Molecular determinants of selectivity and efficacy at the dopamine D3 receptor

Article abstract of DOI:10.1021/jm300482h

The dopamine D3 receptor (D3R) has been implicated in substance abuse and other neuropsychiatric disorders. The high sequence homology between the D3R and D2R, especially within the orthosteric binding site (OBS) that binds dopamine, has made the developm

Full text of DOI:10.1021/jm300482h

Article
pubs.acs.org/jmc
Molecular Determinants of Selectivity and Efficacy at the Dopamine
D3 Receptor
Amy Hauck Newman,*,† Thijs Beuming,‡ Ashwini K. Banala,† Prashant Donthamsetti,§
Katherine Pongetti,∥ Alex LaBounty,∥ Benjamin Levy,† Jianjing Cao,† Mayako Michino,⊥
Robert R. Luedtke,∥ Jonathan A. Javitch,*,§ and Lei Shi*,⊥,#
†Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug AbuseIntramural
Research Program, Baltimore, Maryland, United States
‡Schrodinger Inc., New York, New York, United States
̈
§Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and
Surgeons, New York, New York, United States
∥Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas, United
States
#
⊥Department of Physiology and Biophysics and Institute for Computational Biomedicine, Weill Medical College of Cornell
University, New York, New York, United States
S
* Supporting Information
ABSTRACT: The dopamine D3 receptor (D3R) has been implicated in
substance abuse and other neuropsychiatric disorders. The high sequence
homology between the D3R and D2R, especially within the orthosteric binding
site (OBS) that binds dopamine, has made the development of D3R-selective
compounds challenging. Here, we deconstruct into pharmacophoric elements a
series of D3R-selective substituted-4-phenylpiperazine compounds and use
computational simulations and binding and activation studies to dissect the
structural bases for D3R selectivity and efficacy. We find that selectivity arises
from divergent interactions within a second binding pocket (SBP) separate from
the OBS, whereas efficacy depends on the binding mode in the OBS. Our findings
reveal structural features of the receptor that are critical to selectivity and efficacy
that can be used to design highly D3R-selective ligands with targeted efficacies.
These findings are generalizable to other GPCRs in which the SBP can be targeted by bitopic or allosteric ligands.
of selective D2R, D3R, and D4R antagonists and partial
agonists.1,5,6 The selective distribution of D3R in limbic regions
of the brain, especially the nucleus accumbens,7 has fortified
interest in the D3R as a potential target for drug discovery,
especially for drug addiction.5 Notably, several D3R-selective
agents have been evaluated in animal models of addiction and
other neuropsychiatric disorders,5,8 and clinical trials have been
initiated for smoking cessation with one of these agents
(GSK598809, compound 1 in Figure 1; see ref 9).
Although structural templates differ across laboratories,
structure−activity relationships (SAR) that have been devel-
oped at D3R for antagonists and partial agonists have
traditionally focused on a functionalized tertiary amine (e.g.,
2,3-diCl-4-phenylpiperazine in Figure 1) separated from an
extended arylamide terminus by a requisite 4-atom (n-butyl)
linking chain.5,8 Functionalizing the linker with a 2,3-trans
olefin or a 3-substituent (e.g., OH or F) further increases D3R
selectivity.10−12 Whereas the 4-phenylpiperazine has been
INTRODUCTION
■
The neurotransmitter dopamine is synthesized in and released
from dopaminergic neurons to stimulate G-protein coupled
receptors, thereby affecting movement, cognition, and emotion.
Dopamine receptors are classified into two subfamilies, D1-like
and D2-like, based on sequence, G-protein coupling, and
pharmacology. The D1-like subfamily is comprised of the D1
and D5 receptors that are coupled to stimulatory G-protein α
subunits (Gs/olf) and activate adenylyl cyclase. In contrast, the
D2, D3, and D4 receptors (D2R, D3R, and D4R, respectively),
members of the D2-like subfamily, are coupled to inhibitory G-
protein α subunits (Gi/o), thus inhibiting adenylyl cyclase as
well as modulating other effector pathways.1−3
The high degree of sequence identity within the trans-
membrane (TM) segments of the D2R-like receptors and the
near-identity of the residues that form the binding site in these
receptors4 have made it challenging to create subtype-selective
agents that possess physicochemical properties suitable for in
vivo characterization of the physiological roles of these receptor
subtypes. Nevertheless, significant progress has been made for
D2-subfamily receptor-selective ligands, including the discovery
Received: April 5, 2012
© XXXX American Chemical Society
A
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Figure 1. Representative D2-like compounds.
characterized as the primary recognition element, binding
selectivity between D2R, D3R, and D4R is dependent on the
length of the linker that separates this primary pharmacophore
from the aryl terminus.5,13
acetylcholine receptors, where much emphasis has gone instead
into the development of allosteric agents that target subtype-
specific sites (reviewed in ref 16). It is interesting to note that
the region exploited in the development of these allosteric
compounds is thought to include EL2, position 3.28, and the
extracellular portion of TM7,16,17 all parts of the SBP.
Using a strategy that combines synthetic chemistry, binding
and functional assays, and computational modeling and
simulations, herein we identify components critical to the
binding specificity and efficacy of D3R-targeted compounds.
Despite these successful medicinal chemistry efforts leading
to compounds such as R22 (2) with >100-fold selectivity for
D3R over D2R10 (Figure 1), the structural basis for this binding
specificity has not been elucidated. The recent availability of a
high-resolution structure of the D3R has made structure-based
drug design a possibility, but the very high degree of homology
between D3R and D2R continues to pose a major challenge.
The crystal structure of D3R4 revealed a binding site located in
the upper-half of the transmembrane domain, in which the D2-
like receptor antagonist eticlopride (3) is bound. This
orthosteric binding site (OBS), in which dopamine is also
thought to bind, is enclosed by TMs 3, 5, 6, and 7, consistent
with our accumulated understanding of aminergic GPCRs
based on structure−function studies.14 Of the 18 eticlopride
contact residues in the D3R structure, 17 are identical in the
D2R and one is similar.4 A predicted binding pose of
compound 2 suggested that the 2,3-diCl-4-phenylpiperazine
binds in the OBS in close alignment with eticlopride, with its
butylamide linking chain extending the 2-indole terminus into a
second binding pocket (SBP).4
The discovery of novel D2R/D3R ligands has been reported
recently using computational screening of large compound
libraries in both a D3R homology model and the D3R crystal
structure.15 It is important to note, however, that the two
critical features of drug design, specificity and efficacy, were not
addressed in that study. More importantly, any screening effort
targeted exclusively to the OBS of such similar receptors as the
D2R and D3R will almost certainly fail to identify highly
selective compounds. Similar challenges exist for other GPCRs
that share a common orthosteric agonist, such as the muscarinic
RESULTS AND DISCUSSION
■
The current study was directed toward understanding the
structural bases of the specificity of existing compounds that
would facilitate rational drug design as well as provide general
principles that extend beyond this particular target. To develop
an understanding of drug efficacy in addition to specificity, we
chose to consider not only the inactive structure represented by
the D3R crystal structure but also the active D3R OBS
conformation by taking advantage of recent breakthrough high-
resolution active-state GPCR structures.18,19 Thus, by system-
atically deconstructing the full-length D3R-selective com-
pounds 2, JJC 7−065 (4), and JJC 7−082 (5, Figure 1),
examining both binding and efficacy, and using these data to
validate computational models, we sought to elucidate
structural features responsible both for D3R versus D2R
selectivity and for D3R/D2R efficacy.
Binding Characterization of Compound 2 and Two
Cognate Full-Length Compounds. To investigate the
contribution of each component of 2 to D3R binding and
selectivity, we synthesized compound 4,20,21 an analogue of 2
that lacks the 3-OH group on the linking chain. Compound 4
and 2 bind D3R with similar high affinity (Ki = 1.4 vs 1.1 nM),
whereas compound 4 has somewhat higher affinity than 2 at
B
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Table 1. D3R and D2R Binding Data for the D3R-Selective Compounds and Corresponding Synthons
a
b
c
Compound and data previously described in ref 10. Compound previously described in ref 20. Compound previously described in ref 10,22.
d
e
f
g
Compound previously described in ref 28. Compound previously described in ref 29. Compound previously described in ref 30. Compounds
previously described in ref 12.
D2R (Ki = 103 vs 433 nM, Table 1). Thus, as previously
reported, the 3-OH substituent significantly improves D3R
selectivity over D2R by reducing affinity at D2R rather than by
improving affinity at D3R.10,11 We also synthesized compound
510,22 (Figure 1) by replacing the 2,3-diCl substitution of the
pendant phenyl ring of compound 4 with a 2-OCH3 group,
which is found in many D3R ligands. As for compound 4, the
D3R affinity of compound 5 is high (Ki = 0.32 nM), whereas
D2R affinity is ∼100-fold lower (Table 1).
ing23) (Figure 2a). In D3R, our results show convergence in the
binding of the SP moiety at the interface of TMs 2, 3, and EL1
and EL2 (Figure 2b,c). For all three ligands, the indole ring of
the SP packs against a hydrophobic pocket formed by residues
Val862.61, Leu892.64, Gly94 (EL1), Phe1063.28, and Cys181
(EL2), which interestingly overlaps with the positions of
residues identified previously as responsible for D4R over D2R
selectivity.13,24 We termed this portion of the SBP Ptm23 to
indicate the TMs that form contacts with the SP in this pose.
By contrast, the SP of 2, 4, and 5 did not interact with Ptm23 in
any of the binding poses observed in D2R, and our simulations
suggest that the binding mode of the SP in this receptor is less
well-defined and more widely distributed at the interface of
TMs 1, 2, and 7 of D2R (Figure 2d).
Using representative snapshots from the D3R-compound 2
and D2R-compound 2 trajectories, we carried out WaterMap
calculations25 to characterize the distributions of hydration sites
in the SBP. Water molecules in hydrophobically enclosed
regions such as the Ptm23 are less stable than bulk water due to
less favorable enthalpy (interactions with nonpolar atoms) and/
or entropy (constriction in mobility), and displacement of these
high-energy waters (HEWs) by ligands has been shown to
improve affinity26 and affect selectivity.27 The results indicate
that several HEWs are consistently positioned in the Ptm23 of
both D3R and D2R, while fewer HEWs are found at the
interface of TMs1, 2, and 7 (see the red spheres in Figure 2c,d).
Thus, in D3R, but not in D2R, the indole moiety of 2 occupies
a region from which several HEWs are displaced, consistent
with the higher affinity of 2 for D3R.
On the basis of SAR and previous modeling studies,10,13 we
hypothesized that the 2,3-diCl- or 2-OCH3-substituted-4-
phenylpiperazine termini, defined as the primary pharmaco-
phore (PP), would bind within the OBS of both D3R and
D2R.4 This orientation of the ligand within the binding site
positions the arylamide terminus, or the secondary pharmaco-
phore (SP), in a second binding pocket (SBP) at the interface
of TMs 1, 2, 3, and 7 and the first and second extracellular
loops (EL1 and EL2), which can accommodate the SP in
several poses. While the OBS is completely conserved between
D3R and D2R, the SBP differs substantially, suggesting that the
varied interactions of the ligands with the SBP may be
responsible for D3R over D2R selectivity.4 To evaluate this
hypothesis, we carried out induced-fit docking (IFD), and
subsequent molecular dynamics (MD) simulations to predict
the binding poses for 2, 4, and 5 in D3R-structure-based
models of the D3R and D2R.
In both models, the PP moiety makes extensive interactions
with conserved residues from TMs 3, 5, 6, and 7 of the OBS
and forms the signature salt bridge between the protonated
piperazine amine and Asp3.32(Ballesteros−Weinstein number-
C
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extended the butyl chain to the propionamides (1612 and 1712),
and we also prepared the arylamide terminus compound 18
with an appended butyl linker in the 2-position of the indole
ring system.
All synthons were evaluated for binding to D3R and D2R
stably expressed in HEK 293 cells by displacement of
[
125I]IABN.31 The binding results for the synthons show that
the 4-phenylpiperazine end of the D3R-selective molecules
have moderate to high binding affinities for both the D3R and
D2R with little to no selectivity (Table 1). Notably, as the alkyl
linking chain was extended from the N-phenylpiperazine to the
N-n-butyl analogues, binding affinities for both D3R and D2R
improved from high to low nanomolar levels. Consistent with
previous SAR,10−12,21 this suggests that hydrophobic inter-
actions between the linker and a previously undefined portion
of the receptor contribute to the progressive enhancement of
affinity.
Although it substantially enhanced affinity at both receptors,
increasing the length of the linking chain only very slightly
improved D3R selectivity. Similarly, addition of the 3-OH
group to the linking chain in the absence of the SP enhanced
D3R selectivity only 2-fold. The propionamides compounds 16
and 17 showed the highest D3R selectivities of their respective
synthon series, but their selectivity was still only 3-fold
compared to the ∼100-fold selectivity seen in the full-length
compounds. Not surprisingly, the isolated 2-butyl-indole amide
terminus (18) demonstrated very low affinities competing for
binding of [125I]IABN at both D3R and D2R.
All the 2,3-diCl-synthons showed ∼5−13-fold higher
affinities than their respective 2-OCH3-analogues for both
D3R and D2R. In the 2,3-diCl series, the attachment of the SP
contributes to D3R over D2R selectivity by reducing binding
affinity for D2R without a significant impact on D3R binding
affinity, while for the 2-OCH3 compounds, the addition of the
2-indole amide to the compound with the four-carbon linker
worsened D2R affinity but also enhanced D3R affinity.
Prediction of the Binding Modes of Synthons. To put
the observed SAR in the context of receptor structure, we
predicted the binding modes in the D3R structure using IFD32
for four pairs of synthons from the 2,3-diCl− and 2-OCH3−
series: NH-PP (8 and 9), the N-Me-PP (10 and 11), the N-n-
butyl-PP (14 and 15), and the propionamides (16 and 17). In
our IFD calculations, overall, the piperazine is in the chair
conformation in all poses, and the protonated nitrogen
consistently forms a salt-bridge with Asp1103.32. However, the
modifications on the phenyl ring and on the protonated
piperazine nitrogen lead to distinct binding modes of the 4-
phenylpiperazine core in the OBS. Thus, the phenyl ring in the
OBS can adopt a variety of orientations, with the substituents
pointing toward the intra- or extracellular side (henceforth
referred to as “downward-facing” and “upward-facing” config-
urations, both of which are largely “perpendicular” to the
membrane) or with the ring in the plane of the membrane
(“parallel”). Models of the complexes were selected by
identifying representative members of low-energy clusters of
poses (Supporting Information Figure S1).
Figure 2. Predicted binding modes of the full-length compounds 2, 4,
and 5 in D3R and D2R. (a) The binding mode of 2 (orange stick) in
D3R shows the interaction of the SP with a hydrophobic pocket at the
interface of TMs 2, 3, and EL1 and EL2 (Ptm23). A cone drawn from
the C1 atom of the butyl-linker to the indole ring represents the
orientation of the SP. (b) The binding poses for 2 (orange), 4
(yellow), and 5 (cyan) in D3R are similar and converged. Snapshots of
ligands from the equilibrated stage of MD simulations are super-
imposed by the OBS of the receptor. (c,d) The ensembles of poses for
2, 4, and 5 in D3R (c) and D2R (d) are shown by cones representing
the orientation of the SP. The SP packs against the Ptm23 pocket in
D3R (defined by residues Val862.61, Leu892.64, Phe1063.28, Gly94EL1
,
and Cys181EL2), while it is further away from Ptm23 and more
diffusely distributed in D2R. High energy (>2.5 kcal/mol) hydration
sites in the SBP of D3R and D2R, as determined using WaterMap, are
shown as red spheres. While the compound 2 SP in D3R displaces
several higher-energy waters in Ptm23, in D2R the indole ring only
partially displaces a single HEW site, consistent with the selectivity of
2 for D3R over D2R.
Design, Synthesis, and Binding Affinities of Synthons.
To further characterize the binding modes of each component
of these D3R-selective ligands, we systematically deconstructed
2, 4, and 5 into pharmacophoric components, which we termed
synthons. Specifically, the 2,3-diCl− and 2-OCH3-4-phenyl-
piperazine cores (8 and 9, respectively) were systematically
substituted in methylene group increments to give the N-Me
(1028 and 1129), N-ethanol (12), N-n-propyl (13), and N-n-
butyl (14 and 1530) analogues (Table 1). We then further
For the NH-PP compound of the 2,3-diCl series (8), the
phenyl ring significantly tilts toward the plane of the membrane
(Figure 3a). This “parallel” conformation places the chloro-
substituents facing TM6 where they interact with Phe6.52
,
His6.55, and Val6.56 (Figure 3a). In contrast, for the compounds
with an alkyl group attached to the protonated nitrogen (10,
14, and 16), the phenyl ring is predominantly positioned
D
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All of the 2-OCH3 substituted synthons, from the NH-PP
compound to the propionamide (9, 11, 15, and 17), prefer a
“perpendicular” orientation. In contrast to the 2,3-diCl
substituents in compounds 10, 14, and 16, which are more
likely to be “upward-facing”, the predominant binding mode for
all the alkyl substituted compounds (9, 11, 15, and 17) is
“downward facing” (Supporting Information Figure S1). In this
configuration, the 2-OCH3 methyl group makes hydrophobic
contacts with Ser5.46 and Phe6.52 (Figure 3b,d,f,h); in the less
frequent “upward-facing” configuration, it interacts with Val5.39
and His6.55
.
In both series, the N-methyl group of N-Me-PP (10 and 11)
makes hydrophobic contact with Thr7.39 and Tyr7.43 (Figure
3c,d). When the linker length is increased, the hydrophobic and
saturated butyl group of N-n-butyl-PP (14 and 15) shows
significant flexibility in the docking results. Thus, in addition to
being able to adopt extended orientations that point toward the
divergent TMs 1, 2, 3, and 7 interface and barely contact
individual Ptm23 residues, it can also insert into a hydrophobic
pocket formed by Trp6.48, Phe6.51, Thr7.39, Gly7.42, and Tyr7.43
,
which we termed Ptm67 (Figure 3e,f, Supporting Information
Figure S1). The WaterMap calculations of the D3R crystal
structure show a HEW in Ptm67 displaced by both compounds
14 and 15 (Supporting Information Figure S2), suggesting that
the Ptm67 pocket is the most preferred location of the butyl
chain.
The docking results of compounds 16 and 17 show that
when the synthons are extended even further (Table 1), the
terminal propionamides access Ptm23 (Figure 3g,h). While the
indole moiety of the higher-affinity 2, 4, and 5 displaces the
HEWs in Ptm23, the shorter propionamide synthons 16 and 17
do not extend as deeply toward EL1 and thus do not displace
these HEWs, consistent with their weak D3R-selectivity (Table
1). Interestingly, in both D3R and D2R, the addition of the
propionamide (16 and 17) prevents the butyl groups from
entering the putative high-affinity Ptm67 pocket, thereby
reducing affinity ∼2−4 fold compared to 14 and 15.
The extensive hydrophobic interactions observed between
the longer linkers and TM7 might hinder rearrangements
associated with the conformational transition toward the active
state,18,19 specifically, they may prevent the extracellular
portions of TM7 and TM5 from moving toward each other
(Figure 3i). We predicted, therefore, that there might be a
spectrum of efficacy for the synthons, especially within the 2,3-
diCl series in which the unsubstituted NH-PP (8) has a
significantly different binding mode.
Functional Evaluation of the D3R-Selective Ligands
and Their Synthons. While SAR for D3R binding affinity and
selectivity is well-defined, elucidating SAR for D3R efficacy has
been more challenging.10−12,33 Because it has been difficult to
measure D3R activation reliably using second messenger assays,
particularly with Gi-coupled readouts,34,35 we developed a
BRET-based assay that measures activation of GoA by either
D3R or D2R to determine the efficacies of 2, 4, and 5. This
assay is a modified version of BRET-based assays that have
been described previously,36,37 in which agonist-induced G-
protein activation by coexpressed receptor leads to separation
of GαoA-91-Rluc8 and complemented mVenus-Gβ1γ2, resulting
in a reduction in BRET (Figure 4a). As expected, dopamine
and quinpirole (6) acted as full agonists, whereas sulpiride (7)
increased the BRET signal through its actions as an inverse
agonist (Figure 4b,c). Sulpiride (100 nM) dramatically right-
shifted the potency of dopamine at both D3R and D2R,
Figure 3. Predicted binding modes of compounds 8−11 and 14−17 in
the OBS of the D3R. Predicted binding poses, viewed extracellularly,
of eight bound compounds from the 2,3-diCl (8 (a), 10 (c), 14 (e),
and 16 (g)), and the 2-OCH3 (9 (b), 11 (d), 15 (f), and 17 (h))
series, based on IFD to the crystal structure of D3R. Side chains of the
residues within 3.7 Å of the bound compound are shown. The residues
in the OBS are colored in green, while the SBP residues shown in (e−
h) are colored in cyan. In (e) and (f), two poses for each compound
are shown, with the linker either in an extended form or occupying the
Ptm67 (see text). In (i), a zoom-in view of the OBS shows the
superimposed binding modes of compounds 8 (yellow) and 14
(orange). The hydrophobic interactions between the linker of
compound 14 and Thr7.39 and Tyr7.43, which are absent for compound
8, would prevent the relative rearrangement of the extracellular
portions of TM5 and TM7.
perpendicular to the plane of the membrane, and the chloro-
substituents are reoriented toward the extracellular part of the
binding site (upward-facing) where they interact with the
imidazole ring of His6.55 and are in proximity to Val5.39 (Figure
3c,e,g). However, the phenyl ring of compound 10 still has an
intermediate probability of being in a “parallel” orientation
(Supporting Information Figure S1).
E
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a
Table 2. Emax Data in the Go BRET Assay
D3R
SEM
D2R
SEM
1.62
2
20.56
−10.31
16.00
8.38
5.27
0.61
1.04
3.86
1.58
3.44
4.98
0.64
4.80
1.79
4.36
3.43
3.73
1.78
5.04
3.82
0.65
16.76
3
4
16.97
13.65
87.10
−6.26
40.03
14.02
5.78
1.45
0.76
3.41
4.09
1.12
5
6
79.18
−6.83
57.27
6.27
7
8
9
10
11
12
13
14
15
16
17
18
37.53
7.11
21.08
17.53
14.13
9.40
Figure 4. Evaluation of efficacy for full-length compounds 2, 4, and 5
and their synthons at D3R and D2R. (a) Schematic of the BRET1-
based GoA activation assay in which HEK293T cells transiently express
either wild-type D3R or D2R and GαoA-Rluc8, V1-β1, and V2-γ2. (b)
Dose−response curves for dopamine (DA), quinpirole (QP), and
sulpiride (sulp) are similar for both D3R (left panel) and D2R (right
panel). There is a right shift in the DA dose−response curve in the
presence of 100 nM sulpiride. Dose−response curves are representa-
tive of at least three independent experiments performed with
triplicate samples (error bars, SEM). (c) 2, 4, and 5 display similar
weak partial agonism at D3R and D2R. Whereas compound 9 is
similar to the full length compounds, 8 displays substantial partial
agonism at both receptors. (d) Whereas all synthons from the 2-OCH3
series (white) display similar weak partial agonism, there is a
decreasing trend in efficacy in the 2,3-diCl series (black). Eticlopride
(3) acts as an inverse agonist, while compound 18 has no activity. An x
on the horizontal axis indicates compounds that have not been
synthesized. For (c) and (d), the Emax of a ligand is represented as a
percentage of the Emax of DA at the respective receptor. Emax values
were determined from dose−response curves ranging from 100 pM to
100 μM, as described in the Experimental Methods section, and are
averaged for at least three independent experiments performed with
triplicate samples (error bars, SEM).
22.32
7.37
−0.65
a
Data are represented as a % of the Emax of dopamine.
These data also established that the linking chain and/or SP
reduce efficacy when added to the 2,3-diCl-4-phenylpiperazine
core. Functional analysis of the synthons revealed that the SP is
not necessary for this effect, as increasing the length of the
linker by adding one to three methylenes led to a progressive
decrease in efficacy (Figure 4d and Table 2). Compounds with
a 3-carbon linker or greater, including the full-length D3R
compounds, 2 and 3 had similar efficacies of ∼20%. In contrast,
all of the 2-OCH3 substituted analogues, including the full-
length compound 5, were very weak partial agonists at D3R
(∼10% efficacy) and thus, in this series, the length of the linker
or amide terminus had no effect on efficacy. The SP alone (18)
failed to activate D3R, consistent with the critical role of the PP
in activation. As an additional control, eticlopride, like sulpiride,
acted as an inverse agonist (Figure 4d and Table 2).
consistent with its expected action as a competitive antagonist
(Figure 4b,c).
Comparing Binding Modes in the D3R Active and
Inactive Conformations. Of note, the D3R high-resolution
structure with bound eticlopride represents an inactive
conformation. Therefore, to investigate the structural bases of
the differences in efficacy between dopamine, synthons 8 and 9,
and eticlopride, it was necessary to establish an active
conformational state for the OBS. We used the differences in
the inactive and active structures of the homologous β1 and β2
adrenergic receptors38−40 to guide the construction of an active
conformation of the D3R. Features of the active state near the
OBS include the formation of a bulge around Ser5.46 in TM5,
tilting of the extracellular segments of TMs 6 and 7 toward
TM5, and significantly more H-bonding between the TM5
serines and bound agonist (see Supporting Information). Thus,
starting from the D3R inactive conformation, we rotated the
side chains of Ser5.42 and Ser5.43 in TM5 to face toward the
OBS,41 iteratively docked dopamine in the OBS, and relaxed
the system with MD simulations. In the equilibrated D3R
model bound with dopamine, there is a tightening of the
binding site evidenced by slightly decreased distances from
Ser5.42 or Ser5.46 to either Asp3.32 or Thr7.39 compared to the
conformation bound with the inverse agonist eticlopride, which
parallels the rearrangements observed in the active and inactive
conformations of β2 adrenergic receptors (Supporting
Information Table S1).
Compounds 2, 4, and 5 displayed similar weak partial
agonism at D3R and D2R with efficacies ∼8−20% that of
dopamine (Figure 4c and Table 2). Such similar efficacy
phenotypes contrasted with the substantial differences in the
binding affinities of these compounds for the two receptors as
well as to the completely different binding modes of the SP
within the SBP in D3R and D2R observed in our modeling
studies (Figure 2). This suggested that the differential binding
of the SP of these compounds does not contribute to their
efficacy and, further, that the binding of the PP in the OBS is a
more likely determinant of activation. To address this more
directly, we tested the synthons in the G protein-BRET assay
and discovered that the efficacy of 8, the 2,3-diCl-4-phenyl-
piperazine, was much greater than that of the full-length
compounds in both D3R and D2R, reaching 40−60% that of
dopamine (Figure 4c and Table 2). In contrast, the 2-OCH3-4-
phenylpiperazine (9), displayed only minimal efficacy at both
D3R and D2R, similar to its parent compound 5 (Figure 4c and
Table 2). This supported our hypothesis that the PP binding in
the OBS is critical to determining efficacy but also suggested
that different substituents on the 4-phenylpiperazine affect
activation, presumably by altering the binding pose within the
OBS.
F
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To explore the difference in substituent orientations in the
OBS between the 2,3-diCl− and 2-OCH3− series from the
perspective of the ligands themselves, electrostatic potential
surface calculations were performed on synthons 8 and 9. The
2,3-diCl− and 2-OCH3− substituents result in significantly
different charge distributions on the phenyl ring, specifically on
the face that interacts with TM5 (Figure 5a). Thus, the polar 3-
different interactions with TM5, which impact transitions of the
OBS toward an active conformation.
To explore this difference further, we used IFD to compare
the binding modes of the synthons in the active and inactive
conformations of the OBS. Interestingly, the overall binding
modes of compounds 8 and 9 in the active conformation were
similar to their modes in the inactive conformation, but the
polar 3-position of 8 is better accommodated in the active
conformation due to the rotated TM5 Ser side chains. Indeed,
during multiple 30 ns MD relaxations, a H-bond formed
between the 3-Cl of compound 8 and Ser5.43 is consistently
stable (Figure 5d); in contrast, for compound 9, the serines of
TM5 rotate back to their inactive configuration during the MD
relaxations, consistent with the observation described above
that the 2-OCH3 tilts the 4-phenylpiperazine toward TM5
where it may prevent the formation of the bulge in TM5 that is
associated with the active state (Figure 5e). Moreover, when
the protonated nitrogen is extended by a linker (10−17), the
hydrophobic interactions of the linker formed with TM7
(Thr7.39 and Tyr7.43, see Figure 3) result in an orientation of the
4-phenylpiperazine moiety perpendicular to the membrane,
similar to the poses of these compounds docked to the crystal
structure of D3R (see above), which also prevents the
formation of optimal interactions with the active configuration
of the TM5 serines (Figure 5f,g). Similarly, in the inactive
eticlopride-bound D3R structure, none of the TM5 serines
faces the OBS (Figure 5c).
CONCLUSIONS
■
Given the conservation of the D3R and D2R residues in the
OBS that bind the PP and the linker, it is not surprising that the
synthons lacking the SP have similar affinities (little or no
selectivity) for these two receptor subtypes. Nonetheless,
structure-based SAR of the synthons from the 2,3-diCl and 2-
OCH3 series indicates that substitutions on the terminal phenyl
ring, in combination with the linker, can impact the orientation
of the PP in the OBS, which consequently influences the exact
orientation of the SP. Consistent with this notion, in the 2-
OCH3 series, D3R affinity for the full length compound 5
increases by >7000-fold compared to its PP (9), whereas D2R
affinity improves by <20-fold; in the 2,3-diCl series, D3R
affinities of the full length compounds 2 and 4 increase ∼140-
fold at D3R, but D2R affinity is not appreciably different from
the core PP (8). Thus, the structure of the PP and linker,
although intrinsically lacking receptor selectivity, is critical for
optimal binding of the SP, which on its own binds with
extremely low affinity at both D3R and D2R (Table 1). On the
other hand, in both the 2,3-diCl and 2-OCH3 series, the similar
or higher affinities of the full-length compounds (2, 4, and 5)
for D3R compared to their PP-plus-linker synthons 14 and 15
suggest that the SP is well tolerated in the SBP of D3R. In
contrast, decreased affinities for D2R compared to D3R for the
full-length compounds suggest that the SP cannot bind in the
favored Ptm23 pocket within the D2R SBP. The similar D3R
affinities between compounds 4 and 14 of the 2,3-diCl series,
however, is likely a result of an offsetting effect in which the
contribution of the SP in Ptm23 to binding affinity is countered
by the SP restraining the butyl linker from binding within its
favored Ptm67 pocket. Interestingly, it has been noted in
developing multivalent ligands for muscarinic acetylcholine
receptors that the hydrophobic linkers might be in different
orientations with or without the attached moiety.42
Figure 5. The interactions of compounds 8, 9, 14, and 15 with TM5
serines compared to those of dopamine and eticlopride. In (a), the
electrostatic potentials (kcal/mol) are superimposed onto a surface of
constant electron density (0.001 e/bohr3) with rainbow colors. The
most positive and negative potential regions are in deep-blue/purple
and red, respectively. In (b−g), the binding poses are viewed parallel
to the membrane, with a focus on their interactions with TM5 serines.
The representative poses of dopamine (b) and eticlopride (c) are
taken from the MD trajectories of D3R models in which the OBS are
in active and inactive configurations, respectively. The representative
poses of compounds 8 (d) and 9 (e) are taken from the equilibrated
MD trajectories that were started from IFD results of these
compounds in the D3R model with the OBS in an active configuration
(see text), while those of 14 (f) and 15 (g) are the IFD results on the
same model; similar to 9, the substituents cannot establish any H-
bonding with the TM5 serines. H-bonds are indicated with dotted
lines.
Cl in compound 8 could form a H-bond with TM5 like
dopamine (Figure 5b), while in compound 9, the 2-OCH3 can
only form hydrophobic contacts with TM5, like eticlopride
(Figure 5c). This finding suggests that the profound difference
in efficacy between compounds 8 and 9 is likely due to their
G
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side-chain optimization, a minimization, and a Glide docking stage
using a hard van der Waals potential. The final docking stage was
carried out in the absence of core constraints.
In our examination of the correlation between the binding
mode and efficacy, we found that the PP was the critical
component in receptor activation. Specifically in the 2,3-diCl
series, there is a progressive decrease in efficacy with the
incremental addition of methylenes to the NH-PP (8). In
contrast, in the 2-OCH3 series, all synthons are very weak
partial agonists. These data correspond to our modeling study,
which demonstrates that the binding mode of the 2,3-diCl
compound 8 in the OBS is distinct from the binding mode in
the 2-OCH3 synthon series. Moreover, as linker length
increases in the 2,3-diCl series, the orientation of the
phenylpiperazine moiety becomes more similar to that in the
2-OCH3 series (Supporting Information Figure S1).
Further analysis using a D3R model with the OBS in an
active conformation revealed that like dopamine, compound 8
forms a H-bond with TM5, which is not present for the inverse
agonist eticlopride, is less favorable in the 2,3-diCl synthons
with longer linker length, and cannot form in the 2-OCH3
synthons. In addition, compound 8’s size and its binding mode
in the OBS tend to stabilize a more contracted OBS (Figure
3i). Thus it is likely that interactions with these residues and
corresponding conformational rearrangements are critical for
the observed efficacy of the D3R/D2R partial agonists, in
agreement with studies indicating that the serines in TM5 are
critical for activation in other aminergic receptors.14,43,44
In summary, the molecular identification of the SBP, which
differentiates D3R and D2R, and the role of their nearly
identical OBS in efficacy provide insight into the molecular
basis for both D3R selectivity as well as experimentally
validated computational models to guide the design of
compounds with targeted efficacies. The fact that residues of
the SBP have been shown to play a role in D4R-selectivity24 as
well as in the binding of allosteric ligands16 suggest that this
pocket has been targeted by drugs in multiple GPCRs, for both
bitopic ligands and allosteric modulators, but can now be
exploited more rationally in engineering ligand specificity.
D3R and D2R Homology Models and Molecular Dynamics.
Starting from our D3R and D2R models built previously using the
crystal structure of D3R (PDB: 3PBL) as the template,4 we extended
the extracellular end of TM1 with Modeler 9v245 to better conform to
the lipid bilayer dimension. In the new models, TM1 of the D3R
model starts at residue 23 and of the D2R starts at residue 29. The
new models, bound with eticlopride, were reinserted into the MD
simulation systems and were relaxed with MD for at least 90 ns using
Desmond Molecular Dynamics System (version 3.0, D. E. Shaw
Research, New York, NY, 2011).
The resulting models were used as the starting points for the MD
simulations of the complexes of D3R/D2R and 4-phenylpiperazine
compounds; to characterize further the binding modes of selected
compounds with MD, their binding poses from the docking study with
the D3R crystal structure described above were transferred to the
equilibrated D3R and D2R models.
The MD simulation system includes a lipid bilayer comprised of
∼180 palmitoyoleoylphosphatidylcholine molecules and a water phase
with Na+ and Cl− ions corresponding to a concentration of 150 mM
NaCl. The entire system totally has ∼65000 atoms. We used the all-
atom OPLS_AA force field46 throughout and semi-isotropic pressure
treatment in the isothermal−isobaric ensemble (NPT) stages. When
the receptor−compound complex was inserted back into the
equilibrated system, a relaxation protocol was applied first, which
included two energy minimization stages, first with and then without
restraints on protein heavy atoms, a heating stage to elevate the system
temperature from 10 to 310K in 60 ps, and a protein relaxation stage
to gradually reduce the restraints. In the subsequent production runs, a
constant surface tension of 4000 bar·Å was applied.
WaterMap Calculations. Characterization of solvent thermody-
namics in the D3R and D2R binding sites was performed using the
WaterMap program,25 using default settings, as described previously
for the A2A receptor.26 WaterMap calculations were performed on the
crystal structure of D3R (Supporting Information Figure S2) and on
representative frames from the D3R- and D2R-2 MD trajectories
(Figure 2c,d).
Conformational Search and Electrostatic Potential Surface
Calculation. The conformational search of selected compounds was
EXPERIMENTAL METHODS
performed using MacroModel (version 9.9, Schrodinger, LLC, New
̈
■
York, NY, 2011), with a CHCl3 solvent model. The resulting
conformers were optimized at the quantum mechanical level using
B3LYP-density functional theory and a 6-31G* basis set, as
Molecular Docking and the Selection of the Preferred
Binding Mode. Selection of the preferred binding mode for each
compound was based on molecular docking calculations and the
following analysis with the crystal structure of the D3R (PDB: 3PBL).4
Briefly, the structure was prepared with the Protein Preparation
implemented in Jaguar (version 7.8, Schrodinger, LLC, New York,
̈
NY, 2011), and then ranked by quantum mechanical Relative Energies.
The electrostatic potential was calculated for the lowest-energy
conformer of each compound using Jaguar and was mapped on the
electrostatic density surfaces shown in Figure 5.
Wizard in Maestro (version 9.2, Schrodinger, LLC, New York, NY,
̈
2011) using default options. Ligand docking calculations were
performed with Induced Fit Docking.32 Ligands were docked in the
protonated form. Poses were clustered into upward, downward, and
parallel conformations by determining the angle of the phenyl ring
with the membrane normal. The PP of the alkylated compounds was
able to adopt a large variety of orientations, as revealed by a large
spread of the angle distribution for all compounds. To obtain reliable
predictions, we selected only low energy solutions that occurred
multiple times in the IFD ensemble. The ensemble of poses within 1
kcal/mol of the lowest energy state for each of the compounds was
considered in selecting the final models, and the representative
members of the largest low-energy clusters were selected as the final
prediction (see Supporting Information Figure S1). The ensemble of
poses that survived this protocol is shown in Figure 3.
An Active Configuration of the OBS. On the basis of our
analysis of the available active and inactive adrenergic receptors (see
Supporting Information), to establish an active configuration of the
OBS, we first rotated the side chains of Ser5.42 and Ser5.43 of our D3R
model to face toward the OBS41 and then carried out three rounds of
iterative IFD and MD relaxation; the IFD of each subsequent round
used a representative MD snapshot from the previous round as the
input receptor conformation. Multiple MD trajectories were collected
for each MD round, with a total of ∼50 ns each for the first two
rounds and 120 ns for the third round. In the first round of IFD/MD,
the meta-OH of dopamine was more intracellular relative to the para-
OH, whereas in the second and third rounds, the meta-OH was
consistently more extracellular than the para-OH, consistent with the
topology of the −OH substituents of isoprenaline bound in an β1
adrenergic receptor crystal structure.40
Binding modes of the full-length compounds were predicted using
core-constrained IFD. In this customized protocol, initial poses are
obtained by a separate Glide calculation with softened van der Waals
radii on both protein and ligand (using a scaling factor of 0.50 for
nonpolar atoms). At this stage, core-constraints were used to keep the
PP largely in the orientation shown in Figure 3 (rmsd of the core kept
below 2.0 Å). An ensemble of 20 poses was subsequently submitted to
the second stage of regular IFD calculations, comprised of a Prime
Radioligand Binding Assays. A filtration binding assay was used
to characterize the binding properties of the compounds. For human
D2R (D2long isoform) and D3R stably expressed in HEK 293 cells,
membrane homogenates (50 μL) were suspended in 50 mM Tris-
HCl/150 mM NaCl/10 mM EDTA buffer pH 7.5 and incubated with
H
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50 μL of [125I]IABN at 37 °C for 60 min. Nonspecific binding was
determined using 15 μM (+)-butaclamol. For competition experi-
ments, the radioligand concentration was generally equal to 0.5 times
the Kd value, and the concentration of the competitive inhibitor ranged
over 5 orders of magnitude. Binding was terminated by the addition of
cold wash buffer (10 mM Tris-HCl/150 mM NaCl, pH 7.5) and
filtration over glass-fiber filters (Schleicher and Schuell No. 32). Filters
were washed with 10 mL of cold buffer, and the radioactivity was
measured using a Packard Cobra γ counter. Estimates of the
equilibrium dissociation constant and maximum number of binding
sites were obtained using unweighted nonlinear regression analysis of
data modeled according to the equation describing mass action
binding. Data from competitive inhibition experiments were modeled
using nonlinear regression analysis to determine the concentration of
inhibitor that displaced 50% of the specific binding of the radioligand.
Competition curves were modeled for a single site, and the IC50 values
were converted to equilibrium dissociation constants (Ki values) using
the Cheng−Prusoff correction. Mean Ki values SEM are reported for
at least three independent experiments.
Constructs for Expression Vectors and Transfection. The
human D2shortR construct was previously reported.47 cDNA for human
D3R and GαoA-RLuc8 were kindly provided by D. Sibley (NINDS)
and C. Gales (INSERM), respectively. The following human G-
protein constructs were cloned into the pcDNA3.1 expression vector
(Invitrogen): Gβ1 fused with V1 (N-terminal split of mVenus; residues
1−155) at its N-terminus, and Gγ2 fused with V2 (C-terminal split of
mVenus, residues 156−240) at its N-terminus. The split mVenus
probes were linked to their respective G-protein with an amino acid
linker consisting of GGSAGT. All the constructs were confirmed by
sequencing analysis.
MS (where obtainable), and combustion analysis data, all final
compounds are >95% pure.
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)-1H-indole-
2-carboxamide (4). This compound20 was prepared using the
previously described method21 from 2,3-dichlorophenylpiperazine
linked to the N-phthalimido-protected 1-butylamine using standard
N-alkylation conditions followed by deprotection with hydrazine and
then coupling with 1H-indole-2-carboxylic acid using CDI in THF.
The yield of the amidation step was 65%. The free base was converted
to the HCl salt and recrystallized from hot MeOH/i-PrOH; mp 250−
1
251 °C. H NMR (400 MHz, CDCl3) δ: 1.65−1.73 (m, 4H), 2.47−
2.50 (m, 2H), 2.64 (m, 4H), 3.07 (m, 4H), 3.51−3.54 (m, 2H), 6.83
(m, 1H), 6.91−6.93 (m, 1H), 7.10−7.16 (m, 2H), 7.26−7.31 (m, 2H),
7.43−7.45 (m, 1H), 7.63−7.65 (m, 1H). 13C NMR (100 MHz,
CDCl3) δ: 24.3, 27.6, 39.4, 51.2, 53.3, 57.9, 101.7, 103.2, 111.8, 118.6,
120.7, 121.8, 124.5, 124.6, 127.4, 127.5, 127.6, 130.7, 134.0, 151.1,
167.8. Anal. (C23H26Cl2·HCl·0.25H2O), C, H, N.
1-(2,3-Dichlorophenyl)-4-methylpiperazine (10). This com-
pound was previously reported28 but synthesized using a different
method. Compound 8 (Aqchem, USA; 694 mg, 3 mmol) and
formaldehyde 37% in H2O (1 mL) were mixed in 1,2-dichloroethane
(10 mL) and then treated with sodium triacetoxyborohydride (1.3 g, 2
ee). The mixture was stirred at room temperature (RT) for 12 h and
quenched by adding 1N NaOH (20 mL). The product was extracted
with ethyl acetate (EtOAc). The organic extract was washed with
brine, dried over sodium sulfate (Na2SO4), and concentrated in vacuo.
The crude product was purified by column chromatography using
EtOAc/CHCl3/MeOH (5:5:1) to afford compound 10 (596 mg) in
81% yield. 1H NMR (400 MHz, CDCl3) δ: 2.36 (b s, 3H), 2.61 (br s,
4H), 3.07 (br s, 4H), 6.96 (dd, J = 6.4, 3.2 Hz, 1H), 7.13−7.15 (m,
2H). 13C NMR (100 MHz, CDCl3) δ: 46.20, 51.38, 55.29, 118.74,
124.66, 127.54, 127.60, 134.11, 151.35. GC/MS RT(RIMS): 14.56 min
(244 [M]+ m/z). Anal. (C11H14Cl2N2) C, H, N.
1-(2-Methoxyphenyl)-4-methylpiperazine (11). This com-
pound29 was prepared by a different method as described for
compound 10, employing compound 9 (Aldrich, USA; 576 mg, 3
mmol), formaldehyde 37% in H2O (1 mL), and sodium triacetox-
yborohydride (1.3 g, 6 mmol), to afford 11 (531 mg) in 86% yield. 1H
NMR (400 MHz, CDCl3) δ: 2.36 (s, 3H), 2.63 (br s, 4H), 3.10 (br s,
4H), 3.86 (s, 3H), 6.86 (dd, J = 7.6, 1.2 Hz, 1H), 6.89−7.02 (m, 3H).
13C NMR (100 MHz, CDCl3) δ: 46.32, 50.76, 55.51, 111.28, 118.38,
121.10, 123.04, 141.44, 152.39. GC/MS RT(RIMS): 9.93 min (206
[M]+ m/z). Anal. (C12H18N2O) C, H, N.
Plasmid cDNA was transfected into HEK-293T cells using
polyethylenimine (Polysciences Inc.) in a 1:3 ratio in 10 cm dishes
(BD Falcon). Amounts of receptor and the GαoA, Gβ1, and Gγ2
sensors transfected were optimized by testing various ratios of
plasmids. Cells were maintained in culture with DMEM (GIBCO)
supplemented with 10% FBS, and transfection media was replaced
with fresh media after 24 h. Experiments were performed ∼48 h after
transfection.
BRET1-Based GoA Activation Assay. Cells were washed,
harvested, and resuspended in PBS containing 5 mM glucose at
room temperature. Cells (40 μg of protein per well measured by BCA
protein assay kit, ThermoScientific) were distributed into a 96-well
microplate (Wallac, PerkinElmer Life and Analytical Sciences). After
incubation with coelenterazine h (5 μM) (Dalton Pharma Services) for
8 min, different ligands were injected and incubated for 2 min. Using
the Pherastar FS (BMG Labtech), BRET1 signal was determined by
quantifying and calculating the ratio of the light emitted by mVenus
(510−540 nm) over that emitted by RLuc8 (485 nm). Data were
normalized to vehicle (0%) and dopamine (100%) and nonlinear
regression analysis was performed using the sigmoidal dose−response
function in GraphPad Prism 5.1.
2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethanol (12). This
compound was prepared by the method described for compound
13, employing compound 8 (694 mg, 3 mmol), 2-bromoethanol (375
mg, 3 mmol), and K2CO3 (1.24 g, 9 mmol), to afford compound 12
1
(478 mg) in 58% yield. H NMR (400 MHz, CDCl3) δ: 2.61−2.65
(m, 2H), 2.71 (br s, 4H), 3.07 (br s, 4H), 3.66 (t, J = 5.2 Hz, 2H), 6.95
(dd, J = 6.4, 3.2 Hz, 1H), 7.14−7.16 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ: 51.47, 53.08, 57.85, 59.39, 118.70, 124.75, 127.57, 134.14,
151.23. GC/MS RT (RIMS): 14.01 min (274 [M]+ m/z). Anal.
(C12H16Cl2N2O·0.1H2O) C, H, N.
1
Synthesis. H and 13C NMR spectra were acquired using a Varian
Mercury Plus 400 spectrometer. Chemical shifts are reported and
1
1-(2,3-Dichlorophenyl)-4-propylpiperazine (13). A suspension
of compound 8 (694 mg, 3 mmol), 1-bromopropane (370 mg, 1
equiv), and K2CO3 (1.24 g, 3 equiv) in acetone was stirred at reflux
overnight. Acetone was removed under reduced pressure, and the
residue was diluted with EtOAc, washed with H2O and brine, dried
over Na2SO4, concentrated, and purified by column chromatography
(EtOAc/hexane, 1:1) to afford compound 13 (755 mg) in 92% yield.
1H NMR (400 MHz, CDCl3) δ: 0.93 (t, J = 7.6 Hz, 3H), 1.50−1.60
(m, 2H), 2.36−2.40 (m, 2H), 2.63 (br s, 4H), 3.07 (br s, 4H), 6.96
(dd, J = 6.4, 3.2 Hz, 1H), 7.13−7.15 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ: 12.13, 20.21, 51.48, 53.46, 60.81, 118.70, 124.62, 127.56,
127.61, 134.12, 151.49. GC/MS RT (RIMS): 12.84 min (272 [M]+ m/
z). Anal. (C13H18Cl2N2) C, H, N.
referenced according to deuterated solvent for H spectra (CDCl3,
7.26; DMSO-d6, 2.50), 13C spectra (CDCl3, 77.2), 19F spectra (CFCl3,
0). Combustion analysis was performed by Atlantic Microlab, Inc.
(Norcross, GA) and agrees within 0.4% of calculated values. Melting
point determinations were conducted using a Thomas−Hoover
melting point apparatus and are uncorrected. Anhydrous solvents
were purchased from Aldrich were used without further purification,
except for tetrahydrofuran, which was freshly distilled from sodium-
benzophenone ketyl. All other chemicals and reagents were purchased
from Aldrich Chemical Co., Combi-Blocks, TCI, America, Matrix
Scientific; Lancaster Synthesis, Inc. (Alfa Aesar), and AK Scientific,
Inc. Final compounds (free base) were purified by column
chromatography (EMD Chemicals, Inc.; 230−400 mesh, 60 Å) or
preparative thin layer chromatography (silica gel, Analtech, 1000 μm).
The final products were analyzed as free bases unless otherwise stated.
Yields and reaction conditions are not optimized. Generally, yields and
spectroscopic data refer to the free base. On the basis of NMR, GC-
1-Butyl-4-(2,3-dichlorophenyl)piperazine (14). This com-
pound was prepared by the method described for compound 13,
employing compound 8 (694 mg, 3 mmol), 1-bromobutane (412 mg,
3 mmol), and K2CO3 (1.24 g, 9 mmol) to afford compound 14 (820
I
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Journal of Medicinal Chemistry
Article
mg) in 95% yield. 1H NMR (400 MHz, CDCl3) δ: 0.94 (t, J = 7.6 Hz,
3H), 1.30−1.40 (m, 2H), 1.47−1.55 (m, 2H), 2.39−2.43 (m, 2H),
2.63 (br s, 4H), 3.07 (br s, 4H), 6.96 (dd, J = 6.4, 3.2 Hz, 1H), 7.13−
7.16 (m, 2H). 13C NMR (100 MHz, CDCl3) δ: 14.20, 20.91, 29.18,
51.44, 53.46, 58.59, 118.68, 124.59, 127.54, 127.57, 134.09, 151.45.
GC/MS RT (RIMS): 13.53 min (286 [M]+ m/z). Anal. (C14H20Cl2N2)
C, H, N.
1-Butyl-4-(2-methoxyphenyl)piperazine (15). This com-
pound30 was prepared by the method described for compound 13,
employing compound 9 (576 mg, 3 mmol), 1-bromobutane (412 mg,
3 mmol), and K2CO3 (1.24 g, 9 mmol), to afford compound 15 (690
mg) in 95% yield. 1H NMR (400 MHz, CDCl3) δ: 0.93 (t, J = 7.6 Hz,
3H), 1.30−1.39 (m, 2H), 1.47−1.56 (m, 2H), 2.38−2.42 (m, 2H),
2.65 (br s, 4H), 3.11 (br s, 4H), 3.86 (s, 3H), 6.85 (dd, J = 7.6, 1.2 Hz,
1H), 6.89−7.02 (m, 3H). 13C NMR (100 MHz, CDCl3) δ: 14.24,
20.98, 29.25, 50.82, 53.67, 55.45, 58.77, 111.22, 118.31, 121.09,
122.96, 141.55, 152.39. GC/MS RT (RIMS): 12.03 min (248 [M]+ m/
z). Anal. (C15H24N2O) C, H, N.
NPT, isothermal−isobaric ensemble; OPLS_AA, optimized
potentials for liquid simulations all-atom force field
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N-Butyl-1H-indole-2-carboxamide (18). This compound was
prepared employing 1H-indole-2-carboxylic acid (322 mg, 2 mmol),
CDI (324 mg, 2 mmol), and 1-butylamine (146 mg, 2 mmol) to afford
1
compound 18 (393 mg) in 91% yield; mp 171−173 °C. H NMR
(400 MHz, CDCl3) δ: 0.98 (t, J = 7.2 Hz, 3H), 1.39−1.48 (m, 2H),
1.59−1.67 (m, 2H), 3.47−3.53 (m, 2H), 6.17 (br s, 1H), 6.81−6.82
(m, 1H), 7.12−7.16 (m, 1H), 7.26−7.31 (m, 1H), 7.45 (dd, J = 8.0,
0.8 Hz, 1H), 7.65 (dd, J = 8.0, 0.8 Hz, 1H), 9.42 (br s, 1H). 13C NMR
(100 MHz, CDCl3) δ: 13.93, 20.28, 31.95, 39.65, 101.77, 112.23,
120.68, 121.93, 124.46, 127.74, 131.01, 136.54, 161.93. GC/MS RT
(RIMS): 13.60 min (216 [M]+ m/z). Anal. (C13H16N2O) C, H, N.
ASSOCIATED CONTENT
■
S
* Supporting Information
Analysis of the active vs inactive OBS conformations of β-
adrenergic receptors. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For A.H.N.: phone, 443-740-2887; E-mail, anewman@intra.
nida.nih.gov. For L.S.: phone, 212-746-6348; E-mail, les2007@
med.cornell.edu. for J.A.J.: phone, 212-305-7308; E-mail, jaj2@
columbia.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to R. Abel, W. Sherman, D. Lupyan, C. Gales
̀
,
and R. Stevens for helpful discussion, and R. Mach for the
precursor used for the iodination of [125I]IABN. This work was
supported in part by NIDA Intramural Research Program
(A.H.N.), DA022413 and MH54137 (J.A.J.), DA23957 and
DA13584 (R.R.L.), and DA023694 (L.S.). Computations were
performed on the Ranger at the Texas Advanced Computing
Center (TG-MCB090022).
ABBREVIATIONS USED
■
D2R, dopamine D2 receptor; D3R, dopamine D3 receptor;
OBS, orthosteric binding site; SBP, second binding pocket;
Gs/olf, stimulatory G-protein α subunits; Gi/o, inhibitory G-
protein α subunits; TM, transmembrane; PP, primary
pharmacophore; SP, secondary pharmacophore; EL, extrac-
ellular loop; IFD, induced-fit docking; MD, molecular
dynamics; HEW, high energy water; BRET, bioluminescence
resonance energy transfer; rmsd, root-mean-square deviation;
J
dx.doi.org/10.1021/jm300482h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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