Structure-functional selectivity relationship studies of β-arrestin-biased dopamine D2 receptor agonists

Article abstract of DOI:10.1021/jm300603y

Functionally selective G protein-coupled receptor (GPCR) ligands, which differentially modulate canonical and noncanonical signaling, are extremely useful for elucidating key signal transduction pathways essential for both the therapeutic actions and side

Full text of DOI:10.1021/jm300603y

Article
pubs.acs.org/jmc
Structure−Functional Selectivity Relationship Studies of β‑Arrestin-
Biased Dopamine D2 Receptor Agonists
Xin Chen,†,⊥ Maria F. Sassano,‡,⊥ Lianyou Zheng,†,§ Vincent Setola,‡ Meng Chen,∥ Xu Bai,§
Stephen V. Frye,† William C. Wetsel,∥ Bryan L. Roth,*,‡ and Jian Jin*,†
†Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC
Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
‡Department of Pharmacology and National Institute of Mental Health Psychoactive Drug Screening Program, School of Medicine,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
§Center for Combinatorial Chemistry and Drug Discovery, Jilin University, Changchun, Jilin 130012, China
∥Departments of Psychiatry and Behavioral Sciences, Cell Biology, and Neurobiology, Duke University Medical Center, Durham,
North Carolina 27710, United States
S
* Supporting Information
ABSTRACT: Functionally selective G protein-coupled receptor (GPCR)
ligands, which differentially modulate canonical and noncanonical signaling,
are extremely useful for elucidating key signal transduction pathways essential
for both the therapeutic actions and side effects of drugs. However, few such
ligands have been created, and very little purposeful attention has been
devoted to studying what we term: “structure−functional selectivity
relationships” (SFSR). We recently disclosed the first β-arrestin-biased
dopamine D2 receptor (D2R) agonists UNC9975 (44) and UNC9994 (36),
which have robust in vivo antipsychotic drug-like activities. Here we report the first comprehensive SFSR studies focused on
exploring four regions of the aripiprazole scaffold, which resulted in the discovery of these β-arrestin-biased D2R agonists. These
studies provide a successful proof-of-concept for how functionally selective ligands can be discovered.
be silent and thereby block the actions of an inverse agonist
(e.g., neutral antagonists),10−14 and (3) antagonists can also
induce receptor down regulation15 and receptor internalization
in vitro16 and in vivo,17,18 properties typically associated with
agonists.
INTRODUCTION
■
Classical notions of receptor pharmacology imply that when a
ligand interacts with a receptor, only a unitary outcome is
possible. Accordingly, a full or partial agonist can activate only a
single signal transduction pathway while an antagonist can only
block the actions of an agonist. The key theoretical construct
underlying this model was the concept of intrinsic efficacy.1
According to this conceptualization, a full agonist has maximum
intrinsic efficacy and maximally stimulates all cellular responses
induced by ligand binding. A partial agonist possesses a lower
degree of intrinsic efficacy and partially activates all cellular
responses induced by an agonist. Antagonists, according to this
schema, are neutral entities which possess no intrinsic activity
but are able to block the receptor and preclude activation by
full or partial agonists.1 An extension of this model has been
that a single G protein-coupled receptor (GPCR) interacts with
a single G protein subtype and that full and partial agonists
activate only a single signal transduction pathway.
For many decades, however, it has been clear that these
simplistic notions of GPCR function cannot account for the
myriad of actions induced by agonist and antagonist binding.
This was first convincingly demonstrated for the serotonin and
dopamine families of receptors. For example, it has been
demonstrated that: (1) agonists differentially activate distinct
signal transduction pathways,2−9 (2) antagonists can possess
negative intrinsic activity (e.g., function as inverse agonists) or
Functional selectivity19,20 refers to the process by which
GPCR ligands differentially modulate canonical pathways
involving heterotrimeric large G proteins and noncanonical G
protein-independent pathways involving other signaling
proteins including β-arrestins.6,9,21−24 GPCR ligands with
distinct functional selectivity patterns will be extremely useful
tools for elucidating the key signaling pathways essential for
both the therapeutic actions and the side effects of GPCR
targeted drugs.20 Understanding which signaling pathways
contribute to antipsychotic efficacy and side effects, for
instance, should enable the design of better antipsychotic
drug candidates and may, ultimately, lead to safer and more
effective therapies for patients. Despite the importance of
functionally selective ligands, only a limited number have been
reported.6,19,20,24−27 In addition, to our knowledge, scant
attention has been devoted to studying structure−functional
selectivity relationships (SFSR). We recently reported the first
β-arrestin-biased dopamine D2 receptor (D2R) agonists
Received: May 1, 2012
Published: July 30, 2012
© 2012 American Chemical Society
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UNC9975 (44) and UNC9994 (36), which are simultaneously
inactive toward Gi-regulated cyclic adenosine monophosphate
(cAMP) production and partial agonists for D2R/β-arrestin-2
interactions.28 These β-arrestin-biased D2R agonists displayed
robust antipsychotic drug-like activities in wild-type mice, and
the antipsychotic drug-like activities were significantly attenu-
ated or completely abolished in β-arrestin-2 knockout mice,
suggesting that β-arrestin recruitment and signaling can be a
significant contributor to antipsychotic efficacy.28 Here we
report our SFSR studies that resulted in the discovery of these
β-arrestin-biased D2R agonists. We describe the design,
synthesis, and in vitro and in vivo pharmacological evaluation
of novel compounds that explore four regions of the scaffold
represented by aripiprazole (1), an FDA-approved atypical
antipsychotic drug.24,29,30 These first comprehensive SFSR
studies provide a successful proof-of-concept for how function-
ally selective ligands of GPCRs can be discovered.
A representative synthesis of these compounds is shown in
Scheme 1. Compounds 4, 10, and 11 were produced via
nucleophilic displacement of commercially available 7-(4-
bromobutoxy)-3,4-dihydroquinolin-2(1H)-one with the corre-
sponding 2-subsituted phenylpiperazines in moderate to good
yields. The 2-isopropoxy-,35 2-trifluoromethyl-,36 and 3-
ethoxyphenyl-37 piperazine intermediates were prepared
according to literature procedures. Synthesis of compounds
1−3, 5−9, 12, and 13 were described previously.33,34
The synthesized compounds were evaluated in: (1) D2R
radioligand binding assay to assess their binding affinity to the
receptor, (2) D2R-mediated cAMP accumulation assay, which
measures inhibition of isoproterenol-stimulated cAMP produc-
tion via the Gi-coupled signaling pathway, and (3) D2R-
mediated β-arrestin-2 recruitment Tango assay to determine
their potency and efficacy in activating β-arrestin translocation.
Quinpirole, a full agonist of D2R,38 was used as the positive
control in both cAMP accumulation and β-arrestin-2 recruit-
ment Tango assays. Details of these binding and functional
assays were described in our recent paper.28
Similar to compound 1, which potently activated both D2R-
mediated Gi-regulated cAMP accumulation and β-arrestin-2
recruitment, compounds with or without a 2-subsituent
(compounds 2−10) had high D2R binding affinities (Ki < 10
nM) and high potencies for activating both Gi signaling and β-
arrestin-2 recruitment (Table 1). These compounds did not
display apparent bias for activating either Gi signaling or β-
arrestin-2 recruitment, suggesting that neither the electronic
nature (electron donating or withdrawing, compounds 2, 5−
10) nor the steric bulkiness (compounds 2−4) of the 2-
substituent significantly modulates functional selectivity
profiles. Interestingly, compounds 5 and 6 activated both Gi
and β-arrestin signaling with very high potencies (EC50 < 1
nM) and efficacies (Emax similar to the positive control
quinpirole). On the other hand, compound 2 was a potent,
low efficacy partial agonist (Emax = ∼40%) at both signaling
pathways. In addition, compounds 3, 4, and 10 were about 10-
fold more potent at activating β-arrestin than Gi signaling.
Effects of the substitution patterns were also evaluated. Moving
the ethoxy group from the ortho-position (compound 3) to the
meta- (compound 11) or para-position (compound 12)
resulted in significant decreases in binding affinities and agonist
potencies. However, these modifications did not lead to
significant changes in functional selectivity patterns. Addition-
ally, compound 13, which possesses a 2,3-dimethyl phenyl
group, displayed similar efficacies (e.g., Emax values) for
activating both Gi and β-arrestin pathways (similar to
compound 1), although compound 13 was significantly more
potent at activating β-arrestin than Gi signaling. Overall, the
electronic nature and steric bulkiness of substituents on the
LHS phenyl ring and various patterns of substitution did not
significantly affect patterns of D2R functional selectivity for the
tested compounds.
RESULTS AND DISCUSSION
■
We selected compound 1 as the starting point for the following
reasons: (1) 1 is an FDA-approved drug with excellent
pharmacokinetic (PK) properties including high oral bioavail-
ability and central nervous system (CNS) penetration,28,31 PK
properties likely to be retained by its close analogues,28 (2)
although some structure−activity relationships (SAR) have
been reported,32−34 the SFSR of the scaffold represented by
compound 1 have not been studied, and (3) this core template
is amenable to rapid multidimensional chemical modifications
and optimization, which is ideal for exploring SFSR. To
determine whether modifying various structural motifs of
compound 1 could result in biased compounds that favor either
cAMP or β-arrestin signaling, we intensively investigated the
following four regions of compound 1: (1) the left-hand side
(LHS) phenyl ring with various mono- or disubstitution, (2)
the middle cyclic amino moiety, (3) the central linker, and (4)
the right-hand side (RHS) bicyclic aromatic moiety (Figure 1).
Figure 1. Four regions of compound 1 investigated for SFSR.
SFSR of the LHS Phenyl Moiety. To determine the effects
of substituents in the LHS phenyl ring on D2R functional
selectivity, we designed the compounds outlined in Figure 2.
Because Oshiro and co-workers reported that 2-substituents at
the LHS phenyl were preferred for D2R and enhanced in vivo
activity,33 we explored a number of electron donating or
withdrawing groups at the 2-position (compounds 2−10). In
addition, 3- and 4-substitution and 2,3-disubsitution were also
investigated (compounds 1, 11−13).
SFSR of the Middle Amino Moiety. We next investigated
the middle amino moiety of compound 1. Compounds 18 and
19 (Scheme 2) were designed and synthesized to modulate the
basicity of the inner nitrogen and the ring size of the cyclic
amino motif, respectively. 1,4-Addition of the in situ generated
Grignard reagent to activated pyridinium species gave the
intermediate 14 in 76% yield, which was then converted to the
desired 4-aryl piperidine 16 via hydrogenation and subsequent
deprotection in good overall yield. The nucleophilic displace-
ment of the commercially available 7-(4-bromobutoxy)-3,4-
Figure 2. Compounds designed to explore the LHS phenyl moiety.
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a
Scheme 1. Synthesis of Compounds with Various Substituents on the LHS Phenyl Ring
a
Reagents and conditions: (a) NaI/K2CO3, CH3CN, reflux, 6 h, 40−70%.
a
or β-arrestin signaling although compound 18 was significantly
more potent at activating β-arrestin than Gi signaling (Table 2).
On the other hand, replacing the piperazine group (compound
1) with the homopiperazine group (compound 19), which
leads to substantial conformation changes, resulted in
significant bias for β-arrestin over Gi signaling. Compound 19
was a potent partial agonist at activating β-arrestin-2 recruit-
ment (EC50 = 2.0 nM, Emax = 41%) and was simultaneously
inactive at Gi signaling. The effects of the homopiperazine
moiety on biasing for β-arrestin signaling were also observed
with other analogues (e.g., compounds 44 and 45 in Table 5).
Both compounds 18 and 19 retained high binding affinity to
D2R.
SFSR of the Central Linker. To examine effects of the
central linker on patterns of D2R functional selectivity, we
designed the compounds outlined in Scheme 3, which contain
either shorter or conformationally constrained linkers. Com-
pounds 22−24 were synthesized by a two-step sequence similar
to that previously developed for compound 21.39 Thus, the
commercially available 7-hydroxy-3,4-dihydroquinolin-2(1H)-
one was refluxed with various dibromides or dichlorides to give
the bromo or chloro intermediates, which were then reacted
with commercially available 1-(2,3-dichlorophenyl)piperazine
hydrochloride (20) to afford the target compounds 22−24.
Synthesis of compounds 25 and 26 started from the piperazine
20 reacting with 1-bromo-3-(bromomethyl)benzene and 1-
bromo-4-(bromomethyl)benzene to give the intermediates 27
and 28, which were then treated with 7-hydroxy-3,4-
dihydroquinolin-2(1H)-one to afford the target compounds
25 and 26, respectively.
Table 1. SFSR of the LHS Phenyl Moiety
β-arrestin
cAMP
D2R Ki
(nM)
EC50
(nM)
Emax
(%)
EC50
(nM)
Emax
Cmpd
R
(%)
1
2,3-(Cl)2
2-OCH3
2-OEt
2-OiPr
2-H
3.9
0.3
2.8
3.1
4.3
5.5
3.7
2.9
5.9
4.2
21
4.0
0.6
0.8
2.0
0.4
0.8
2.0
0.8
3.2
2.5
10
62
46
66
63
87
89
82
77
81
79
40
82
74
1.0
2.5
7.9
25
51
40
46
43
90
93
68
78
56
77
76
69
65
2
3
4
5
0.8
0.3
7.9
5.0
16
6
2-F
7
2-Cl
8
2-CN
9
2-CH3
2-CF3
3-OEt
4-OEt
2,3-(CH3)2
10
11
12
13
25
158
251
200
53
50
8.1
5.0
a
Ki, EC50, and Emax values are the average of at least two duplicate
experiments with standard deviations (SD) values that are 3-fold less
than the average.
dihydroquinolin-2(1H)-one with the intermediate 16 afforded
the targeted compound 18 in good yield. Synthesis of the
intermediate 17 and the targeted compound 19 was reported in
our previous paper.28
Results of these compounds in D2R binding, cAMP
accumulation, and β-arrestin-2 recruitment assays are summar-
ized in Table 3. Compound 21, which contains a shorter linker
(3-carbon versus 4-carbon in compound 1), had slightly higher
efficacy at activating β-arrestin-2 recruitment than Gi signaling
although compound 21 had a lower binding affinity and agonist
These compounds were evaluated in the D2R radioligand
binding, cAMP accumulation, and β-arrestin-2 recruitment
assays to assess their binding affinities and patterns of D2R
functional selectivity. Replacing the piperazine group (com-
pound 1) with the more basic piperidine group (compound 18)
did not result in significant changes in the efficacy of either Gi
a
Scheme 2. Synthesis of Compounds for Exploring the Middle Amino Moiety
a
Reagents and conditions: (a) ClCOOCH2Ph, CuI, THF, −10 °C; (b) i-PrMgCl, THF, −20 °C, 76%; (c) H2, RhCl(PPh3)3, toluene, 70 °C, 4 d,
95%; (d) 6 N HCl, reflux, 93%; (e) NaI/K2CO3, CH3CN, reflux, 6 h, 60% for 18, 62% for 19.
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a
Table 2. SFSR of the Middle Amino Moiety
a
Ki, EC50, and Emax values are the average of at least two duplicate experiments with standard deviations (SD) values that are 3-fold less than the
average.
a
Scheme 3. Synthesis of Compounds for Exploring the Central Linker
a
Reagents and conditions: (a) K2CO3, EtOH, reflux, overnight, 35−80%; (b) NaI/K2CO3, CH3CN, reflux, 6 h, 40−70%; (c) Et3N, CH3CN, reflux, 4
h, 70−75%; (d) CuCl, Cs2CO3, TMHD, NMP, 120 °C, 7.5 h, 40−50%.
potency than compound 1. Interestingly, the cyclohexyl group
containing compound 22, the acetylene containing compound
23, the meta-benzyl group containing compound 25, and the
para-benzyl group containing compound 26 were all biased
ligands for β-arrestin: partial agonists at β-arrestin-2 recruit-
ment and simultaneously inactive at Gi signaling. However, all
four compounds suffered from significant lower binding
affinities and agonist potencies. On the other hand, the
cyclopropyl group containing compound 24 did not display
significant bias for activating β-arrestin-2 recruitment over Gi
signaling. Taken together, these results suggest that the central
linker plays an important role in modulating functional
selectivity and potency of these compounds. This finding is
consistent with previously reported results: the linker flexibility
significantly affects ligand binding affinities and intrinsic
efficacies.32−34,40−42
SFSR of the RHS Bicyclic Aromatic Moiety. We next
explored the RHS bicyclic aromatic moiety of compound 1. We
selected six different bicyclic aromatic groups (outlined in
Scheme 4) to evaluate their effects on patterns of D2R
functional selectivity. The previously described two-step
nucleophilic displacement sequence for synthesizing com-
pounds 29 and 3028 was applied to the preparation of
compounds 31−34 (Scheme 4). The corresponding commer-
cially available phenols were reacted with butyl-1,4-dibromide
to give the bromo intermediates, which were then reacted with
the piperazine 20 to yield the target compounds 31−34.
Results of these compounds in D2R binding, cAMP
accumulation, and β-arrestin-2 recruitment assays are summar-
ized in Table 4. In contrast to compound 1, compound 29,
which contains a quinoline-2-one moiety, exhibited a higher
efficacy for activating D2R mediated β-arrestin translocation
than D2R-mediated Gi activity. Similar effects were observed
with compound 30, which contains a dihydro-1,8-naphthyridin-
2-one moiety. Importantly, replacing the dihydroquinolin-2-one
(1) with the dihydroisoquinolinone (compound 31), indazole
(compound 32), benzimidazolone (compound 33), or
benzothiazole (compound 34) resulted in apparent bias for
D2R-mediated β-arrestin over Gi signaling. These four
compounds were partial agonists at D2R-mediated β-arrestin-
2 recruitment, with moderate to high potencies and
simultaneously inactive at D2R-mediated Gi signaling. In
general, the RHS bicyclic aromatic moiety plays a critical role
in modulating functional selectivity of these analogues. All six
investigated RHS aryl groups exhibited some degree of bias for
β-arrestin over Gi signaling.
Synthesis and in Vitro Pharmacological Evaluation of
Combination Compounds. After exploring the above four
regions of compound 1 and studying their SFSR, we next
designed and synthesized a number of combination compounds
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a
Table 3. SFSR of the Central Linker
a
Ki, EC50, and Emax values are the average of at least two duplicate experiments with standard deviations (SD) values that are 3-fold less than the
average.
a
Scheme 4. Synthesis of Compounds with Various RHS Bicyclic Aromatic Groups
a
Reagents and conditions: (a) K2CO3, EtOH, reflux, 6 h, 30−80%; (b) NaI/K2CO3, CH3CN, reflux, 6 h, 40−70%.
(outlined in Scheme 5), which combined the structural motifs
that contribute to the bias for β-arrestin recruitment into single
molecules. We selected all three middle amino groups, 3- and 4-
carbon linkers, and all six RHS aromatic groups for this study.
The synthetic routes for these combination compounds were
shown in Scheme 5. These compounds (35−45) were prepared
following the same synthetic approach developed for
compounds 29−34 using the corresponding amino groups,
central linkers, and RHS aromatic groups.
We next evaluated compounds 35−45 in the D2R radio-
ligand binding, cAMP accumulation, and β-arrestin-2 recruit-
ment assays (results are summarized in Table 5). As expected,
these combination compounds were all significantly biased for
D2R-mediated β-arrestin over Gi signaling. With the exception
of compound 40, all compounds were agonists at D2R
mediated β-arrestin-2 recruitment with moderate to high
potencies and simultaneously inactive at D2R-mediated Gi
signaling. Most notably, compounds 36 and 37 were the
most biased for β-arrestin, with Emax values similar to the
positive control quinpirole. Notably, compounds 44 and 45 had
extremely high potencies (EC50 < 5 nM) but relatively low
efficacies (Emax = 40−50%) at D2R-mediated β-arrestin
translocation. These unique patterns of D2R-mediated func-
tional selectivity profiles will be extremely useful for elucidating
which signaling pathways contribute to antipsychotic efficacies
and side effects.
From these SFSR studies, we observed the following general
trends: (1) the electronic nature (e.g., electron donating or
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a
Table 4. SFSR of the RHS Bicyclic Aromatic Moiety
a
Ki, EC50, and Emax values are the average of at least two duplicate experiments with standard deviations (SD) values that are 3-fold less than the
average.
a
Scheme 5. Synthesis of Combination Compounds
a
Reagents and conditions: (a) K2CO3, EtOH, reflux, 6 h, 30−80%; (b) NaI/K2CO3, CH3CN, reflux, 6 h, 40−70%.
withdrawing), steric bulkiness, and substitution pattern of
substituents on the LHS phenyl ring did not, apparently, have
significant effects on patterns of D2R functional selectivity, (2)
the homopiperazine group as a middle amino moiety reduced
efficacy for activating both β-arrestin and Gi pathways, (3)
several conformationally constrained central linkers could lead
to significant bias for D2R-mediated β-arrestin-2 over Gi
activities. However, these central linkers resulted in significant
losses of potency, and (4) a number of RHS aromatic groups
such as benzothiazole, dihydroisoquinolinone, indazole, and
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a
Table 5. SFSR of Combination Compounds
a
Ki, EC50, and Emax values are the average of at least two duplicate experiments with standard deviations (SD) values that are 3-fold less than the
average.
benzimidazolone led to significant bias for D2R-mediated β-
arrestin-2 over Gi activities. Importantly, we observed that
subtle structural changes could result in substantial changes in
functional selectivity.
We subsequently confirmed that compounds 19, 36, and 44
were β-arrestin-biased agonists in several orthologous β-
arrestin-2 translocation and signaling assays.28
In addition, we determined selectivity of compounds 19, 35,
36, and 44 against a number of dopamine and serotonin
receptors. Compound 19 displayed high affinities to D2 and D3
receptors and low affinities to D1, D4, and D5 receptors, while
compound 35 displayed high affinities to D2, D3, and D4
receptors and low affinities to D1 and D5 receptors (Supporting
Information Table S1). At serotonin receptors, although
compounds 19 and 35 displayed moderate to high binding
affinities (Ki’s: 0.6−138 nM) for 5-HT2A, 5-HT2B, 5-HT2C, and
5-HT1A (Supporting Information Table S1), compound 19 was
significantly less potent in functional assays (Ca2+ mobilization
fluorometric imaging plate reader (FLIPR) or cAMP
biosensor).28 Similarly, compounds 36 and 44 displayed
much lower functional potencies compared to their binding
affinities to 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT1A receptors.28
Selectivity of compounds 36 and 44 against dopamine D1−D5
receptors was reported previously.28
In Vivo Behavioral Studies in Mice. We previously
reported that compounds 36 and 44 displayed robust
antipsychotic drug-like activities and did not induce catalepsy
in wild-type mice.28 In β-arrestin-2 knockout mice, however,
the antipsychotic drug-like activities of these compounds were
significantly attenuated or completely abolished.28 To extend
and confirm these findings, we evaluated the effect of two
additional β-arrestin-biased D2R agonists (compound 19 and
35) on phencyclidine (PCP)-induced hyperlocomotion43 in β-
arrestin-2 knockout mice and the wild-type littermate
controls.44 Compound 19 (2 mg/kg, intraperitoneal (ip)
administration) markedly inhibited PCP-induced hyperloco-
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Figure 3. Compounds 19 and 35 exhibit potent antipsychotic-like activities in mouse hyperlocomotion studies, which are completely abolished or
significantly attenuated in β-arrestin-2 knockout mice. Locomotor activities shown as 5 min binned intervals for wild-type (WT) or β-arrestin-2
knockout (β-ARR2 KO) littermate mice given vehicle or 2.0 mg/kg 19 or 35 (ip), followed 30 min later with 6 mg/kg phencyclidine (PCP, ip). n =
8−13 WT and β-ARR2 KO pairs/group.
1
respectively) at ambient temperature. All H and 13C chemical shifts
motion in wild-type mice. Importantly, this significant
antipsychotic-like activity of compound 19 was completely
abolished in β-arrestin-2 knockout mice (Figure 3). Similarly,
compound 35 (2 mg/kg, ip) significantly reduced PCP-induced
are reported in ppm (δ) relative to CDCl3 (7.26 and 77.16,
respectively) or CD3OD (3.30 and 49.00, respectively).45 HPLC
data for all compounds were acquired using an Agilent 6110 series
system with a UV detector set to 220 nm. Samples were injected (<10
hyperlocomotion in wild-type mice and this antipsychotic-like
μL) onto an Agilent Eclipse Plus 4.6 mm × 50 mm, 1.8 μm, C18
activity was significantly attenuated in β-arrestin-2 knockout
mice (Figure 3). Taken together, these results suggest that β-
arrestin recruitment and signaling can be a significant
contributor to antipsychotic efficacy.
column at room temperature (rt) at a flow rate of 1.0 mL/min. A
linear gradient from 10% to 100% (v/v) B over 5.0 min followed by
2.0 min at 100% B with a mobile phase of (A) H2O + 0.1% acetic acid
and (B) MeOH + 0.1% acetic acid was used. Mass spectra (MS) data
were acquired in positive ion mode using an Agilent 6110 series single
quadrupole mass spectrometer with an electrospray ionization (ESI)
source. High-resolution (positive ion) mass spectrum (HRMS) for
compound 35 was acquired using a Shimadzu LCMS-IT-Tof time-of-
flight mass spectrometer. HRMS (positive ion) for compounds 37, 38,
41, and 42 were recorded on Agilent 6210 ESI-LCT-TOF mass
spectrometer with dual source for reference and sample. HPLC was
used to establish the purity of targeted compounds. All compounds
that were evaluated in biological assays had >95% purity using the
HPLC methods described above.
CONCLUSION
■
In summary, we designed and synthesized a series of novel
compounds for exploring four regions of the scaffold
represented by compound 1. Comprehensive evaluation of
these compounds in D2R radioligand binding, cAMP
accumulation, and β-arrestin-2 recruitment assays revealed a
number of important SFSR findings. Combining the best
structural motifs identified from these studies into single
molecules resulted in the discovery of extremely β-arrestin-
biased D2R agonists 35−37 and high potency and low efficacy
β-arrestin-biased D2R agonists 19 and 44. Findings from our in
vivo studies of these β-arrestin-biased D2R agonists in wild-type
and β-arrestin-2 knockout mice suggest that β-arrestin
recruitment and signaling can be a significant contributor to
antipsychotic efficacy. Our combined medicinal chemistry and
pharmacological profiling approach provides the biomedical
community a successful proof-of-concept for how functionally
selective ligands can be discovered.
Compounds 1−3, 5−9, 12, and 13 were synthesized as previously
reported.31,33
7-(4-(4-(2-Isopropoxyphenyl)piperazin-1-yl)butoxy)-3,4-di-
hydroquinolin-2(1H)-one (4). A mixture of intermediate 7-(4-
bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (119 mg, 0.4 mmol)
and NaI (119.6 mg, 0.8 mmol) in CH3CN was heated to reflux for 30
min and then cooled to rt. Compound 1-(2-isopropoxyphenyl)-
piperazine (97.0 mg, 0.44 mmol) and anhydrous K2CO3 (121.6 mg,
0.88 mmol) were added to the mixture. The resulting mixture was
heated to reflux and stirred for 6 h. Precipitated crystals were filtered
off, and the filtrate was evaporated under reduced pressure. The
residue was extracted with EtOAc. The combined EtOAc layers were
washed with brine, dried over anhydrous Na2SO4, concentrated in
vacuo, and purified by flash chromatography on silica gel column
(elution with DCM/MeOH = 30:1) to give compound 4 as light-
yellow solid (75.3 mg, 43%). 1H NMR (400 MHz, CDCl3): δ 8.59 (br
s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.41 (app t, J = 7.6 Hz, 1H), 7.06 (d, J
= 8.4 Hz, 1H), 7.02−6.96 [m, 2H, containing a d at δ 7.00 (J = 8.4
Hz)], 6.49 (s, 1H), 6.46 (d, J = 8.4 Hz, 1H), 4.99−4.86 (m, 2H), 4.80
(sept, J = 6.0 Hz, 1H), 4.63−4.49 (m, 2H), 4.01−3.92 (m, 2H), 3.76−
3.61 (m, 4H), 3.31−3.20 (m, 2H), 2.85 (app t, J = 7.4 Hz, 2H), 2.56
(app t, J = 7.4 Hz, 2H), 2.20−2.08 (m, 2H), 1.91−1.81 (m, 2H), 1.56
(d, J = 6.0 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 171.7, 158.2,
151.1, 138.5, 132.3, 128.8, 128.2, 124.7, 121.4, 116.2, 114.7, 108.9,
EXPERIMENTAL SECTION
■
Chemistry General Procedures. Unless stated to the contrary,
where applicable, the following conditions apply: all commercial grade
reagents were used without further purification. MeCN and CH2Cl2
were distilled from CaH2 under a N2 atmosphere before use, THF was
distilled from Na/benzophenone under N2. All other dry solvents were
of anhydrous quality purchased from Sigma-Aldrich. Brine (NaCl),
NaHCO3, and NH4Cl refer to saturated aqueous (satd aq) solutions.
Column chromatography was performed on silica gel G (200−300
mesh) with reagent grade solvents. Melting points were uncorrected.
NMR spectra were recorded on a Varian spectrometer (400 or 300
MHz for 1H NMR and 100 MHz or 75 MHz for 13C NMR,
7148
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102.6, 72.7, 67.2, 57.3, 49.8, 48.0, 31.2, 26.4, 24.7, 21.9, 20.9. HPLC
99%, RT 4.39 min. MS (ESI) m/z 438.3 [M + H]+.
2.07 (m, 2H), 1.99−1.85 (m, 2H). HPLC: 99%, RT 2.090 min. MS
(ESI) m/z 230.0 [M + H]+.
7-(4-(4-(2-(Trifluoromethyl)phenyl)piperazin-1-yl)butoxy)-
3,4-dihydroquinolin-2(1H)-one (10). Compound 10 (188 mg) was
prepared as white solid by the same procedure as preparing 4, yield
62%. 1H NMR (400 MHz, CDCl3): δ 8.17 (br s, 1H), 7.62 (d, J = 8.0
Hz, 1H), 7.52 (app t, J = 7.8 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.23
(app t, J = 7.6 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.52 (dd, J = 2.4, 8.4
Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 4.01−3.92 (m, 2H), 3.18−2.93 (m,
4H), 2.92−2.44 [m, 10H, containing an app t at δ 2.89 (J = 7.6 Hz)
and an app t at δ 2.61 (J = 7.6 Hz)], 1.93−1.71 (m, 4H). 13C NMR
(101 MHz, CDCl3): δ 171.8, 158.7, 138.3, 133.0, 129.3, 128.8, 127.5
(q, JCF = 29.0 Hz), 127.3 (q, JCF = 5.4 Hz), 125.2, 124.3, 124.2 (q, JCF
= 274.4 Hz), 115.9, 108.8, 102.3, 67.9, 58.2, 53.5, 53.0, 31.2, 27.3, 24.8,
23.1. HPLC 99%, RT 4.36 min. MS (ESI) m/z 448.3 [M + H]+.
7-(4-(4-(3-Ethoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydro-
quinolin-2(1H)-one (11). Compound 11 (48 mg) was prepared as
white solid by the same procedure as preparing compound 4, yield
57%. 1H NMR (400 MHz, CDCl3): δ 7.76 (s, 1H), 7.15 (t, J = 8.1 Hz,
1H), 7.04 (d, J = 8.1 Hz, 1H), 6.56−6.49 (m, 2H), 6.47 (s, 1H), 6.40
(d, J = 8.1 Hz, 1H), 6.29 (s, 1H), 4.06−3.91 (m, 4H), 3.25−3.14 (m,
4H), 2.90 (t, J = 7.4 Hz, 2H), 2.67−2.56 (m, 6H), 2.46 (t, J = 7.2 Hz,
2H), 1.88−1.77 (m, 2H), 1.76−1.66 (m, 2H), 1.40 (t, J = 6.7 Hz, 3H).
13C NMR (101 MHz, CDCl3): δ 171.6, 160.1, 158.8, 152.8, 138.2,
7-(4-(4-(2,3-Dichlorophenyl)piperidin-1-yl)butoxy)-3,4-dihy-
droquinolin-2(1H)-one (18). Compound 18 (106.8 mg) was
prepared as white solid by the same procedure as preparing compound
4 from intermediate 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-
one (119 mg, 0.4 mmol) and compound 16 (117.3 mg, 0.44 mmol),
1
yield 60%. H NMR (300 MHz, CDCl3): δ 7.85 (s, 1H), 7.33 (d, J =
8.1 Hz, 1H), 7.23−7.17 (m, 2H), 7.05 (d, J = 8.1 Hz, 1H), 6.54 (dd, J
= 2.1 Hz, 8.1 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 3.96 (t, J = 6.0 Hz,
2H), 3.15−3.05 (m, 3H), 2.90 (t, J = 7.3 Hz, 2H), 2.62 (t, J = 7.3 Hz,
2H), 2.50 (t, J = 7.2 Hz, 2H), 2.30−2.10 (m, 2H), 1.91−1.72 (m, 8H).
HPLC: 99%, RT 2.504 min. MS (ESI) m/z 447.2 [M + H]+.
Intermediate 17 and compounds 19 and 21 were prepared
according to previous procedures.27,37,39
trans-7-((4-((4-(2,3-Dichlorophenyl)piperazin-1-yl)methyl)-
cyclohexyl)methoxy)-3,4-dihydroquinolin-2(1H)-one (22). A
mixture of 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (105 mg, 0.65
mmol), 1,4-bis(bromomethyl)cyclohexane (523 mg, 1.9 mmol), and
anhydrous K2CO3 (89 mg, 0.65 mmol) was dissolved in EtOH, and
the solution was heated to reflux for 6 h. The solution was diluted with
water and extracted with EtOAc. The combined organic layers were
washed with satd aq NaHCO3, brine, dried over anhydrous Na2SO4,
concentrated in vacuo, and purified by flash chromatography on silica
gel column to give 7-((4-(bromomethyl)cyclohexyl)methoxy)-3,4-
dihydroquinolin-2(1H)-one (70 mg, 30%, 0.2 mmol) as a white solid,
which was redissolved in CH3CN. To this mixture was added NaI (60
mg, 0.4 mmol), and the reaction mixture was heated to reflux for 30
min and then cooled to rt. The commercial available compound 20
(81 mg, 0.3 mmol) and anhydrous K2CO3 (110 mg, 0.8 mmol) were
added to the mixture. The resulting mixture was heated to reflux and
stirred for 6 h. Precipitated crystals were filtered off, and the filtrate
was evaporated under reduced pressure. The residue was extracted
with EtOAc. The combined EtOAc layers was washed with brine, dried
over anhydrous Na2SO4, concentrated in vacuo, and purified by flash
chromatography on silica gel column (elution with DCM/MeOH =
50:1) to give trans-7-((4-((4-(2,3-dichlorophenyl)piperazin-1-yl)-
methyl)cyclohexyl)methoxy)-3,4-dihydroquinolin-2(1H)-one (22) as
a white solid (63 mg, yield 63%). 1H NMR (300 MHz, CDCl3): δ 8.06
(s, 1H), 7.15−7.13 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 6.97−6.94 (m,
1H), 6.52 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 3.74−
3.71 (m, 2H), 3.07−3.05 (m, 4H), 2.92−2.87 (m, 2H), 2.64−2.59 (m,
6H), 2.24 (d, J = 6.9 Hz, 2H), 1.83 (d, J = 6.9 Hz, 2H), 1.53−1.69 (m,
4H), 1.10−0.94 (m, 4H). HPLC: 99%, RT 2.652 min. MS (ESI) m/z
502.2 [M + H]+.
129.9, 128.9, 115.9, 108.9, 108.8, 105.1, 103.2, 102.3, 68.1, 63.5, 58.4,
53.4, 49.19, 31.3, 27.4, 24.8, 23.6, 15.1. HPLC 99%, RT 4.26 min. MS
(ESI) m/z 424.3 [M + H]+.
(4-(2,3-Dichlorophenyl)pyridin-1(4H)-yl)(phenyl)methanone
(14). To a −30 °C solution of 1-bromo-2,3-dichlorobenzene (10 g, 44
mmol) in THF (120 mL) was added i-PrMgCl (2.0 M in THF, 35 mL,
70 mmol) at a rate such that the temperature <−20 °C. Meanwhile, to
a −10 °C solution of CuI (420 mg, 2.2 mmol) in THF (120 mL) was
added pyridine (7.1 mL, 88 mmol) and then benzyl carbonochloridate
(9.7 mL, 68 mmol) such that the temperature <0 °C. To this
heterogeneous mixture was added the initially formed Grignard at a
rate such that the temperature <0 °C. The resulting solution was
stirred at 0 °C for 30 min and then allowed to warm to rt. The reaction
was then quenched with 10% aq NH4Cl. EtOAc was added, and the
blue aqueous layer was removed. The organic layer was washed with
10% aq NH4Cl, 1 N HCl, and a 20% aq NaCl solution. The organic
layer was then concentrated, and the residue was dissolved and
crystallized from MeOH. The slurry was filtered and the filtercake
washed with MeOH to give benzyl 4-(2,3-dichlorophenyl)pyridine-
1
1(4H)-carboxylate (14) (12 g, 76%) as an off-white solid. H NMR
(300 MHz, CDCl3): δ 7.42−7.21 (m, 8H), 7.04 (d, J = 9.2 Hz, 1H),
6.92 (d, J = 6.4 Hz, 1H), 5.25 (s, 2H), 5.02 (d, J = 6.4 Hz, 1H), 4.93
(d, J = 9.2 Hz, 1H), 4.73−4.71 (m, 1H). HPLC: 99%, RT 4.069 min.
MS (ESI) m/z 360.0 [M + H]+.
7-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)but-2-ynyloxy)-
3,4-dihydroquinolin-2(1H)-one (23). Compound 23 (280 mg) was
prepared as yellow solid by the same procedure as preparing 22, yield
1
79%. H NMR (400 MHz, CDCl3): δ 7.62 (s, 1H), 7.20−7.11 (m,
(4-(2,3-Dichlorophenyl)piperidin-1-yl)(phenyl)methanone
(15). To a solution of intermediate 15 (7.0 g, 19.5 mmol) in toluene
(150 mL) was added RhCl(PPh3)3 (2.1 g, 2.0 mmol) as a slurry in
toluene (50 mL). The reaction was subjected to an atmosphere of H2
at 40 psi and heated to 70 °C. After 6 h, the reaction was filtered
through silica gel and washed with 1:9 EtOAc/toluene. The filtrate was
dissolved in toluene, concentrated in vacuo, and purified by flash
chromatography on silica gel column (elution with PE/EtOAc = 50:1)
to give benzyl 4-(2,3-dichlorophenyl)piperidine-1-yl)(phenyl)-
methanone (15) (6.8 g, 96%) as an oil. 1H NMR (300 MHz,
CDCl3): δ 7.41−7.28 (m, 5H), 7.26−7.25 (m, 1H), 7.18 (t, J = 7.8 Hz,
1H), 7.13−7.10 (m, 1H), 5.16 (s, 2H), 4.35 (d, J = 12.0 Hz, 2H),
3.24−3.19 (m, 1H), 2.93 (t, J = 12.6 Hz, 2H), 1.86 (d, J = 13.0 Hz,
2H), 1.59 (t, J = 10.5 Hz, 2H). HPLC: 99%, RT 3.881 min. MS (ESI)
m/z 364.0 [M + H]+.
2H), 7.07 (d, J = 8.3 Hz, 1H), 6.96 (dd, J = 7.1, 2.5 Hz, 1H), 6.62 (dd,
J = 8.3, 2.5 Hz, 1H), 6.39 (d, J = 2.4 Hz, 1H), 4.72 (t, J = 1.7 Hz, 2H),
3.41 (t, J = 1.7 Hz, 2H), 3.08 (bs, 4H), 2.93−2.85 (m, 2H), 2.78−2.68
(m, 2H), 2.63−2.57 (m, 2H). HPLC: 99%, RT 2.403 min. MS (ESI)
m/z 444.1 [M + H]+.
trans-7-((2-((4-(2,3-Dichlorophenyl)piperazin-1-yl)methyl)-
cyclopropyl)methoxy)-3,4-dihydroquinolin-2(1H)-one (24).
Compound 24 (82 mg) was prepared as white solid by the same
procedure as prepaing 22, yield 48%. 1H NMR (400 MHz, CDCl3): δ
7.69 (s, 1H), 7.19−7.09 (m, 2H), 7.03 (d, J = 8.2 Hz, 1H), 6.98−6.91
(m, 1H), 6.57 (d, J = 8.4 Hz, 1H), 6.35 (s, 1H), 3.89 (s, 2H), 3.04 (s,
4H), 2.88 (t, J = 7.3 Hz, 2H), 2.80−2.56 (m, 6H), 2.46 (s, 2H), 0.65
(s, 2H), 0.48 (s, 2H). 13C NMR (101 MHz, CDCl3): δ 171.6, 159.2,
151.5, 138.1, 134.2, 128.7, 127.6, 127.6, 124.6, 118.7, 115.8, 109.1,
102.5, 72.3, 62.8, 53.7, 51.4, 31.3, 24.8, 18.0, 9.5, 2.1. HPLC: 99%, RT
2.541 min. MS (ESI) m/z 460.1 [M + H]+.
1-(3-Bromobenzyl)-4-(2,3-dichlorophenyl)piperazine (27). A
mixture of 1-(2,3-dichlorophenyl)piperazine hydrochloride (20) (294
mg, 1.1 mmol), 1-bromo-3-(bromomethyl)benzene (250 mg, 1
mmol), and anhydrous triethylamine (253 mg, 2.5 mmol) was
dissolved in CH3CN, and the solution was heated to reflux for 4 h.
4-(2,3-Dichlorophenyl)piperidine Hydrochloride (16). To 6N
HCl (30 mL) was added a solution of compound 15 (5.2 g, 14 mmol)
in THF (10 mL). The mixture was heated to reflux for 3 h and then
concentrated in vacuo. The residue was washed with Et2O to give 4-
(2,3-dichlorophenyl)piperidine hydrochloride (16) (3.5 g, 92%) as a
1
white solid. H NMR (300 MHz, CDCl3): δ 7.48−7.42 (m, 1H),
7.36−7.31 (m, 2H), 3.51−3.41 (m, 3H), 3.33−3.15 (m, 3H), 2.11−
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1
The solution was diluted with water and extracted with EtOAc. The
combined organic layers were washed with sd aq NaHCO3, brine,
dried over anhydrous Na2SO4, concentrated in vacuo, and purified by
flash chromatography on silica gel column (elution with PE/EtOAc =
8:1) to give 1-(3-bromobenzyl)-4-(2,3-dichlorophenyl)piperazine (27)
(220 mg, 74%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 7.54
(s, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.21 (d, J =
7.8 Hz, 1H), 7.17−7.10 (m, 2H), 6.97−6.94 (m, 1H), 3.55 (s, 2H),
3.07 (br, 4H), 2.64 (br, 4H). HPLC: 99%, RT 2.542 min. MS (ESI)
m/z 399.1 [M + H]+.
7-(3-((4-(2,3-Dichlorophenyl)piperazin-1-yl)methyl)-
phenoxy)-3,4-dihydroquinolin-2(1H)-one (25). To a solution of
7-hydroxy-3,4-dihydroquinolin-2(1H)-one (196 mg, 1.2 mmol) in
NMP was added Cs2CO3 (391 mg, 1.2 mmol). The slurry was
degassed by evacuating and filling the reaction flask with N2 three
times. Compound 27 (240 mg, 0.6 mmol) and TMHD (11 mg, 0.06
mmol) were added, followed by the addition of CuCl (60 mg, 0.6
mmol). The reaction mixture was degassed by evacuating and filling
the reaction flask with N2 three times and then warmed to 120 °C
under N2 for 7.5 h. The reaction mixture was cooled to rt and diluted
with Et2O. The slurry was filtered, and the filtercake was washed with
Et2O. Combined filtrates were washed with 2 N HCl, 0.6 N HCl, 2 M
NaOH, and 10% aq NaCl. The resulting organic layer was dried,
concentrated, and purified by flash chromatography on a silica gel
column (elution with PE/EtOAc = 1:1) to give 7-(3-((4-(2,3-
dichlorophenyl)piperazin-1-yl)methyl)phenoxy)-3,4-dihydro quinolin-
2(1H)-one (Compound 25) (off-white solid, 110 mg, 38%). 1H NMR
(300 MHz, CDCl3): δ 7.62 (s, 1H), 7.31 (d, J = 7.2 Hz, 2H), 7.15−
7.05 (m, 4H), 6.96−6.89 (m, 2H), 6.62 (dd, J = 2.1 Hz, 8.1 Hz, 1H),
6.41 (d, J = 2.1 Hz, 1H), 3.57 (s, 2H), 3.06 (m, 4H), 2.94 (t, J = 7.5
Hz, 2H), 2.66−2.61 (m, 5H). HPLC: 99%, RT 2.637 min. MS (ESI)
m/z 482.1 [M + H]+. Mp: 182−183 °C.
42%. H NMR (300 MHz, CDCl3): δ 9.53 (s, 1H), 9.24 (s, 1H),
7.17−7.13 (m, 2H), 6.97−6.91 (m, 2H), 6.66−6.60 (m, 2H), 3.95 (t, J
= 5.7 Hz, 2H), 3.20−3.01 (m, 4H), 2.80−2.61 (m, 4H), 2.52 (t, J = 7.5
Hz, 2H), 1.82−1.73 (m, 4H). HPLC: 99%, RT 2.325 min. MS (ESI)
m/z 435.1 [M + H]+.
6-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butoxy)benzo[d]-
thiazole (34). Compound 34 (light-yellow soild, 98 mg) was
prepared by the same procedure as preparing 22, yield 60%. 1H
NMR (300 MHz, CDCl3): δ 8.97 (s, 1H), 7.79 (d, J = 8.7 Hz, 1H),
7.61 (s, 1H), 7.16−7.08 (m, 3H), 6.97−6.94 (m, 1H), 4.11 (t, J = 5.7
Hz, 2H), 3.09 (br, 4H), 2.68 (brs, 4H), 2.55−2.50 (m, 2H), 1.92−1.76
(m, 4H). HPLC: 99%, RT 2.651 min. MS (ESI) m/z 436.3 [M + H]+.
Melting point: 93−94.5 °C.
5-(4-(4-(2,3-Dichlorophenyl)piperidin-1-yl)butoxy)benzo[d]-
thiazole (35). Compound 35 (144 mg) was prepared as light-yellow
solid by the same procedure as preparing 22, yield 66%. 1H NMR (400
MHz, CDCl3): δ 8.97 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 2.4
Hz, 1H), 7.31 (dd, J = 7.6, 1.8 Hz, 1H), 7.23−7.12 (m, 2H), 7.09 (dd,
J = 8.8, 2.5 Hz, 1H), 4.10 (t, J = 6.3 Hz, 2H), 3.16−3.01 (m, 3H),
2.55−2.45 (m, 2H), 2.14 (t, J = 10.9 Hz, 2H), 1.96−1.68 (m, 8H). 13C
NMR (101 MHz, CDCl3): δ 158.6, 155.0, 154.8, 133.2, 131.9, 128.2,
127.5, 125.6, 125.5, 122.1, 116.6, 106.6, 68.3, 58.7, 54.4, 39.9, 32.1,
27.4, 23.7. HPLC: 99%, RT 2.689 min. MS (ESI) m/z 435.3 [M +
H]+. HRMS m/z [M + H]+ calcd for C22H25Cl2N2OS 435.1065, found
435.1039. Melting point:79−81 °C.
Synthesis of compound 36 was described previously.27
5-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propoxy)benzo-
[d]thiazole (37). Compound 37 (light-yellow solid, 209 mg) was
prepared by the same procedure as preparing 22, yield 62%. 1H NMR
(400 MHz, CDCl3): δ 8.97 (s, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.62 (d, J
= 2.4 Hz, 1H), 7.15 (dd, J = 7.0, 4.7 Hz, 2H), 7.10 (dd, J = 8.8, 2.5 Hz,
1H), 6.97 (dd, J = 6.6, 2.8 Hz, 1H), 4.15 (t, J = 6.3 Hz, 2H), 3.11 (bs,
4H), 2.81−2.61 (m, 6H), 2.15−2.05 (m, 2H). 13C NMR (101 MHz,
CDCl3): δ 158.5, 155.1, 154.8, 151.3, 134.2, 127.7, 127.6, 125.7, 124.8,
122.2, 118.8, 116.5, 106.7, 66.7, 55.3, 53.4, 51.3, 26.7. HPLC: 99%, RT
2.607 min. MS (ESI) m/z 422.0 [M + H]+. HRMS m/z [M + H]+
calcd for C20H22Cl2N3OS 422.0861, found 422.0885. Melting point:
127−129 °C.
1-(4-Bromobenzyl)-4-(2,3-dichlorophenyl)piperazine (28).
Compound 28 (220 mg) was prepared as white solid from 20 (294
mg, 1.1 mmol), 1-bromo-4-(bromomethyl) benzene (250 mg, 1.0
mmol), and anhydrous triethylamine (253 mg, 2.5 mmol) by the same
procedure as preparing 27, yield 74%. 1H NMR (300 MHz, CDCl3): δ
7.46 (d, J = 8.1 Hz, 2H), 7.27−7.23 (m, 2H), 7.15−7.13 (m, 2H),
6.96−6.93 (m, 2H), 3.54 (s, 2H), 3.06 (br, 4H), 2.63 (m, 4H). HPLC:
99%, RT 2.541 min. MS (ESI) m/z 398.9 [M + H]+.
5-(3-(4-(2,3-Dichlorophenyl)-1,4-diazepan-1-yl)propoxy)-
benzo[d]thiazole (38). Compound 38 (light-brown solid, 98 mg)
was prepared by the same procedure as preparing 22, yield 60%. H
1
7-(4-((4-(2,3-Dichlorophenyl)piperazin-1-yl)methyl)-
phenoxy)-3,4-dihydroquinolin-2(1H)-one (26). Compound 26
(120 mg) was prepared as off-white solid by the same procedure as
preparing 25, yield 35%. 1H NMR (300 MHz, CDCl3): δ 7.67 (s, 1H),
7.32 (d, J = 8.7 Hz, 2H), 7.16−7.09 (m, 3H), 6.98−6.94 (m, 3H),
6.64−6.61 (m, 1H), 6.41 (d, J = 3.0 Hz, 1H), 3.56 (s, 2H), 3.07 (m,
4H), 2.94 (t, J = 6.6 Hz, 2H), 2.66−2.61 (m, 6H). HPLC: 99%, RT
2.626 min. MS (ESI) m/z 482.1 [M + H]+. Melting point: 185−186
°C.
NMR (400 MHz, CD3OD): δ 9.91 (s, 1H), 8.10 (t, J = 8.7 Hz, 1H),
7.65 (s, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.29−7.15 (m, 3H), 4.31 (t, J =
5.2 Hz, 2H), 3.87−3.71 (m, 2H), 3.67−3.43 (m, 6H), 3.42−3.29 (m,
2H), 2.48−2.24 (m, 4H). 13C NMR (101 MHz, CD3OD): δ 162.1,
160.9, 152.9, 134.9, 129.1, 128.6, 126.4, 125.8, 125.2, 122.1, 119.5,
119.2, 103.6, 67.2, 56.5, 56.3, 54.6, 53.4, 50.9, 25.8, 25.5. HPLC: 99%,
RT 2.601 min. MS (ESI) m/z 436.3 [M + H]+. HRMS m/z [M + H]+
calcd for C21H24Cl2N3OS 436.1017, found 436.1038. mp: 93−94 °C.
5-(4-(4-(2,3-Dichlorophenyl)-1,4-diazepan-1-yl)butoxy)-
benzo[d]thiazole (39). Compound 39 (light-brown solid, 98 mg)
Compounds 29 and 30 were synthesized as previously
described.27,32
1
was prepared by the same procedure as preparing 22, yield 60%. H
7-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butoxy)-3,4-dihy-
droisoquinolin-1(2H)-one (31). Compound 31 (215 mg) was
prepared as white solid by the same procedure as preparing 22, yield
65%. 1H NMR (300 MHz, CDCl3): δ 7.58 (d, J = 2.7 Hz, 1H), 7.14−
7.11 (m, 3H), 7.01−6.94 (m, 2H), 6.18 (brs, 1H), 4.05 (t, J = 6.3 Hz,
2H), 3.56−3.51 (m, 2H), 3.07 (m, 4H), 2.90 (t, J = 8.1 Hz, 2H), 2.66
(m, 4H), 2.49−2.46 (m, 2H), 1.86−1.68 (m, 4H). HPLC: 99%, RT
2.374 min. MS (ESI) m/z 448.3 [M + H]+.
NMR (300 MHz, CDCl3): δ 8.97 (s, 1H), 7.80 (d, J = 8.7 Hz, 1H),
7.60 (d, J = 3.0 Hz, 1H), 7.11−7.07 (m, 3H), 7.01−6.98 (m, 1H), 4.10
(t, J = 5.7 Hz, 2H), 3.76−3.68 (m, 2H), 3.33−3.27 (m, 4H), 2.96−
2.95 (m, 4H), 2.73−2.71 (m, 2H), 2.06−2.04 (m, 2H), 1.92−1.80 (m,
2H). HPLC: 99%, RT 2.702 min. MS (ESI) m/z 450.1 [M + H]+.
Melting point: 82−84 °C.
5-(4-(4-(2,3-Dichlorophenyl)piperidin-1-yl)butoxy)-1H-
benzo[d]imidazol-2(3H)-one (40). Compound 40 (61 mg) was
prepared as off-white solid by the same procedure as preparing 22,
6-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butoxy)-1H-inda-
zole (32). Compound 32 (off-white solid, 35 mg) was prepared by the
1
1
yield 40%. H NMR (300 MHz, CDCl3): δ 9.59 (brs, 1H), 9.29 (brs,
same procedure as preparing 22, yield 45%. H NMR (300 MHz,
1H), 7.33−7.30 (m, 2H), 7.20−7.13 (m, 1H), 6.92 (d, J = 8.7 Hz,
1H), 6.65−6.59 (m, 2H), 3.94−3.91 (m, 2H), 3.15−3.04 (m, 3H),
2.51−2.49 (m, 2H), 2.15(t, J = 11.1 Hz, 2H), 1.90−1.74 (m, 8H).
HPLC: 99%, RT 2.332 min. MS (ESI) m/z 434.0 [M + H]+. Melting
point: 180−182 °C.
6-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propoxy)-1H-in-
dazole (41). Compound 41 (90 mg) was prepared by the same
procedure as preparing 22, yield 71%. 1H NMR (400 MHz, CDCl3): δ
CDCl3): δ 7.97 (s, 1H), 7.61 (d, J = 8.7 Hz 1H), 7.17−7.15 (m, 2H),
6.96 (dd, J = 3.0 Hz, 6.6 Hz, 1H), 6.86−6.81 (m, 2H), 4.05 (t, J = 5.4
Hz, 2H), 3.16 (s, 4H), 2.79 (s, 4H), 2.64−2.60 (m, 2H), 1.89−1.83
(m, 4H). HPLC: 99%, RT 2.491 min. MS (ESI) m/z 419.2 [M + H]+.
Melting point: 101−103 °C.
5-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butoxy)-1H-
benzo[d]imidazol-2(3H)-one (33). Compound 33 (54 mg) was
prepared as white solid by the same procedure as preparing 22, yield
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10.38 (bs, 1H), 7.97 (s, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.20−7.08 (m,
2H), 6.95 (dd, J = 7.1, 2.5 Hz, 1H), 6.90−6.78 (m, 2H), 4.08 (t, J = 6.3
Hz, 2H), 3.09 (bs, 4H), 2.77−2.61 (m, 6H), 2.12−2.00 (m, 2H). 13C
NMR (101 MHz, CDCl3): δ 159.2, 151.3, 141.5, 135.0, 134.2, 127.6,
127.5, 124.7, 121.7, 118.7, 118.1, 113.5, 91.7, 66.6, 55.3, 53.5, 51.4,
26.8. HPLC: 99%, RT 2.462 min. MS (ESI) m/z 405.2 [M + H]+.
HRMS m/z [M + H]+ calcd for C22H26Cl2N3OS 405.1249, found
405.1273. Melting point: 159−161 °C.
7-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propoxy)-3,4-di-
hydroisoquinolin-1(2H)-one (42). Compound 42 (88 mg) was
prepared as a white solid by the same procedure as preparing 22, yield
52%. 1H NMR (400 MHz, CDCl3): δ 7.60 (d, J = 2.7 Hz, 1H), 7.18−
7.09 (m, 3H), 7.01 (dd, J = 8.3, 2.8 Hz, 1H), 6.96 (dd, J = 6.2, 3.4 Hz,
1H), 6.15 (bs, 1H), 4.10 (t, J = 6.4 Hz, 2H), 3.54 (td, J = 6.6, 2.8 Hz,
2H), 3.08 (bs, 4H), 2.93 (t, J = 6.6 Hz, 2H), 2.73−2.57 (m, 6H),
2.06−1.97 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 166.4, 158.3,
151.5, 134.1, 131.1, 129.9, 128.5, 127.7, 127.6, 124.7, 120.4, 118.8,
112.0, 66.7, 55.2, 53.5, 51.4, 40.7, 27.7, 26.9. HPLC: 99%, RT 2.612
min. MS (ESI) m/z 434.1 [M + H]+. HRMS m/z [M + H]+ calcd for
C22H26Cl2N3O2 434.1402, found 434.1425. Melting point: 162−164
°C.
harvested onto 0.3% PEI-soaked Filtermax GF/A filters (Wallac) and
washed three times with ice-cold 50 mM Tris, pH 7.4, using a Perkin-
Elmer Filtermate 96-well harvester. The filters were subsequently dried
and placed on a hot plate (100 °C), and Melitilex-A (Wallac)
scintillant was applied. The filters were then removed from the hot
plate and allowed to cool. The filters were counted on a Wallac TriLux
microbeta counter (3 min/well). Residual [3H]N-methylspiperone
binding to filtered membranes was plotted as a function of log
[reference] or log [D2 test ligand], and the data were regressed using
the one-site competition model built into Prism 4.0 (GraphPad
software).
D2-Mediated cAMP Assay. HEK293T cells coexpressing the cAMP
biosensor GloSensor-22F (Promega) and hD2 L receptors were seeded
(20000 cells/20 μL/well) into white, clear-bottom, tissue culture
plates in HBSS, 20 mM Hepes, pH 7.4. After 30 min of recovery, cells
were treated with 10 μL of 3× test or reference drug prepared in
HBSS, 20 mM Hepes, pH 7.4. After 30 min, cells were treated with 10
μL of 1200 nM (4×) isoproterenol in 8% (4×) GloSensor reagent.
Luminescence per well per second was read on a Wallac TriLux
microbeta plate counter. Data were normalized to the isoproterenol
response (100%) and the maximal quinpirole-induced inhibition
thereof (0%) and regressed using the sigmoidal dose−response
function built into GraphPad Prism 4.0. Notably, HEK293T cells
expressing the GloSensor-22F alone (no hD2) were assayed in parallel
and displayed no inhibition of isoproterenol-stimulated cAMP, either
by quinpirole or by the test compounds, suggesting that the effect
observed in hD2 L-expressing cells was due to compound acting via the
recombinant receptor.
D2 β-Arrestin Recruitment Tango Assay. Recruitment of β-arrestin
to agonist-stimulated D2 L receptors was performed using a previously
described “Tango”-type assay.46 Briefly, HTLA cells stably expressing
β-arrestin-TEV protease and a tetracycline transactivator-driven
luciferase were plated in 15 cm dishes in DMEM containing 10%
FBS and transfected (via calcium phosphate) with 16 μg of a D2V2-
TCS-tTA construct.46 The next day, cells were plated in white, clear-
bottom, 384-well plates (Greiner; 15000 cells/well, 50 μL/well) in
DMEM containing 1% dialyzed FBS. The following day, the cells were
challenged with 10 μL/well of reference agonist (6 μM) or D2 test
ligand (6 μM) prepared in HBSS, 20 mM Hepes, pH 7.4, and 6%
DMSO (final ligand concentrations are 1 μM, final DMSO
concentration is 1%). After 18 h, the medium was removed and
replaced with 1× BriteGlo reagent (Promega), and luminescence per
well was read using a TriLux plate reader (1 s/well). Data were
normalized to vehicle (0%) and quinpirole (100%) controls and
regressed using the sigmoidal dose−response function built into
GraphPad Prism 4.0.0
Experimental Procedures for in Vivo Studies in Mice.
General Procedures. All experiments were approved by the Institu-
tional Animal Care and Use Committees at the University of North
Carolina, Chapel Hill, and Duke University. Wild-type and β-arrestin-2
knockout mice were housed under standard conditions −14 h light/
dark cycle (lights on 0600 h) with food and water provided ad libitum.
Adult, age-matched male and female wild-type and β-arrestin-2
knockout drug-naive mice were used for all behavioral testing.
Locomotor Activity of Compounds 19 and 35. Wild-type and β-
arrestin-2 knockout mice were treated with vehicle or 2 mg/kg
compounds 19 or 35 (ip) and were immediately placed into an open
field. Thirty minutes later, the animals were administered 6 mg/kg
PCP (ip) and were immediately returned to the open field for 90 min.
Horizontal activity, measured as distance traveled, was recorded over 5
min segments for the duration of testing. RMANOVA for the first 30
min of testing (baseline) revealed a significance within subject effects
of time [F(5300) = 38.344, p < 0.001]; the time by genotype, time by
treatment, or the time by treatment by genotype interactions were not
significant (Figure 2).
7-(3-(4-(2,3-Dichlorophenyl)-1,4-diazepan-1-yl)propoxy)-
3,4-dihydroisoquinolin-1(2H)-one (43). Compound 43 (125 mg)
was prepared as a white solid by the same procedure as preparing 22,
1
yield 71%. H NMR (300 MHz, CDCl3): δ 7.60 (d, J = 2.1 Hz, 1H),
7.14−7.08 (m, 3H), 7.02−7.00 (m, 2H), 6.02 (br., 1H), 4.10 (t, J = 6.3
Hz, 2H), 3.56−3.53 (m, 2H), 3.31−3.27 (m, 4H), 2.96−2.88 (m, 6H),
2.77−2.72 (m, 2H), 2.01(m, 4H). HPLC: 99%, RT 2.578 min. MS
(ESI) m/z 448.1 [M + H]+. Melting point: 116−117 °C.
Synthesis of compound 44 was described previously.27
7-(4-(4-(2,3-Dichlorophenyl)-1,4-diazepan-1-yl)butoxy)-
quinolin-2(1H)-one (45). Compound 45 (50 mg) was prepared as a
1
white solid by the same procedure as preparing 22, yield 46%. H
NMR (300 MHz, CDCl3): δ 11.54 (br, 1H), 7.71 (d, J = 6.9 Hz, 1H)
7.44 (d, J = 8.1 Hz, 1H), 7.09−7.07 (m, 2H), 7.01−6.97 (m, 1H),
6.82−6.78 (m, 2H), 6.53 (d, J = 9.3 Hz, 1H), 4.10 (t, J = 6.6 Hz, 2H),
3.30 (d, J = 5.1 Hz, 4H), 2.87−2.81 (m, 4H), 2.62 (t, J = 7.5 Hz, 2H),
2.01−1.99 (m, 2H), 1.92−1.82 (m, 2H), 1.76−1.68 (m, 2H). HPLC:
99%, RT 2.461 min. MS(ESI) m/z 460.2 [M + H]+. mp: 55−57 °C.
Experimental Procedures for in Vitro Biochemical Assays.
General Procedures. Experimental procedures for the radioligand
binding assays for the GPCRs listed in Supporting Information Table
S1 (including D1, D3, D4, D5, 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT1A)
are available online through the Psychoactive Drug Screening Program
(PDSP) Web site: http://pdsp.med.unc.edu/. The PDSP Assay
Protocol book is freely available at http://pdsp.med.unc.edu/
UNC−CH%20Protocol%20Book.pdf. The dopamine D2 radioligand
binding, cAMP biosensor, and β-arrestin recruitment Tango assays are
detailed below.
CHO−D2 Membrane Preparation and Radioligand Binding
Assay. CHO−D2 membrane preparation. Cells stably expressing
human D2 L receptors (CHO−D2 L) were plated in 15 cm dishes (in
DMEM containing 10% FBS) and grown to 90% confluence. Then
cells were washed with PBS, pH 7.4, and harvested by scraping into
PBS, pH 7.4. Harvested cells were centrifuged at 1000g for 10 min and
then hypotonically lysed by resuspension into ice-cold 50 mM Hepes,
1% BSA, pH 7.4. Membranes were isolated by centrifugation at 21000g
for 20 min. The supernatant was removed, and the membrane pellets
were stored at −80 °C until used for radioligand binding assays.
Radioligand Binding Assay. Membranes prepared as above were
resuspended to 1 μg protein/μL (measured by Bradford assay using
BSA as standard), and 50 μL was added to each well of a
polypropylene 96-well plate containing (per well) 50 μL of buffer
(20 mM Hepes, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 100 mM
N-methyl-D-gluconate, pH 7.4), 50 μL of 1.5 nM [3H]N-
methylspiperone (final concentration 0.3 nM), and reference or D2
test ligands at various concentrations ranging from 50 pM to 50 μM
(final concentrations ranging from 10 pM to 10 μM, triplicate
determinations for each concentration of D2 test ligand). After a 1.5 h
incubation in the dark at room temperature, the reactions were
A second analysis was run to analyze the locomotor activity of the
mice following PCP treatment over the entire 90 min following
injection. A RMANOVA noted a significant within subject effects of
time [F(171020) = 64.520, p < 0.001] and significant time by
genotype [F(171020) = 1.592, p < 0.012] and time by treatment
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interactions [F(341020) = 2.316, p < 0.001]. Because these analyses
indicated significant genotype and treatment effects, the RMANOVA
was run within each treatment condition as a function of genotype. For
the PCP-treated mice, no genotype differences were discerned across
time (Figure 2). For mice treated with 19, there was a significant
within subjects effect of time [F(17340) = 26.012, p < 0.001] and time
by genotype interaction [F(17340) = 1.901, p < 0.017]. The
Bonferroni test reported that 19 suppressed PCP-induced locomotion
in the WT relative to the β-arrestin 2 knockout mice at 50, 55, 60, 65,
75, and 105 min (ps <0.042). For mice treated with 35, there was a
significant within subject effect of time [F(17272) = 14.194, p < 0.001]
and time by genotype interaction [F(17272) = 1.729, p < 0.038]. The
Bonferroni test found that 35 suppressed PCP-induced locomotion in
the WT relative to the β-arrestin 2 knockout mice at 90 min (p <
0.050). Hence, these data show that at 2 mg/kg 19 is more efficacious
than 35 in reducing PCP-stimulated locomotion in wild-type mice;
neither compound affected PCP-induced activity in the β-arrestin-2
knockout mice.
(5) Ghosh, D.; Snyder, S. E.; Watts, V. J.; Mailman, R. B.; Nichols, D.
E. 9-Dihydroxy-2,3,7,11b-tetrahydro-1H-naph[1,2,3-de]isoquinoline: a
potent full dopamine D1 agonist containing a rigid beta-phenyldop-
amine pharmacophore. J. Med. Chem. 1996, 39, 549−555.
(6) Kilts, J. D.; Connery, H. S.; Arrington, E. G.; Lewis, M. M.;
Lawler, C. P.; Oxford, G. S.; O’Malley, K. L.; Todd, R. D.; Blake, B. L.;
Nichols, D. E.; Mailman, R. B. Functional selectivity of dopamine
receptor agonists. II. Actions of dihydrexidine in D2L receptor-
transfected MN9D cells and pituitary lactotrophs. J. Pharmacol. Exp.
Ther. 2002, 301, 1179−1189.
(7) Mottola, D. M.; Kilts, J. D.; Lewis, M. M.; Connery, H. S.;
Walker, Q. D.; Jones, S. R.; Booth, R. G.; Hyslop, D. K.; Piercey, M.;
Wightman, R. M.; Lawler, C. P.; Nichols, D. E.; Mailman, R. B.
Functional selectivity of dopamine receptor agonists. I. Selective
activation of postsynaptic dopamine D2 receptors linked to adenylate
cyclase. J. Pharmacol. Exp. Ther. 2002, 301, 1166−1178.
(8) Beaulieu, J. M.; Tirotta, E.; Sotnikova, T. D.; Masri, B.; Salahpour,
A.; Gainetdinov, R. R.; Borrelli, E.; Caron, M. G. Regulation of Akt
signaling by D2 and D3 dopamine receptors in vivo. J. Neurosci. 2007,
27, 881−885.
(9) Beaulieu, J. M.; Sotnikova, T. D.; Marion, S.; Lefkowitz, R. J.;
Gainetdinov, R. R.; Caron, M. G. An Akt/beta-arrestin 2/PP2A
signaling complex mediates dopaminergic neurotransmission and
behavior. Cell 2005, 122, 261−273.
(10) Chidiac, P.; Hebert, T.; Valiquette, M.; Dennis, M.; Bouvier, M.
Inverse agonist activity of beta-adrenergic antagonists. Mol. Pharmacol.
1993, 45, 490−499.
(11) Barker, E. L.; Westphal, R. S.; Schmidt, D.; Sanders-Bush, E.
Constitutively active 5-hydroxytryptamine2C receptors reveal novel
inverse agonist activity of receptor ligands. J. Biol. Chem. 1994, 269,
11687−11690.
(12) Rauser, L.; Savage, J. E.; Meltzer, H. Y.; Roth, B. L. Inverse
agonist actions of typical and atypical antipsychotic drugs at the human
5-hydroxytryptamine(2C) receptor. J. Pharmacol. Exp. Ther. 2001, 299,
83−89.
(13) Gilliland, S. L.; Alper, R. H. Characterization of dopaminergic
compounds at hD2short, hD4.2 and hD4.7 receptors in agonist-
stimulated [35S]GTPgammaS binding assays. Naunyn Schmiedebergs
Arch. Pharmacol. 2000, 361, 498−504.
(14) Burstein, E. S.; Ma, J.; Wong, S.; Gao, Y.; Pham, E.; Knapp, A.
E.; Nash, N. R.; Olsson, R.; Davis, R. E.; Hacksell, U.; Weiner, D. M.;
Brann, M. R. Intrinsic efficacy of antipsychotics at human D2, D3, and
D4 dopamine receptors: identification of the clozapine metabolite N-
desmethylclozapine as a D2/D3 partial agonist. J. Pharmacol. Exp.
Ther. 2005, 315, 1278−1287.
(15) Peroutka, S. J.; Snyder, S. H. Long-term antidepressant
treatment decreases spiroperidol-labeled serotonin receptor binding.
Science 1980, 210, 88−90.
(16) Berry, S.; Shah, M.; Khan, N.; Roth, B. Rapid agonist-induced
internalization of the 5-hydroxytryptamine2A receptor occurs via the
endosome pathway in vitro. Mol. Pharmacol. 1996, 50, 306−313.
(17) Willins, D. L.; Alsayegh, L.; Berry, S. A.; Backstrom, J. R.;
Sanders-Bush, E.; Friedman, L.; Khan, N.; Roth, B. L. Serotonergic
antagonist effects on trafficking of serotonin 5-HT2A receptors in vitro
and in vivo. Ann. N.Y. Acad. Sci. 1998, 861, 121−127.
(18) Willins, D.; Berry, S.; Alsayegh, L.; Backstrom, J.; Sanders-Bush,
E.; Roth, B. Clozapine and other 5-hydroxytryptamine2A receptor
antagonists alter the subcellular distribution of 5-hydroxytryptamine2A
receptors in vitro and in vivo. Neuroscience 1999, 91, 599−606.
(19) Urban, J. D.; Clarke, W. P.; von Zastrow, M.; Nichols, D. E.;
Kobilka, B.; Weinstein, H.; Javitch, J. A.; Roth, B. L.; Christopoulos, A.;
Sexton, P. M.; Miller, K. J.; Spedding, M.; Mailman, R. B. Functional
selectivity and classical concepts of quantitative pharmacology. J.
Pharmacol. Exp. Ther. 2007, 320, 1−13.
ASSOCIATED CONTENT
* Supporting Information
■
S
Radioligand binding affinities of compounds 19 and 35 at select
dopamine and serotonin receptors. 1H and 13C NMR spectra of
compounds 35 and 37. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For J.J.: phone, 919-843-8459; fax, 919-843-8465; E-mail,
jianjin@unc.edu. For B.L.R.: phone, 919-966-7535; fax, 919-
843-5788; E-mail, bryan_roth@med.unc.edu.
Author Contributions
⊥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NIH (U19MH082441S1 and U19MH082441)
for financial support and Dr. Weihe Zhang for critical reading of
synthetic procedures and characterization data.
ABBREVIATIONS USED
■
GPCR, G protein-coupled receptor; SFSR, structure−func-
tional selectivity relationships; D2R, dopamine D2 receptor;
cAMP, cyclic adenosine monophosphate; PK, pharmacokinetic;
CNS, central nervous system; SAR, structure−activity relation-
ship; LHS, left-hand side; RHS, right-hand side; ip, intra-
peritoneal
REFERENCES
■
(1) Furchgott, R. F. The use of haloalkylamines in the differentiation
of receptors and in the determination of dissociation constants of
agonist-receptor complexes. In Advances in Drug Research; Harper, N.;
Simmonds, A., Eds.; Academic Press: New York, 1966; Vol. 3, pp 21−
55.
(2) Roth, B. L.; Chuang, D. M. Multiple mechanisms of serotonergic
signal transduction. Life Sci. 1987, 41, 1051−1064.
(3) Berg, K. A.; Maayani, S.; Goldfarb, J.; Scaramellini, C.; Leff, P.;
Clarke, W. P. Effector pathway-dependent relative efficacy at serotonin
type 2A and 2C receptors: evidence for agonist-directed trafficking of
receptor stimulus. Mol. Pharmacol. 1998, 54, 94−104.
(4) Parrish, J.; Braden, M.; Gundy, E.; Nichols, D. E. Differential
phospholipase C activation by phenylalkylamine serotonin 5-HT2A
receptor agonists. J. Neurochem. 2005, 95, 1575−1584.
(20) Mailman, R. B. GPCR functional selectivity has therapeutic
impact. Trends Pharmacol. Sci. 2007, 28, 390−396.
(21) Luttrell, L. M.; Ferguson, S. S.; Daaka, Y.; Miller, W. E.;
Maudsley, S.; Della Rocca, G. J.; Lin, F.; Kawakatsu, H.; Owada, K.;
Luttrell, D. K.; Caron, M. G.; Lefkowitz, R. J. Beta-arrestin-dependent
7152
dx.doi.org/10.1021/jm300603y | J. Med. Chem. 2012, 55, 7141−7153

Journal of Medicinal Chemistry
Article
formation of beta2 adrenergic receptor−Src protein kinase complexes.
Science 1999, 283, 655−661.
functional uroselective alpha(1)-adrenoceptor antagonists. J. Med.
Chem. 1997, 40, 2674−2687.
(37) Fu, X. T.; Lei; Yuan, M.; Shi, J. Design, synthesis and
vasorelaxant activity of R,S-1-(substituted phenyl)-4-[3-(naphtha-1-yl-
oxy)-2-hydroxypropyl]-piperazine derivatives. Yaoxue Xuebao 2007,
42, 735−740.
(38) Tsuruta, K.; Frey, E. A.; Grewe, C. W.; Cote, T. E.; Eskay, R. L.;
Kebabian, J. W. Evidence that LY-141865 specifically stimulates the D-
2 dopamine receptor. Nature 1981, 292, 463−465.
(39) Banno, K.; Fujioka, T.; Kikuchi, T.; Oshiro, Y.; Hiyama, T.;
Nakagawa, K. Studies on 2(1H)-quinolinone derivatives as neuroleptic
agents I. Synthesis and biological activities of (4-phenyl-1-piperazinyl)-
propoxy-2(1H)-quinolinone derivatives. Chem. Pharm. Bull. 1988, 36,
4377−4388.
(40) Kiss, B.; Horvath, A.; Nemethy, Z.; Schmidt, E.; Laszlovszky, I.;
Bugovics, G.; Fazekas, K.; Hornok, K.; Orosz, S.; Gyertyan, I.; Agai-
Csongor, E.; Domany, G.; Tihanyi, K.; Adham, N.; Szombathelyi, Z.
Cariprazine (RGH-188), a dopamine D(3) receptor-preferring, D(3)/
D(2) dopamine receptor antagonist−partial agonist antipsychotic
candidate: in vitro and neurochemical profile. J. Pharmacol. Exp. Ther.
2010, 333, 328−340.
(41) van Wijngaarden, I.; Kruse, C. G.; van der Heyden, J. A.; Tulp,
M. T. 2-Phenylpyrroles as conformationally restricted benzamide
analogues. A new class of potential antipsychotics. 2. J. Med. Chem.
1988, 31, 1934−1940.
(42) van Wijngaarden, I.; Kruse, C. G.; van Hes, R.; van der Heyden,
J. A.; Tulp, M. T. 2-Phenylpyrroles as conformationally restricted
benzamide analogues. A new class of potential antipsychotics. 1. J.
Med. Chem. 1987, 30, 2099−2104.
(43) Bohn, L. M.; Lefkowitz, R. J.; Gainetdinov, R. R.; Peppel, K.;
Caron, M. G.; Lin, F. T. Enhanced morphine analgesia in mice lacking
beta-arrestin 2. Science 1999, 286, 2495−2498.
(22) Lefkowitz, R. J.; Shenoy, S. K. Transduction of receptor signals
by beta-arrestins. Science 2005, 308, 512−517.
(23) Abbas, A.; Roth, B. L. Arresting serotonin. Proc. Natl. Acad. Sci.
U.S.A 2008, 105, 831−832.
(24) Urban, J. D.; Vargas, G. A.; von Zastrow, M.; Mailman, R. B.
Aripiprazole has functionally selective actions at dopamine D2
receptor-mediated signaling pathways. Neuropsychopharmacology
2007, 32, 67−77.
(25) Violin, J. D.; Lefkowitz, R. J. [beta]-Arrestin-biased ligands at
seven-transmembrane receptors. Trends Pharmacol. Sci. 2007, 28,
416−422.
(26) Gesty-Palmer, D.; Flannery, P.; Yuan, L.; Corsino, L.; Spurney,
R.; Lefkowitz, R. J.; Luttrell, L. M. A beta-arrestin-biased agonist of the
parathyroid hormone receptor (PTH1R) promotes bone formation
independent of G protein activation. Sci. Transl. Med. 2009, 1, 1ra1.
(27) Butini, S.; Gemma, S.; Campiani, G.; Franceschini, S.; Trotta, F.;
Borriello, M.; Ceres, N.; Ros, S.; Coccone, S. S.; Bernetti, M.; De
Angelis, M.; Brindisi, M.; Nacci, V.; Fiorini, I.; Novellino, E.; Cagnotto,
A.; Mennini, T.; Sandager-Nielsen, K.; Andreasen, J. T.; Scheel-Kruger,
J.; Mikkelsen, J. D.; Fattorusso, C. Discovery of a new class of potential
multifunctional atypical antipsychotic agents targeting dopamine D3
and serotonin 5-HT1A and 5-HT2A receptors: design, synthesis, and
effects on behavior. J. Med. Chem. 2009, 52, 151−169.
(28) Allen, J. A.; Yost, J. M.; Setola, V.; Chen, X.; Sassano, M. F.;
Chen, M.; Peterson, S.; Yadav, P. N.; Huang, X.-P.; Feng, B.; Jensen,
N. H.; Che, X.; Bai, X.; Frye, S. V.; Wetsel, W. C.; Caron, M. G.;
Javitch, J. A.; Roth, B. L.; Jin, J. Discovery of Beta-Arrestin-Biased
Dopamine D2 Ligands for Probing Signal Transduction Pathways
Essential for Antipsychotic Efficacy. Proc. Natl. Acad. Sci. U.S.A. 2011,
108, 18488−18493.
(29) Lawler, C. P.; Prioleau, C.; Lewis, M. M.; Mak, C.; Jiang, D.;
Schetz, J. A.; Gonzalez, A. M.; Sibley, D. R.; Mailman, R. B.
Interactions of the novel antipsychotic aripiprazole (OPC-14597) with
dopamine and serotonin receptor subtypes. Neuropsychopharmacology
1999, 20, 612−627.
(44) Nordquist, R. E.; Risterucci, C.; Moreau, J. L.; von Kienlin, M.;
Kunnecke, B.; Maco, M.; Freichel, C.; Riemer, C.; Spooren, W. Effects
of aripiprazole/OPC-14597 on motor activity, pharmacological models
of psychosis, and brain activity in rats. Neuropharmacology 2008, 54,
405−416.
(45) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts
of common laboratory solvents as trace impurities. J. Org. Chem. 1997,
62, 7512−7515.
(46) Barnea, G.; Strapps, W.; Herrada, G.; Berman, Y.; Ong, J.; Kloss,
B.; Axel, R.; Lee, K. J. The genetic design of signaling cascades to
record receptor activation. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 64−
69.
(30) Mailman, R. B.; Murthy, V. Third generation antipsychotic
drugs: partial agonism or receptor functional selectivity? Curr. Pharm.
Des. 2010, 16, 488−501.
(31) Mallikaarjun, S.; Salazar, D. E.; Bramer, S. L. Pharmacokinetics,
Tolerability, and Safety of Aripiprazole following Multiple Oral Dosing
in Normal Healthy Volunteers. J. Clin. Pharmacol. 2004, 44, 179−187.
(32) Johnson, D. S.; Choi, C.; Fay, L. K.; Favor, D. A.; Repine, J. T.;
White, A. D.; Akunne, H. C.; Fitzgerald, L.; Nicholls, K.; Snyder, B. J.;
Whetzel, S. Z.; Zhang, L.; Serpa, K. A. Discovery of PF-00217830: Aryl
piperazine napthyridinones as D2 partial agonists for schizophrenia
and bipolar disorder. Bioorg. Med. Chem. Lett. 2011, 21, 2621−2625.
(33) Oshiro, Y.; Sato, S.; Kurahashi, N.; Tanaka, T.; Kikuchi, T.;
Tottori, K.; Uwahodo, Y.; Nishi, T. Novel antipsychotic agents with
dopamine autoreceptor agonist properties: synthesis and pharmacol-
ogy of 7-[4-(4-phenyl-1-piperazinyl)butoxy]-3,4-dihydro-2(1H)-qui-
nolinone derivatives. J. Med. Chem. 1998, 41, 658−667.
(34) Vangveravong, S.; Zhang, Z.; Taylor, M.; Bearden, M.; Xu, J.;
Cui, J.; Wang, W.; Luedtke, R. R.; Mach, R. H. Synthesis and
characterization of selective dopamine D(2) receptor ligands using
aripiprazole as the lead compound. Bioorg. Med. Chem. 2011, 19,
3502−3511.
(35) Martin, G. E.; Elgin, R. J.; Mathiasen, J. R.; Davis, C. B.;
Kesslick, J. M.; Baldy, W. J.; Shank, R. P.; Distefano, D. L.; Fedde, C.
L.; Scott, M. K. Activity of Aromatic Substituted Phenylpiperazines
Lacking Affinity for Dopamine Binding Sites in a Preclinical Test of
Antipsychotic Efficacy. J. Med. Chem. 1989, 32, 1052−1056.
(36) Elworthy, T. R.; Ford, A. P. D. W.; Bantle, G. W.; Morgans, D.
J.; Ozer, R. S.; Palmer, W. S.; Repke, D. B.; Romero, M.; Sandoval, L.;
Sjogren, E. B.; Talamas, F. X.; Vazquez, A.; Wu, H.; Arredondo, N. F.;
Blue, D. R.; DeSousa, A.; Gross, L. M.; Kava, M. S.; Lesnick, J. D.;
Vimont, R. L.; Williams, T. J.; Zhu, Q. M.; Pfister, J. R.; Clarke, D. E.
N-Arylpiperazinyl-N′-propylamino derivatives of heteroaryl amides as
7153
dx.doi.org/10.1021/jm300603y | J. Med. Chem. 2012, 55, 7141−7153