at 37 °C, 5% CO2 for an additional
4 h. A 1 μL aliquot of compound (at the appropriate dilution in
DMSO) was added to each well and incubated for 10 min. A 100 μL
aliquot of 1 nM NPS final concentration (EC80) was added to every
well prepared in Opti-MEM supplemented with 0.2% BSA and 0.005%
Tween-20 (Sigma). After incubation for 20 min, all of the medium was
removed, and the plate was placed on ice for 5 min. The Cellul’erk kit
(Cisbio) instructions were then followed. Briefly, 50 μL of 1× lysis
solution was added per well and incubated at room temperature with
gentle rocking for 15 min. Lysed cells (16 μL) were transferred to
Greiner white medium binding half-well plates. A 1× solution of
deuterium-conjugated anti-ERK1/2 antibody (2 μL per well) was
added and incubated for 2 h at room temperature in the dark. A 1×
solution of europium cryptate-conjugated anti−ERK1/2 antibody (2
μL per well) was added and incubated overnight at ambient
temperature in the dark. The plate was read using HTRF settings
on an EnVision reader (PerkinElmer). The reported ERK assay
activities are mean IC50 values with SDs from one experiment run in
duplicate.
Calcium Assay. The CHO-NPSR cell line used in the cAMP assay
was also used in this calcium mobilization assay. The suspended cells
were plated at 3 μL/well with 2000 cells in black tissue-culture-treated
clear-bottom 1536-well plates. After overnight incubation at 37 °C, 5%
CO2, 3 μL of the calcium dye (no-wash High Performance PBX
Calcium Assay Kit, BD Biosciences) was loaded into each well, and the
plates were incubated at 37 °C, 5% CO2 for 1 h followed by incubation
for 10 min with 23 nL of compound prepared in DMSO solution. The
assay plates were then placed onto the FDSS-7000 kinetic fluorescence
plate reader for measurement of the changes in intracellular free
calcium. The basal fluorescence signal was recorded for 6 s at 1 Hz
followed by the addition of 1 μL of NPS stimulation buffer (1× PBS,
0.1% BSA, 0.05% Tween-20, 500 μM Ro 20-1724, EC80 of NPS) and
continuous recording at 1 Hz for 4 min. This assay was done routinely
as compounds were generated. The reported data are mean IC50 values
with SDs from two independent experiments, each run in duplicate.
[
125I]Tyr10-hNPS Binding Assay. The assay was carried out as
described earlier with minor modification.3 Y10-NPS labeled with 125I
was bought from NEN PerkinElmer (Boston, MA). CHO cells stably
expressing human NPSR were seeded into 24-well plates and cultured
until they reached 90−95% confluency. The cells were washed once
with 1 mL of PBS and then incubated with radioligand with or without
2-Methylimidazo[1,2-a]pyridine (10b). 2-Amino-1-(prop-2-yn-
1-yl)pyridinium bromide 9 (6.0 g, 28 mmol) was mixed with
copper(I) iodide (0.537 g, 2.82 mmol) and PdCl2(PPh3)2 (0.495 g,
0.705 mmol) in DMF (50 mL). The mixture was treated with
H
dx.doi.org/10.1021/jm400904m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
triethylamine (12 mL, 87 mmol) and stirred overnight, during which
the light-orange solution turned brown. The reaction mixture was
concentrated, adsorbed onto silica gel, and then purified by flash silica
gel chromatography (0 to 100% DCM/EtOAc) to provide 2-
reversed-phase HPLC. The fractions were treated with sat. aq.
NaHCO3, extracted with EtOAc, dried (MgSO4), filtered, concen-
trated, redissolved in DCM, and converted to the purported HCl salt
by treatment with excess 1 M HCl in Et2O followed by concentration.
The residue was redissolved in DCM and treated with Et2O to cause
precipitation. The solid obtained was filtered and washed with Et2O to
obtain 3-(diphenylphosphoryl)-2-methylimidazo[1,2-a]pyridine HCl
1
methylimidazo[1,2-a]pyridine (2.69 g, 20.4 mmol, 70.5% yield). H
NMR (400 MHz, CDCl3): δ 2.46 (s, 3H), 6.72 (m, 1H), 7.11 (ddd, J
= 9.0, 6.8, 1.4 Hz, 1H), 7.33 (s, 1H), 7.54 (m, 1H), 8.03 (dt, J = 6.8,
1.2 Hz, 1H).
1
(30 mg, 0.08 mmol, 5.9% yield). H NMR (400 MHz, DMSO-d6): δ
3-(Diphenylphosphino)imidazo[1,2-a]pyridine (11a). Chlor-
odiphenylphosphine (1.40 g, 6.35 mmol) and iodotrimethylsilane
(1.27 g, 6.35 mmol) were mixed in toluene (4 mL) and stirred for
about 2 h. This mixture was transferred to a premixed solution of
imidazo[1,2-a]pyridine (500 mg, 4.23 mmol) and triethylamine (2.35
mL, 16.9 mmol) in pyridine (10 mL). The reaction mixture was
allowed to stir overnight, concentrated in vacuo, and concentrated
with toluene (×2) to remove pyridine. The crude mixture was
dissolved in DCM and purified by silica gel chromatography (0 to
100% EtOAc/DCM) to provide 3-(diphenylphosphino)imidazo [1,2-
1.82 (d, J = 1.2 Hz, 3H), 7.24 (m, 1H), 7.68 (m, 11H), 7.85 (d, J = 8.6
Hz, 1H), 8.84 (d, J = 6.7 Hz, 1H). LC/MS: method 1, retention time
3.734 min. HRMS (m/z): calcd for C20H18N2OP+ (M + H)+ 333.1151,
found 333.1157.
3-(Diphenylphosphorothioyl)-2-methylimidazo[1,2-a]-
pyridine (12d) (General Procedure A). Chlorodiphenylphosphine
(3.89 mL, 26.4 mmol) and iodotrimethylsilane (5.29 g, 26.4 mmol)
were stirred in toluene (10 mL) for 2 h. This mixture was added to a
premixed solution of 2-methylimidazo[1,2-a]pyridine 10b (2.33 g,
17.6 mmol) and triethylamine (9.78 mL, 70.5 mmol) in pyridine (10.0
mL), and the resulting mixture was stirred for 12 h. Sulfur (0.565 g,
17.6 mmol) was added, and the mixture was stirred for another 6 h,
concentrated, concentrated again with toluene (to remove pyridine),
and diluted with benzene. The solids (presumed to be Et3N and
pyridine salts) were filtered, and the filtrate was adsorbed over silica
and subjected to purification by flash silica gel chromatography (0 to
75% EtOAc in DCM) to provide 3-(diphenylphosphorothioyl)-2-
1
a]pyridine (550 mg, 1.82 mmol, 43.0% yield). H NMR (400 MHz,
DMSO-d6): δ 6.94 (td, J = 6.8, 1.4 Hz, 1H), 7.36 (m, 12H), 7.71 (m,
1H), 8.16 (m, J = 6.8, 2.1, 1.1, 1.1 Hz, 1H). LC/MS: method 1,
retention time 4.500 min. HRMS (m/z): calcd for C19H16N2P+ (M +
H)+ 303.1046, found 303.1049.
3-(Diphenylphosphoryl)imidazo[1,2-a]pyridine HCl (12a). 3-
(Diphenylphosphino)imidazo[1,2-a]pyridine 11a (125 mg, 0.413
mmol) was dissolved in THF and treated with excess 30% hydrogen
peroxide (300 mg, 2.65 mmol). The mixture was stirred for 16 h and
then diluted with EtOAc and washed with water. The organic layer was
dried (MgSO4), filtered, concentrated, and purified by reversed-phase
HPLC. The TFA salt obtained was dissolved in CH2Cl2, treated with
excess HCl (4 M in Et2O), and then concentrated in vacuo (twice) to
obtain the HCl salt of 3-(diphenylphosphoryl)imidazo[1,2-a]pyridine
1
methylimidazo[1,2-a]pyridine (3.02 g, 8.67 mmol, 49.2% yield). H
NMR (400 MHz, DMSO-d6): δ 1.57 (m, 3H), 6.96 (m, J = 6.7, 6.7,
1.0, 0.7 Hz, 1H), 7.44 (m, 1H), 7.60 (m, 4H), 7.68 (m, 3H), 7.78 (m,
4H), 8.35 (dt, J = 6.8, 1.2 Hz, 1H).
3-(Diphenylphosphorothioyl)-1,2-dimethyl-1H-imidazo[1,2-
a]pyridin-1-ium Methyl Sulfate (13b) (General Procedure B). 3-
(Diphenylphosphorothioyl)-2-methylimidazo[1,2-a]pyridine 12d (90
mg, 0.26 mmol) and dimethyl sulfate (0.05 mL, 0.52 mmol) were
added to dioxane (2 mL). The mixture was heated in a sealed tube at
100 °C for 16 h and then cooled. The solid obtained was filtered,
washed with diethyl ether, and air-dried to obtain 3-(diphenylphos-
phorothioyl)-1,2-dimethyl-1H-imidazo[1,2-a]pyridin-1-ium methyl
sulfate (50 mg, 0.11 mmol, 41% yield). 1H NMR (400 MHz,
DMSO-d6): δ 1.67 (m, 3H), 3.91 (s, 3H), 7.54 (t, J = 7.0 Hz, 1H),
7.68 (td, J = 7.6, 3.5 Hz, 4H), 7.77 (m, 2H), 7.87 (m, 4H), 8.15 (t, J =
8.3 Hz, 1H), 8.39 (m, 1H), 8.66 (d, J = 6.8 Hz, 1H). LC/MS: method
1, retention time 4.545 min. HRMS (m/z): calcd for C21H20N2PS+
(M)+ 363.1079, found 363.1080.
1
(45 mg, 0.13 mmol, 31% yield). H NMR (400 MHz, DMSO-d6): δ
7.39 (m, 1H), 7.63 (m, 4H), 7.75 (m, 6H), 7.87 (m, 1H), 7.94 (s, 1H),
8.00 (m, 1H), 8.78 (d, J = 6.8 Hz, 1H). LC/MS: method 1, retention
time 3.693 min. HRMS (m/z): calcd for C19H16N2OP (M + H)+
319.0995, found 319.0995.
3-(Diphenylphosphorothioyl)imidazo[1,2-a]pyridine HCl
(12b). 3-(Diphenylphosphino)imidazo [1,2-a]pyridine 11a (125 mg,
0.413 mmol) was dissolved in THF and treated with excess sulfur (50
mg, 1.6 mmol). The mixture was stirred for 16 h and then diluted with
EtOAc and washed with water. The organic layer was dried (MgSO4),
filtered, concentrated, and purified by reversed-phase HPLC. The TFA
salt obtained was dissolved in CH2Cl2, treated with excess HCl (4 M
in Et2O), and then concentrated in vacuo (twice) to obtain the HCl
salt of 3-(diphenylphosphorothioyl)imidazo[1,2-a]pyridine (50 mg,
0.135 mmol, 33% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.19 (t, J
= 6.8 Hz, 1H), 7.38 (br s, 1H), 7.66 (m, 7H), 7.80 (m, 4H), 7.89 (d, J
= 9.2 Hz, 1H), 8.52 (d, J = 6.7 Hz, 1H). LC/MS: method 1, retention
time 4.845 min. HRMS (m/z): calcd for C19H16N2PS (M + H)+
335.0766, found 335.0771.
3-(Diphenylphosphorothioyl)-1-methyl-1H-imidazo[1,2-a]-
p y r i d i n - 1 - i u m M e t h y l S u l f a t e ( 1 3 a ) . 3 -
(Diphenylphosphorothioyl)imidazo[1,2-a]pyridine 12b (150 mg,
0.449 mmol) was reacted with dimethyl sulfate (0.086 mL, 0.897
mmol) according to general procedure B to obtain 3-(diphenylphos-
phorothioyl)-1-methyl-1H-imidazo[1,2-a]pyridin-1-ium methyl sulfate
1
(70 mg, 0.15 mmol, 34% yield). H NMR (400 MHz, DMSO-d6): δ
4.02 (s, 3H), 7.65 (m, 5H), 7.77 (m, 2H), 7.86 (m, 4H), 8.08 (dd, J =
2.0, 0.4 Hz, 1H), 8.21 (m, 1H), 8.3 (m, 1H), 8.72 (m, 1H). LC/MS:
method 1, retention time 4.468 min. HRMS (m/z): calcd for
C20H18N2PS+ (M)+ 349.0923, found 349.0925.
3-(Diphenylphosphoryl)-2-methylimidazo[1,2-a]pyridine
HCl (12c). Compound 12c could be prepared from 2-methylimidazo-
[1,2-a]pyridine 10b using the procedure to make 12a or by the
following procedure: 3-Bromo-2-methylimidazo[1,2-a]pyridine (290
mg, 1.37 mmol, not completely pure) was dissolved in THF (5 mL),
and the solution was cooled to −15 °C under nitrogen.
Isopropylmagnesium chloride·lithium chloride (2 mL, 2 mmol) was
added, and the mixture was allowed to warm to 10 °C, at which point
it was determined by TLC (1:1 DCM/EtOAc) that the halogen−
metal exchange was complete. A solution of chlorodiphenylphosphine
(300 mg, 1.36 mmol) in THF (1 mL) was added via syringe, and the
reaction mixture was allowed to gradually warm to rt. The reaction was
quenched by addition of sat aq. NH4Cl, and the mixture was extracted
with EtOAc. The organic layer was separated and purified by silica gel
chromatography (0 to 100% EtOAc/DCM). The product, which was
Imidazo[1,2-a]pyridin-3-yldiphenylmethanol (18a). 3-
Bromoimidazo[1,2-a]pyridine (172 mg, 0.873 mmol) 17a was
dissolved in THF (5 mL), and the solution was cooled to −15 °C
under nitrogen. Isopropylmagnesium chloride·lithium chloride (1.5
mL, 1.5 mmol) was added, and the mixture was gradually warmed to
10 °C. A solution of benzophenone (175 mg, 0.960 mmol) in THF (1
mL) was added via syringe, and the solution was warmed to rt. The
reaction was quenched by addition of sat aq. NH4Cl, and the resulting
solution was extracted with EtOAc. The organic layer was separated,
concentrated, and purified by reversed-phase HPLC, which provided
the TFA salt. This was converted to its free base by dissolution in
EtOAc followed by treatment with sat aq. NaHCO3. The organic
phase was dried (MgSO4), filtered, and concentrated. The oily residue
was dissolved in minimal CH2Cl2, treated with Et2O, and sonicated to
cause precipitation of a solid, which was filtered and air-dried to
provide imidazo[1,2-a]pyridin-3-yldiphenylmethanol (80 mg, 0.27
1
not completely pure as judged by H NMR analysis, was added to
THF,treated with excess 30% hydrogen peroxide, and stirred
overnight. Water was added, and the mixture was extracted with
EtOAc and concentrated, after which the residue was purified by
I
dx.doi.org/10.1021/jm400904m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mmol, 18% yield). 1H NMR (400 MHz, CDCl3): δ 3.05 (s, 1H), 6.63
(m, 1H), 7.01 (s, 1H), 7.18 (m, 1H), 7.35 (m, 8H), 7.61 (m, 1H), 8.06
(m, 1H). LC/MS: method 1, retention time 4.143 min. HRMS (m/z):
calcd for C20H17N2O+ (M + H)+ 301.1335, found 301.1337.
(m, 1H), 7.68 (m, 4H), 7.77 (m, 2H), 7.91 (m, 4H), 8.19 (m, 1H),
8.44 (m, 1H), 8.70 (d, J = 6.8 Hz, 1H). LC/MS: method 1, retention
time 5.160 min. HRMS (m/z): calcd for C27H24N2PS+ (M)+ 439.1392,
found 439.1398.
3-(Diphenylphosphorothioyl)-2-methyl-1-phenethylimidazo[1,2-
(2-Methylimidazo[1,2-a]pyridin-3-yl)diphenylmethanol
(18b). 3-Bromo-2-methylimidazo[1,2-a]pyridine (220 mg, 1.04 mmol,
not completely pure) was dissolved in THF (5 mL), and the solution
was cooled to −15 °C under nitrogen. Isopropylmagnesium chloride·
lithium chloride (2.7 mL, 2.70 mmol) was added, and the mixture was
allowed to warm to 10 °C. A solution of benzophenone (208 mg,
1.141 mmol) in THF (1 mL) was added via syringe, and the solution
warmed to rt. The reaction was quenched by addition of sat. aq.
NH4Cl, and the resulting solution was extracted with EtOAc. The
organic layer was separated and then purified by silica gel
chromatography (0 to 100% EtOAc in DCM) to provide (2-
methylimidazo[1,2-a]pyridin-3-yl)diphenylmethanol (100 mg, 0.318
mmol, 30.5% yield). 1H NMR (400 MHz, DMSO-d6): δ 1.38 (s, 3H),
6.65 (m, 1H), 6.93 (s, 1H), 7.14 (ddd, J = 9.0, 6.7, 1.4 Hz, 1H), 7.23
(m, 4H), 7.33 (m, 6H), 7.43 (dt, J = 9.0, 1.2 Hz, 1H), 8.11 (dt, J = 7.0,
1.3 Hz, 1H). LC/MS: method 1, retention time 4.290 min. HRMS
(m/z): calcd for C21H19N2O+ (M + H)+ 315.1492, found 315.1495.
3-(Hydroxydiphenylmethyl)-1,2-dimethylimidazo[1,2-a]-
pyridin-1-ium Trifluoroacetate (19b) (General Procedure C). A
solution of (2-Methylimidazo[1,2-a]pyridin-3-yl)diphenylmethanol
(32 mg, 0.102 mmol) and dimethyl sulfate (15 μL, 0.157 mmol) in
dioxane (3.0 mL) was heated in a sealed tube overnight. An oily
residue suspended in dioxane was observed. The mixture was
concentrated, purified with reversed-phase HPLC (25 to 70%
MeCN in water, 0.1% TFA) to provide 3-(hydroxydiphenylmethyl)-
1,2-dimethylimidazo[1,2-a]pyridin-1-ium trifluoroacetate (25 mg,
0.057 mmol, 56% yield) . The methyl sulfate counteranion was
1
a]pyridin-1-ium Bromide (20b). H NMR (400 MHz, DMSO-d6): δ
1.41 (d, J = 1.4 Hz, 3H), 3.07 (t, J = 6.6 Hz, 2H), 4.66 (t, J = 6.8 Hz,
2H), 7.11 (m, 2H), 7.23 (m, 3H), 7.49 (td, J = 7.1, 1.3 Hz, 1H), 7.66
(m, 4H), 7.78 (m, 6H), 8.04 (m, 1H), 8.22 (m, 1H), 8.65 (m, 1H).
LC/MS: method 1, retention time 5.180 min. HRMS (m/z): calcd for
C28H26N2PS+ (M)+ 453.1549, found 453.1555.
3-(Diphenylphosphorothioyl)-2-methyl-1-(1-oxo-2-phenylethyl)-
1
imidazo[1,2-a]pyridin-1-ium Bromide (20c). H NMR (400 MHz,
DMSO-d6): δ 1.62 (s, 3H), 6.37 (s, 2H), 7.66 (m, 7H), 7.79 (m, 3H),
7.91 (m, 4H), 8.11 (dd, J = 8.4, 1.4 Hz, 2H), 8.18 (m, 1H), 8.44 (m,
1H), 8.76 (d, J = 6.8 Hz, 1H). LC/MS: method 1, retention time
5.391 min. HRMS (m/z): calcd for C28H24N2OPS+ (M)+ 467.1341,
found 467.1346.
3-(Diphenylphosphorothioyl)-2-methyl-1-(3-phenylpropyl)-
1
imidazo[1,2-a]pyridin-1-ium Trifluoroacetate (20d). H NMR (400
MHz, DMSO-d6): δ 1.65 (m, 3H), 2.05 (qd, J = 8.0, 7.7 Hz, 2H), 2.71
(m, 2H), 4.42 (t, J = 7.7 Hz, 2H), 7.17 (m, 5H), 7.52 (tt, J = 7.0, 0.6
Hz, 1H), 7.64 (m, 4H), 7.74 (m, 2H), 7.83 (m, 4H), 8.13 (m, 1H),
8.41 (m, 1H), 8.62 (d, J = 7.0 Hz, 1H). LC/MS: method 1, retention
time 5.407 min. HRMS (m/z): calcd for C29H28N2PS+ (M)+ 467.1705,
found 467.1709.
1-Cinnamyl-3-(diphenylphosphorothioyl)-2-methylimidazo[1,2-
1
a]pyridin-1-ium Bromide (20e). H NMR (400 MHz, DMSO-d6): δ
1.75 (d, J = 1.4 Hz, 3H), 5.28 (m, 2H), 6.43 (m, 1H), 6.86 (d, J = 16.0
Hz, 1H), 7.32 (m, 3H), 7.44 (m, 2H), 7.59 (td, J = 7.1, 1.3 Hz, 1H),
7.68 (m, 4H), 7.78 (m, 2H), 7.91 (m, 4H), 8.20 (ddd, J = 9.2, 7.2, 1.2
Hz, 1H), 8.48 (m, 1H), 8.70 (m, 1H). LC/MS: method 1, retention
time 5.744 min. HRMS (m/z): calcd for C29H26N2PS+ (M)+ 465.1549,
found 465.1553.
3-(Diphenylphosphorothioyl)-2-methyl-1-(4-phenylbutyl)-
imidazo[1,2-a]pyridin-1-ium Bromide (20f). 1H NMR (400 MHz,
DMSO-d6): δ 1.73 (m, 7H), 2.62 (m, 2H), 4.43 (m, 2H), 7.18 (m,
3H), 7.28 (m, 2H), 7.55 (td, J = 7.0, 1.4 Hz, 1H), 7.68 (m, 4H), 7.78
(m, 2H), 7.89 (m, 4H), 8.15 (ddd, J = 9.1, 7.1, 1.2 Hz, 1H), 8.42 (dd, J
= 9.1, 1.1 Hz, 1H), 8.64 (m, 1H). LC/MS: method 1, retention time
5.638 min. HRMS (m/z): calcd for C30H30N2PS+ (M)+ 481.1868,
found 481.1871.
1
assumed to be exchanged for trifluoroacetate. H NMR (400 MHz,
DMSO-d6): δ 1.49 (s, 3H), 3.88 (s, 3H), 7.37 (m, 11H), 7.60 (m, 1H),
7.99 (m, 1H), 8.25 (d, J = 9.2 Hz, 1H), 8.58 (d, J = 7.0 Hz, 1H). LC/
MS: method 1, retention time 4.321 min. HRMS (m/z): calcd for
C22H21N2O+ (M)+ 329.1648, found 329.1656.
3-(Hydroxydiphenylmethyl)-1-methylimidazo[1,2-a]pyridin-
1-ium Trifluoroacetate (19a). Imidazo[1,2-a]pyridin-3-yldiphenyl-
methanol 18a (48 mg, 0.16 mmol) and dimethyl sulfate (25 μL, 0.26
mmol) were reacted according to general procedure C to obtain 3-
(hydroxydiphenylmethyl)-1-methylimidazo[1,2-a]pyridin-1-ium tri-
1
fluoroacetate (35 mg, 0.082 mmol, 51% yield). H NMR (400 MHz,
3-(Diphenylphosphorothioyl)-2-methyl-1-(5-phenylpentyl)-
DMSO-d6): δ 7.41 (m, 11H), 7.56 (s, 1H), 8.05 (ddd, J = 9.1, 7.1, 1.2
Hz, 1H), 8.24 (dt, J = 9.2, 1.1 Hz, 1H), 8.39 (m, 1H). LC/MS:
method 1, retention time 4.074 min. HRMS (m/z): calcd for
C21H19N2O+ (M)+ 315.1492, found 315.1497.
1
imidazo[1,2-a]pyridin-1-ium Trifluoroacetate (20g). H NMR (400
MHz, DMSO-d6): δ 1.39 (m, 2H), 1.61 (m, 2H), 1.70 (s, 3H), 1.76
(m, 2H), 2.57 (t, J = 7.4 Hz, 2H), 4.39 (t, J = 7.7 Hz, 2H), 7.16 (m,
3H), 7.26 (m, 2H), 7.55 (m, 1H), 7.68 (td, J = 7.6, 3.5 Hz, 4H), 7.78
(m, 2H), 7.89 (ddd, J = 14.7, 8.3, 1.3 Hz, 4H), 8.15 (m, 1H), 8.43 (m,
1H), 8.65 (d, J = 7.0 Hz, 1H). LC/MS: method 1, retention time
5.738 min. HRMS (m/z): calcd for C31H32N2PS+ (M)+ 495.2030,
found 495.2033.
(E)-3-(Diphenylphosphorothioyl)-1-(3-(3-methoxyphenyl)allyl)-2-
methylimidazo[1,2-a]pyridin-1-ium Trifluoroacetate (21a). 1H
NMR (400 MHz, DMSO-d6): δ 1.75 (d, J = 1.4 Hz, 3H), 3.75 (s,
3H), 5.27 (m, 2H), 5.75 (s, 1H), 6.86 (m, 2H), 7.01 (m, 2H), 7.26 (m,
1H), 7.59 (m, 1H), 7.69 (m, 4H), 7.78 (td, J = 7.6, 1.6 Hz, 2H), 7.91
(ddd, J = 14.7, 8.4, 1.4 Hz, 4H), 8.20 (m, 1H), 8.48 (d, J = 9.2 Hz,
1H), 8.70 (m, 1H). LC/MS: method 1, retention time 5.305 min.
HRMS (m/z): calcd for C30H28N2OPS+ (M)+ 495.1661, found
495.1661.
(E)-1-(3-(2-Chlorophenyl)allyl)-3-(diphenylphosphorothioyl)-2-
methylimidazo[1,2-a]pyridin-1-ium Trifluoroacetate (21b). 1H
NMR (400 MHz, DMSO-d6): δ 1.76 (d, J = 1.4 Hz, 3H), 5.39 (m,
2H), 6.46 (dt, J = 15.8, 6.2 Hz, 1H), 7.10 (d, J = 16.0 Hz, 1H), 7.33
(m, 2H), 7.46 (m, 1H), 7.65 (m, 6H), 7.78 (m, 2H), 7.91 (m, 4H),
8.21 (ddd, J = 8.9, 7.3, 1.2 Hz, 1H), 8.50 (dd, J = 9.2, 1.2 Hz, 1H), 8.70
(m, 1H). LC/MS: method 1, retention time 5.440 min. HRMS (m/z):
calcd for C29H25ClN2PS+ (M)+ 499.1168, found 499.1170.
3-(Diphenylphosphorothioyl)-1,2-dimethyl-1H-indole (15b).
Chlorodiphenylphosphine, iodotrimethylsilane, commercially available
1,2-dimethyl-1H-indole, and triethylamine were reacted according to
general procedure A to obtain 3-(diphenylphosphorothioyl)-1,2-
1
dimethyl-1H-indole in 40% yield. H NMR (400 MHz, DMSO-d6):
δ 2.17 (d, J = 1.4 Hz, 3H), 3.72 (s, 3H), 6.36 (dt, J = 8.0, 1.0 Hz, 1H),
6.78 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.10 (ddd, J = 8.2, 7.0, 1.2 Hz,
1H), 7.53 (m, 5H), 7.59 (m, 2H), 7.82 (m, 4H). LC/MS: method 1,
retention time 6.915 min. HRMS (m/z): calcd for C22H21NPS+ (M +
H)+ 362.1127, found 362.1134.
3-(Diphenylphosphorothioyl)-1-methyl-1H-indole (15a).
Chlorodiphenylphosphine, iodotrimethylsilane, 1-methyl-1H-indole,
triethylamine, and sulfur were reacted according to general procedure
A to produce 3-(diphenylphosphorothioyl)-1-methyl-1H-indole in
1
39% yield. H NMR (400 MHz, DMSO-d6): δ 3.85 (s, 3H), 7.05
(ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 7.25 (ddd, J = 8.3, 7.1, 1.3 Hz, 1H),
7.30 (dt, J = 8.0, 1.0 Hz, 1H), 7.44 (d, J = 4.5 Hz, 1H), 7.56 (m, 7H),
7.74 (m, 4H). LC/MS: method 1, retention time 6.793 min. HRMS
(m/z): calcd for C21H19NPS+ (M + H)+ 348.0970, found 348.0974.
Compounds 20a−g and 21a−k. These compounds were
prepared by general procedure B or C.
1-Benzyl-3-(diphenylphosphorothioyl)-2-methylimidazo[1,2-a]-
(E)-1-(3-(3-Chlorophenyl)allyl)-3-(diphenylphosphorothioyl)-2-
methylimidazo[1,2-a]pyridin-1-ium Trifluoroacetate (21c). 1H
NMR (400 MHz, DMSO-d6): δ 1.75 (m, 3H), 5.29 (m, 2H), 6.53
1
pyridin-1-ium Trifluoroacetate (20a). H NMR (400 MHz, DMSO-
d6): δ 1.64 (m, 3H), 5.76 (m, 2H), 7.30 (m, 2H), 7.39 (m, 3H), 7.60
J
dx.doi.org/10.1021/jm400904m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(dt, J = 15.9, 6.1 Hz, 1H), 6.82 (d, J = 15.8 Hz, 1H), 7.36 (m, 3H),
7.54 (m, 1H), 7.60 (m, 1H), 7.69 (m, 4H), 7.78 (m, 2H), 7.90 (m,
4H), 8.20 (m, 1H), 8.46 (m, 1H), 8.71 (d, J = 7.0 Hz, 1H). LC/MS:
method 1, retention time 5.520 min. HRMS (m/z): calcd for
C29H25ClN2PS+ (M)+ 499.1168, found 499.1163.
lational Sciences, 9800 Medical Center Drive, Rockville, MD
20850.
Notes
The authors declare no competing financial interest.
(E)-1-(3-(4-Chlorophenyl)allyl)-3-(diphenylphosphorothioyl)-2-
methylimidazo[1,2-a]pyridin-1-ium Trifluoroacetate (21d). 1H
NMR (400 MHz, DMSO-d6): δ 1.75 (d, 3H), 5.28 (m, 2H), 6.47
(m, 1H), 6.84 (d, J = 16.0 Hz, 1H), 7.41 (m, 2H), 7.47 (m, 2H), 7.59
(td, J = 7.1, 1.2 Hz, 1H), 7.69 (m, 4H), 7.78 (m, 2H), 7.91 (m, 4H),
8.20 (ddd, J = 9.2, 7.2, 1.2 Hz, 1H), 8.47 (m, 1H), 8.70 (m, 1H). LC/
MS: method 1, retention time 5.503 min. HRMS (m/z): calcd for
C29H25ClN2PS+ (M)+ 499.1168, found 499.1166.
ACKNOWLEDGMENTS
■
This research was supported by the Molecular Libraries
Initiative of the National Institutes of Health Roadmap for
Medical Research and the Intramural Research Programs of the
National Institute on Alcohol Abuse and Alcoholism, NIH. We
thank Dr. Reinscheid for generously providing the NPSR clone
originally used to generate the CHO-NPSR cell line; Sam
Michael for assistance in robotic screening; and William Leister,
Paul Shinn, Jeremy Smith, Danielle van Leer, and James Bougie
for compound management.
(E)-3-(Diphenylphosphorothioyl)-2-methyl-1-(3-(4-
(trifluoromethyl)phenyl)allyl)imidazo[1,2-a]pyridin-1-ium Trifluor-
1
oacetate (21e). H NMR (400 MHz, DMSO-d6): δ 1.75 (d, J = 1.4
Hz, 3H), 5.32 (dd, J = 6.7, 0.9 Hz, 2H), 6.62 (dt, J = 16.1, 6.0 Hz, 1H),
6.92 (d, J = 16.0 Hz, 1H), 7.59 (td, J = 7.0, 1.2 Hz, 1H), 7.68 (m, 8H),
7.78 (m, 2H), 7.91 (m, 4H), 8.20 (ddd, J = 8.9, 7.3, 1.2 Hz, 1H), 8.47
(dd, J = 9.2, 1.0 Hz, 1H), 8.71 (m, 1H). LC/MS: method 1, retention
time 5.592 min. HRMS (m/z): calcd for C30H25F3N2PS+ (M)+
533.1427, found 533.1426.
(E)-1-(3-(4-Bromophenyl)allyl)-3-(diphenylphosphorothioyl)-2-
methylimidazo[1,2-a]pyridin-1-ium Trifluoroacetate (21f). 1H NMR
(400 MHz, DMSO-d6): δ 1.74 (d, J = 1.4 Hz, 3H), 5.26 (dd, J = 6.1,
1.2 Hz, 2H), 6.47 (m, 1H), 6.81 (d, J = 16.2 Hz, 1H), 7.40 (d, J = 8.6
Hz, 2H), 7.57 (m, 3H), 7.68 (m, 4H), 7.78 (m, 2H), 7.90 (m, 4H),
8.19 (m, 1H), 8.46 (dd, J = 9.2, 1.2 Hz, 1H), 8.70 (dd, J = 6.9, 1.1 Hz,
1H). LC/MS: method 1, retention time 5.555 min. HRMS (m/z):
calcd for C29H25BrN2PS+ (M)+ 543.0660, found 543.0657.
ABBREVIATIONS USED
■
NPS, neuropeptide S; NPSR, neuropeptide S receptor; cAMP,
cyclic adenosine monophosphate; MAPK, mitogen-activated
protein kinase; SAR, structure−activity relationship; qHTS,
quantitative high-throughput screening; HTRF, homogeneous
time-resolved fluorescence; ERK, extracellular signal-regulated
kinase; Tyr, tyrosine; G-protein, guanosine nucleotide-binding
protein; ICV, intracerebroventricular; IP, intraperitoneal; mpk,
milligrams per kilogram; DAMGO, [D-Ala2, N-MePhe4, Gly-
ol]-enkephalin.
(E)-1-(3-(3-Bromophenyl)allyl)-3-(diphenylphosphorothioyl)-2-
methylimidazo[1,2-a]pyridin-1-ium Trifluoroacetate (21g). 1H
NMR (400 MHz, CDCl3): δ 1.79 (d, J = 0.4 Hz, 3H), 5.35 (m,
2H), 6.20 (m, 1H), 6.57 (d, J = 15.1 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H),
7.26 (m, 2H), 7.40 (m, 1H), 7.47 (t, J = 1.7 Hz, 1H), 7.63 (m, 6H),
7.96 (m, 5H), 8.13 (m, 1H), 8.81 (d, J = 6.8 Hz, 1H). LC/MS:
method 1, retention time 5.552 min. HRMS (m/z): calcd for
C29H25BrN2PS+ (M)+ 543.0660, found 543.0662.
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(E)-3-(Diphenylphosphorothioyl)-2-methyl-1-(3-p-tolylallyl)-
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ASSOCIATED CONTENT
■
S
* Supporting Information
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http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author
*Phone: 301-217-9198. Fax: 301-217-5736. E-mail: maruganj@
mail.nih.gov. Address: National Center for Advancing Trans-
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(35) Calculated properties for 20e as per ChemBioDraw Ultra 12.0:
ClogP 3.33, tPSA 6.25. Measured solubility of 20e in PBS: kinetic
solubility >150 μM, equilibrium solubility ∼45 μM.
(21) McCoy, J. G.; Marugan, J. J.; Liu, K.; Zheng, W.; Southall, N.;
Heilig, M.; Austin, C. A. Selective Modulation of Gq/Gs pathways by
L
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