Development of a bivalent dopamine D2 receptor agonist

Article abstract of DOI:10.1021/jm2009919

Bivalent D₂ agonists may function as useful molecular probes for the discovery of novel neurological therapeutics. On the basis of our recently developed bivalent dopamine D₂ receptor antagonists of type 1, the bivalent agonist 2 was

Full text of DOI:10.1021/jm2009919

Article
pubs.acs.org/jmc
Development of a Bivalent Dopamine D2 Receptor Agonist
Julia Kuhhorn,† Angela Gotz,† Harald Hubner,† Dawn Thompson,‡ Jennifer Whistler,‡
̈
̈
̈
and Peter Gmeiner*,†
†Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstraße 19, D-91052 Erlangen,
Germany
‡Ernest Gallo Clinic & Research Center and Department of Neurology, University of California, San Francisco, Emeryville,
California 94608, United States
S
* Supporting Information
ABSTRACT: Bivalent D2 agonists may function as useful molecular probes for
the discovery of novel neurological therapeutics. On the basis of our recently
developed bivalent dopamine D2 receptor antagonists of type 1, the bivalent
agonist 2 was synthesized when a spacer built from 22 atoms was employed.
Compared to the monovalent control compound 6 containing a capped spacer,
the bis-aminoindane derivative 2 revealed substantial steepening of the
competition curve, indicating a bivalent binding mode. Dimer-specific Hill
slopes were not a result of varying functional properties because both the
dopaminergic 2 and the monovalent control agent 6 proved to be D2 agonists
substantially inhibiting cAMP accumulation and inducing D2 receptor
internalization. Investigation of the heterobivalent ligands 8 and 9, containing
an agonist and a phenylpiperazine-based antagonist pharmacophore, revealed moderate steepening of the displacement curves
and antagonist to very weak partial agonist properties.
revealed substantial steepening of the competition curve,
indicating a bivalent binding mode. This behavior depended
on the structure and on the length of the linker arm when the
dimeric 1,4-DAP 1 containing a 22-atom spacer gave the
steepest curve and a Hill slope of 2.
INTRODUCTION
■
Over the past decade, pharmacological, biochemical, and
biophysical evidence has accumulated suggesting that G-
protein-coupled receptors (GPCRs) can cross-react to form
homo- or heterodimers or higher-order oligomers.1−3 In some
cases, it was shown that dimerization is essential for receptor
function and, moreover, can also affect ligand pharmacology,
signal transduction, and cellular trafficking.4,5 Concerning the
dopamine D2 receptor, different studies indicate the existence
of homomeric6−9 and heteromeric complexes.10,11 According to
very recent findings, dopamine D2 receptor homodimers might
be of particular importance in the pathophysiology of
schizophrenia.12 Thus, bivalent antagonists of type 1 can
serve as promising pharmacological tools for the discovery of
atypical antipsychotics. For the treatment of Parkinsons's
disease, dyskinesia, hyperprolactinemia, and restless legs
syndrome, D2-like agonists are needed, suggesting that bivalent
agonists may function as useful molecular probes for the
development of novel neurological therapeutics.
In a very recent paper, we reported the synthesis and the
biological evaluation of type 1 bivalent ligands, incorporating
the privileged structure of 1,4-disubstituted aromatic piper-
idines/piperazines (1,4-DAPs)13−15 and triazolyl-linked spacer
units, differing in length and structure.16 Radioligand binding
assays revealed that the bivalent ligands exhibited a distinct
binding profile compared with monovalent control ligands and
compounds composed of a pharmacophore with a complete
spacer arm and a structurally distorted second pharmacophore.
In particular, for the D2 subtype, some of our bivalent ligands
Taking advantage of these structural insights, we were
intrigued by whether bivalent dopamine receptor agonists or
partial agonists can be designed in an analogous manner.
Competition assays between agonists and radiolabeled
antagonists are often characterized by shallow curves with
Hill coefficients between 0.5 and 0.7. For the bivalent agonists,
a steepening of the competition curves and Hill slopes of at
least 1 were expected. The optimized spacer properties and
spacer length (22 atoms) should be conserved. However, the
pharmacophore should be displaced by structural moieties
that were expected to promote agonist properties. N-(4-
arylcarboxamido)butyl substituted propylaminoindanes are
known as strong partial to full D2 receptor agonists11,17 and
were applied in the present study as agonist pharmacophores of
the bivalent ligand 2. Besides the incorporation of a typical full
agonist, we envisioned to investigate bivalent ligands carrying
N-(4-arylcarboxamido)butyl substituted phenylpiperazines of
the class of 1,4-DAPs, which have been described as partial
agonists and antagonists.18−21 In this paper, we describe the
synthesis of the homobivalent ligands 2 and 3 and monovalent
fragments containing one-half of the linker (Chart 1).
Received: July 25, 2011
Published: October 17, 2011
© 2011 American Chemical Society
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According to the recently established binding mode of the
above-described pharmacophores,20,24 simultaneous interac-
tion of both recognition elements of a bivalent ligand can be
accomplished assuming a dopamine D2 receptor dimer
interacting via the TM1/H8 interface. When the human β2-
adrenergic receptor crystal structure32 was employed as a
template, a conceptual dopamine D2 receptor dimer model was
generated with the TM1/H8 interface (Figure 1). Incorporation
Chart 1. General Approach
Figure 1. Molecular docking studies demonstrate a potential binding
orientation of the bivalent ligand 3 in a D2 receptor dimer model (top
view). A homology model of the human D2long receptor was generated
using the human β2-adrenergic receptor crystal structure as template.
The helices TM1 and H8 provide the interface of the D2 receptor
dimer.
of the target compound 3 into the binding pockets of both
protomers corroborated our assumption that a bivalent binding
mode can be adopted if the 22-atom spacer adopts an extended
conformation.
Moreover, heterobivalent ligands, combining agonist and
antagonist pharmacophores, are discussed. Diagnostic radio-
ligand binding experiments are also reported revealing Hill
slopes that clearly indicated a bivalent binding mode, especially
for the bis-aminoindane 2. Further investigations were directed
toward determining dimer specific signaling and internalization
properties.
Chemical Synthesis. Homodimeric dopamine receptor
ligands with a linker arm built from 22 atoms were synthesized
when the spacer length resulted from SAR studies on recently
described bivalent GPCR ligands.33,34 As a dopamine surrogate,
we employed N-propylaminoindane and o-methoxyphenylpi-
perazine units that were attached via an amidobutyl linker to an
arene moiety. For comparative purposes, monovalent fragments
containing one-half of the spacer as well as heterobivalent
ligands incorporating the N-propylaminoindane pharmaco-
phore and a phenylpiperazine-derived pharmacophore of the
bivalent ligands 1 and 3 were prepared. To facilitate the
introduction of different pharmacophores at the end of a
general synthetic route, we intended to proceed via a common
intermediate. The synthesis started from methyl vanillate,
which was subjected to O-alkylation with 5-chloro-1-pentyne to
receive the central building block 4 in 94% yield (Scheme 1).
Taking advantage of click chemistry, the preparation of the
triazole based precursor of the bivalent target compounds was
performed by cooper(I)-catalyzed [3 + 2] azide−alkyne
cycloaddition (CuAAC)35 of 1,8-diazidooctane and the alkyne
4. Subsequent ester cleavage in the presence of methanolic
potassium hydroxide gave access to the carboxylic acid 5.
Finally, HATU-promoted coupling of the molecular scaffold 5
with N-propylaminoindanyl or o-methoxyphenylpiperazinyl
substituted butylamine17,21 resulted in formation of the bivalent
ligands 2 and 3, respectively, in excellent yields. For the
preparation of the monovalent ligands 6 and 7 containing
capped spacers, the central building block 4 was reacted with
butylazide. Subsequent cleavage of the ester function and amide
coupling using TBTU furnished the monovalent products 6
and 7 in 73−83% yield. The synthesis of the heterobivalent
probe 8 started again from the alkyne 4, which was saponified
RESULTS AND DISCUSSION
■
Design. The biological properties of dopaminergic phar-
macophores are encoded by an aromatic headgroup, which
controls intrinsic activity, and an amine moiety, which is
responsible for the formation of a reinforced hydrogen bond
to the crucial residue Asp.3,32 Thus, the N-propylaminoindane
and the phenylpiperazine scaffolds were chosen to simulate
the endogenous biogenic amine.13 A lipophilic appendage,
which is arranged in a defined remote position by a linker
unit, was expected to be necessary to enhance ligand affinity.
The lipophilic appendage, which is usually represented by a
functionalized arene moiety, resides within a hydrophobic
microdomain at the extracellular end of TM2, TM3, and TM7
and specifically interacts with residues of the extracellular loop
2 (EL2).22−24 A formal elongation of the lipophilic moiety is
suggested to lead to the “entrance region” of the receptor and
from there to the binding pocket of a neighboring protomer.
According to our recent findings,16 the para-position of an
aromatic moiety terminating the lipophilic appendage is
perfectly applicable as attachment point for a 22-atom spacer.
In various GPCR dimers, interaction interfaces are formed by
lipid-exposed surfaces of different TMs, with TM1, TM4, and/
or TM5 gaining the most support as dimer interfaces.25−30
Employing cystein cross-linking experiments, Guo et al.8,9,31
demonstrated for the dopamine D2 receptor that TM4/5 forms
a symmetrical dimer interface while TM1 and the cytoplasmatic
helical domaine H8 are part of a second symmetrical interface.
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a
Scheme 1
a
Reagents and conditions: (a) 5-chloro-1-pentyne, K2CO3, KI, CH3CN, reflux, 20 h (94%); (b) (1) 1,8-diazidooctane, sodium ascorbate, CuSO4, 1:4
MeOH/CH2Cl2, room temp, 24 h; (2) 2 M KOH, MeOH, 60 °C, 2 h (97%); (c), (N-indan-2-yl-N-propyl)butan-1,4-diamine or 4-[4-(2-
methoxyphenyl)piperazin-1-yl]butan-1-amine, HATU, DIPEA, DMF, 0 °C to room temp, 1−2 h (81−87%); (d) (1) butylazide, sodium ascorbate,
CuSO4, 1:2:2 H2O/tBuOH/CH2Cl2, room temp, 16 h (79%); (2) 2 M KOH, MeOH, 60 °C, 2 h (97%); (3) (N-indan-2-yl-N-propyl)butan-1,4-
diamine or 4-[4-(2-methoxyphenyl)piperazin-1-yl]butan-1-amine, TBTU, DIPEA, DMF, CH2Cl2, 0 °C to room temp, 20 h (73−83%); (e) (1) 2 M
KOH, MeOH, 60 °C, 1.5 h (89%); (2) (N-indan-2-yl-N-propyl)butan-1,4-diamine or 4-[4-(2-methoxyphenyl)piperazin-1-yl]butan-1-amine, TBTU,
DIPEA, DMF, CH2Cl2, 0 °C to room temp, 16 h (86−97%); (3) N-[4-[indan-2-yl(propyl)amino]butyl]-3-methoxy-4-(pent-4-yn-1-
yloxy)benzamide, 1,8-diazidooctane, sodium ascorbate, CuSO4, 1:4 MeOH/CH2Cl2, room temp, 2 h (28%); (4) 3-methoxy-N-[4-[4-(2-
methoxyphenyl)piperazin-1-yl]butyl]-4-(pent-4-yn-1-yloxy)benzamide, sodium ascorbate, CuSO4, 1:4 MeOH/CH2Cl2, room temp, 16 h (82%); (f)
N-[4-[indan-2-yl(propyl)amino]butyl]-3-methoxy-4-(pent-4-yn-1-yloxy)benzamide, 1-[4-[3-[1-(8-azidooctyl)-1H-1,2,3-triazole-4-yl]propoxy]-3-
methoxyphenyl)methyl]-4-(2-methoxyphenyl)piperazine, sodium ascorbate, CuSO4, 1:4 MeOH/CH2Cl2, room temp, 3 h (74%).
and coupled to N-propylaminoindanyl substituted butylamine.
The resulting intermediate was subjected to CuAAC using a 5-
fold excess of 1,8-diazidooctane to afford a triazole derivative
incorporating one pharmacophore in 28% yield. Subsequent
cycloaddition with the phenylpiperazinylbutyl based pharma-
cophore equivalent afforded the heterobivalent ligand 8.
Synthesis of the heterobivalent ligand 9 proceeded via an
intermediate containing a phenylpiperazinylmethyl-derived
pharmacophore14 when the final triazole formation was
performed using an excess of the appropriately functionalized
aminoindane pharmacophore.
Receptor Binding. In vitro binding affinity of the homo-
and heterobifunctional molecular probes36−39 along with their
monovalent control agents and the reference drug haloperidol
was measured by displacement of the radioligand [3H]-
spiperone from D2long, D2short, D3, and D4.4 receptors stably
expressed in Chinese hamster ovary cells (CHO). D1 receptor
binding affinities were determined utilizing striatal membranes
and the D1 selective radioligand [3H]SCH 23390. The
characterization of the test compounds was initiated by
carefully comparing the homobivalent ligands 2 and 3 with
their monovalent counterparts 6 and 7. The aminoindane
nanomolar range were observed for the bivalent test compound
2 (Ki = 16 nM). Ki values of 1.8 and 0.71 nM for 2 and 6,
respectively, indicated that D3 binding was high for both the
dimeric and the monomeric ligand. The binding pattern observed
for the bivalent amidobutylphenylpiperazine 3 displayed 6- to
9-fold lower receptor binding for D2long, D2short, D3, and D4
compared to the monovalent analogue 7.
In the course of our investigations of bivalent GPCR ligands,
we also intended to evaluate the pharmacological properties of
heterobivalent dopamine receptor ligands, combining an
agonist with an antagonist/partial agonist pharmacophore.
Thus, dopamine receptor binding studies of the heterobivalent
ligands 8 and 9 clearly revealed significant receptor recognition
with Ki between 8.8 and 25 nM for the D2-like subtypes. To
gain diagnostic insights into the binding mode of our bivalent
test compounds, we analyzed the profiles of the competition
curves in detail. Competitive binding curves that follow the law
of mass action have a slope of 1. A Hill slope (nH) less than 1 is
considered a reflection of negative cooperativity or the ability
of a ligand to bind to G-protein-precoupled receptors with
higher affinity than uncoupled receptors. Thus, competition
assays between agonists and radiolabeled antagonists are often
characterized by shallow curves with Hill coefficients between
0.5 and 0.7. Antagonists tend to have Hill slope values close to
1, since they fail to differentiate between the precoupled and
derivatives 2 and 6 gave significant specific binding for D2long
2short, D3, and D4 and clearly reduced D1 affinity (Table 1).
For both variants of the D2 subtype, Ki values in the double-digit
,
D
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the uncoupled receptor population, thereby following the
general law of mass action for single site competition.40 For the
bivalent ligand 1, a steepening of the competition curves was
observed, compared to the respective monomer, leading to a
Hill slope of 2.0 for the interactions with D2long and D2short. This
is indicative of a positive cooperative binding, which is usually
observed as an allosteric effect inducing a conformational
crosstalk within one receptor protomer or modulation of the
interaction between two protomers of a receptor dimer.41,42
Bivalent ligands addressing two adjacent binding sites of
receptor dimers will also induce such cooperativity because
bivalent binding of the second pharmacophore is significantly
accelerated because of the vicinity of ligand and the thus
facilitated enrichment of local concentration. Thus, bivalent
binding leads to the liberation of 2 equiv of radioligand and a
substantial steepening of the competition curve. Examination of
the binding curves of the monomeric ligand 6 at D2long and
D2short revealed shallow Hill slopes between 0.5 and 0.6,
indicating agonistic properties. Interestingly, careful analysis of
the competition experiments that we conducted with the
dimeric analogue 2 revealed steepening of the competition
curves. Hill slopes of 1.3 were calculated for the interaction
of D2long and D2short with the bivalent ligand 2 (Figure 2).
Figure 2. Representative binding curves of the homobivalent ligand 2
and its monovalent control agent 6 at the human D2long receptor
displaying different curve shapes resulting in Hill slopes (absolute
value of nH) of 1.3 (for 2) and 0.5 (for 6) indicating a bivalent and a
monovalent binding mode, respectively.
Two different hypotheses can be formulated to explain the
steepening of the competition curve: (1) a claim that the bis-
aminoindane 2 exhibits a bivalent binding mode, leading to
the liberation of 2 equiv of radioligand, or (2) a change of
functional activity from the monovalent ligand 6 to the bivalent
ligand 2 from agonist to antagonist properties. To gain further
insights, functional experiments determining the activation
profiles of 6 and 2 were envisioned.
In contrast to the results obtained for the aminoindane
derivatives, the Hill slope of the bivalent phenylpiperazine 3
(nH = 1.0−1.3) differs only slightly from the value for the
monovalent control ligand 7 (nH = 0.8−0.9), which may be a
result of a coexistence of binding modes displacing 1 equiv of
[3H]spiperone and those liberating 2 equiv of radioligand.
Analyses of the heterobivalent ligand 8 revealed a steepening
of the competition curves (nH = 1.5 at D2long and D2short
)
compared to their monovalent building blocks 6 and 7 (nH =
0.5−0.8), while the heterobivalent ligand 9 gave Hill slopes
close to 1 for the interaction with D2long and D2short
.
Interestingly, a strongly increased Hill coefficient of 1.6 was
calculated for the interaction of 9 with D3 when compared to
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Table 2. Intrinsic Activity of the Bivalent Compounds 1−3, 8, 9 and the Monomeric Controls 6, 7, 10 Determined at the D2long
Receptor by Measuring the Inhibition of cAMP Accumulation and the Propensity To Induce Internalization in Comparison
with the Reference Compound Quinpirole
compd
g
2
3
1
6
7
10
8
9
quinpirole
a
cAMP
b
d
d
d
d
d
Eff
84 ± 8.3
440 ± 64
+
<3
<3
88 ± 4.2
13 ± 6.3
+
<3
<3
<3
13 ± 5.6
380 ± 120
−
101 ± 0.87
140 ± 45
+
c
EC50
internalization
e
−
−
−
−
−
f
Hill slope (nH)
1.3 ± 0.06
1.2 ± 0.1
2.0 ± 0.1
0.5 ± 0.02
0.8 ± 0.05
1.1 ± 0.0
1.5 ± 0.06
1.2 ± 0.07
a
b
Bioluminescence based cAMP-Glo assay using a D2long expressing CHO cell line. Ligand efficacy ± SEM in % compared to the full agonist
c
d
quinpirole. EC50 ± SEM in nM derived from the mean curves of three to nine experiments each done in triplicate. Data from two independent
e
experiments done in triplicate. Ligand effects on D2long receptor internalization: + = ligand stimulates receptor internalization; − = no ligand
f
mediated receptor internalization. Hill slopes ± SEM are derived from binding curves recorded for the determination of Ki values (Table 1); the
g
original nH was negative but is displayed as absolute value. 10: 1-[[4-[3-(1-butyl-1H-1,2,3-triazol-4-yl)propoxy]-3-methoxyphenyl]methyl]-4-(2-
methoxyphenyl)piperazine.
the values of the monovalent fragment 6 (nH = 0.9) and the
respective monovalent ligand of 1 (1-[[4-[3-(1-butyl-1H-1,2,3-
triazol-4-yl)propoxy]-3-methoxyphenyl]methyl]-4-(2-
methoxyphenyl)piperazine, 10), which contains one-half of the
linker (nH = 1.2).
Functional Experiments. To better understand the bio-
logical properties of our bivalent dopaminergics, intrinsic
activities of the ligands were determined by measuring the
ability of the test compounds to modulate D2long receptor
mediated inhibition of cAMP accumulation and to induce
internalization of FLAG-tagged D2long receptors.43
suggesting that at physiological doses within a native system with
lower D2 expression levels, the heterodimer 9 might be less likely
to cause down-regulation of the D2 receptor via internalization,43
which has been shown to be important for regulating the balance
of D1 and D2 receptor signaling in vivo.44
When a bioluminescence based cAMP assay was employed,
substantial D2 agonist activity was observed for the bivalent
ligand 2 and its monovalent control agent 6 with 84% and 88%
ligand efficacy, respectively (Table 2). A significantly higher
EC50 value was calculated for 2 compared to 6, indicating that
the bivalent binding requires a higher receptor occupation to
exert signaling. On the other hand, the phenylpiperazinyl-
methyl substituted ligands 1 and 10, incorporating an
antagonist pharmacophore, as well as the phenylpiperazinylbu-
tyl derivatives 3 and 7 were not able to inhibit cAMP
accumulation.
Figure 3. Ligand induced D2long receptor internalization. FLAG-tagged
D
2long receptors stably expressed in HEK293 cells were incubated with
M1 antibody followed by treatment for 30 min with 2, 3, 6, and 7 each
at 10 μM. Treatment with 2 and 6 potently induced D2long receptor
internalization (indicated with yellow arrow). No internalization was
observed after incubation with 3 and 7. Representative pictures of each
condition are shown (n = 3).
The test compounds were evaluated for their propensity to
induce internalization of the FLAG-tagged D2long receptor. In
agreement with the outcome of the cAMP accumulation assay,
the homobivalent ligands 1 and 3 as well as the corresponding
monomers 10 and 7 were not able to induce D2 internalization,
whereas the aminoindane derivatives 2 and 6 potently induced
translocation of the receptor from the plasma membrane to the
cytosol, indicating receptor internalization (Figure 3). The
heterobivalent ligands 8 and 9 linking an agonist with an
antagonist pharmacophore were also investigated for their
ability to induce activation and internalization. Measuring the
inhibition of cAMP accumulation revealed D2 antagonist
behavior for 8 and low partial agonist properties for 9 (eff =
13%). Both heterobivalent test compounds did not induce D2
receptor internalization. Obviously, recognition of the phenyl-
piperazine derived antagonist moieties of the heterobivalent
ligands leads to an inhibition of D2 internalization and ligand
efficacy of the aminoindane pharmacophore. Thus, for this series,
there did not appear to be a functional dissociation or “ligand
bias” for cAMP accumulation versus receptor internalization at
saturating ligand concentration. However, while both dimers 2
and 9 had similar potencies for cAMP accumulation, the
heterodimer 9 was not able to induce receptor internalization,
CONCLUSION
■
On the basis of our recently developed bivalent dopamine D2
receptor antagonists of type 1, the bivalent agonist 2 was
developed. Compared with monovalent control compounds
containing capped spacers, both ligands revealed substantial
steepening of the competition curve, indicating a bivalent
binding mode. Dimer-specific Hill slopes were not a result of
varying functional properties because both the dopaminergic 2
and the monovalent control agent 6 were able to substantially
inhibit cAMP accumulation and to induce D2 receptor
internalization. Investigation of the heterobivalent ligands 8
and 9, containing an agonist and a phenylpiperazine-based
antagonist pharmacophore, revealed moderate steepening of
the displacement curves and antagonist to very weak partial
agonist properties in both functional assays. Thus, recognition
of the phenylpiperazine derived antagonist moieties of the
heterobivalent ligands leads to an inhibition of D2 internal-
ization and ligand efficacy of the aminoindane pharmacophore
indicating functional crosstalk between two physically interact-
ing D2 protomers.
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181 μmol) in anhydrous DMF (1 mL) was added. The reaction
mixture was stirred for 10 min. Then DIPEA (22 μL, 135 μmol) was
added very slowly, and after that a solution of (N-indan-2-yl-N-
propyl)butane-1,4-diamine17 (28 mg, 113 μmol) in anhydrous DMF
(2 mL) was added dropwise. The mixture was stirred for 1 h at room
temperature before aqueous NaHCO3 was added. The aqueous layer
was extracted with CH2Cl2. Then the combined organic layers were
washed three times with brine, dried (Na2SO4), and evaporated. The
residue was purified by flash chromatography (CH2Cl2/MeOH, 98:2
to 96:4 + 0.5% TEA) to give 2 as a colorless oil (44.1 mg, 87% yield).
EXPERIMENTAL SECTION
■
Chemistry. Dry solvents and reagents were of commercial quality
and were used as purchased. MS were run on a JEOL JMS-GC Mate II
spectrometer by EI (70 eV) with solid inlet or a Bruker Esquire 2000
by APC or ionization. HR-EIMS experiments were run on a JEOL
JMS-GC Mate II using Peak-Matching (M/ΔM > 5000). NMR spectra
were obtained on a Bruker Avance 360 or a Bruker Avance 600
spectrometer relative to TMS in the solvents indicated (J in Hz).
Melting points were determined with a MEL-TEMP II melting point
apparatus (Laboratory Devices, U.S.) in open capillaries and given
uncorrected. IR spectra were performed on a Jasco FT/IR 410
spectrometer. Purification by flash chromatography was performed
using silica gel 60; TLC analyses were performed using Merck 60 F254
aluminum sheets and analyzed by UV light (254 nm). Analytical
HPLC was performed on Agilent 1100 HPLC systems employing
a VWL detector. As column, a ZORBAX ECLIPSE XDB-C18
(4.6 mm × 150 mm, 5 μm) was used. HPLC purity was measured
using following binary solvent systems: system A, eluent, CH3OH in
0.1% aqueous trifluoroacetic acid, 10−100% CH3OH in 15 min, 100%
for 3 min, flow rate of 1.0 mL/min, λ = 254 nm; system B, eluent,
CH3CN in 0.1% aqueous trifluoroacetic acid, 10% CH3CN for 2 min
to 95% CH3CN in 18 min, 95% for 1 min, flow rate of 1.0 mL/min,
λ = 254 nm. The purity of all test compounds and key intermediates
was determined to be >95%.
IR 2933, 1637, 1547, 1506, 1462, 1269, 1225, 1128, 1030, 744 cm−1
.
1H NMR (600 MHz, CDCl3) δ 0.87 (t, J = 7.4 Hz, 6H), 1.22−1.32
(m, 8H), 1.49 (m, 4H), 1.55−1.68 (m, 8H), 1.76−1.90 (m, 4H), 2.23
(m, 4H), 2.50 (m, 4H), 2.57 (t, J = 7.2 Hz, 4H), 2.87 (dd, J = 15.1, 8.7
Hz, 4H), 2.92 (t, J = 7.4 Hz, 4H), 3.00 (dd, J = 15.5, 7.9 Hz, 4H), 3.45
(m, 4H), 3.65 (m, 2H), 3.89 (s, 6H), 4.06 (t, J = 6.4 Hz, 4H), 4.28 (t,
J = 7.0 Hz, 4H), 6.57 (m, 2H), 6.77 (d, J = 8.3 Hz, 2H), 7.09−7.17 (m,
8H), 7.21 (dd, J = 8.3, 1.9 Hz, 2H), 7.30 (s, 2H), 7.42 (d, J = 1.9 Hz,
2H). 13C NMR (150 MHz, CDCl3) δ 12.0, 19.8, 22.1, 25.0, 26.2, 27.8,
28.7, 30.2, 40.1, 50.1, 51.0, 53.3, 56.1, 63.0, 67.9, 111.1, 111.7, 119.2,
120.9, 124.4, 126.3, 127.6, 141.7, 147.0, 149.3, 151.0, 167.2. HPLC
system A (254 nm) purity >99% (tR = 12.1 min); system B (254 nm)
purity 98% (tR = 12.7 min). APCI-MS calcd m/z for C66H92N10O6,
1121.5; found 1122 (M + H)+.
Radioligand Binding Studies. Receptor binding studies were
carried out as described previously.46 In brief, competition binding
experiments with the human D2long, D2short, D3, and D4.4 receptors were
run on membrane preparations from CHO cells stably expressing the
corresponding receptor. Assays were run with membranes at protein
concentrations per well of 1−10 μg/mL and [3H]spiperone (specific
activity of 84 Ci/mmol, PerkinElmer, Rodgau, Germany) at final
concentrations of 0.1−0.4 nM according to the individual KD values in
binding buffer (50 mM Tris, 1.0 mM EDTA, 5.0 mM MgCl2, 100 μg/
mL bacitracin, and 5 μg/mL soybean trypsin inhibitor at pH 7.4). The
KD values were 0.040−0.18, 0.090−0.24, 0.10−0.36, and 0.15−0.30
nM for the D2long, D2short, D3, and D4 receptor, respectively. The
corresponding Bmax values were in the range of 450−1270 fmol/mg for
Methyl 3-Methoxy-4-(pent-4-yn-1-yloxy)benzoate (4). A
suspension of methyl 4-hydroxy-3-methoxybenzoate (4.0 g, 22.0
mmol), 5-chloropent-1-yne (5.86 mL, 54.9 mmol), K2CO3 (9.1 g, 65.9
mmol), and KI (3.6 g, 22.0 mmol) in anhydrous CH3CN (60 mL) was
stirred at reflux temperature for 20 h. Then the reaction mixture was
allowed to cool to room temperature, and the solvent was evaporated.
The residue was dissolved in H2O and extracted with CH2Cl2. The
combined organic layers were dried (MgSO4) and evaporated. The
residue was purified by flash chromatography (hexane/EtOAc, 4:1
to 2:1) to give 4 as a white solid (5.13 g, 94% yield), mp 67 °C. IR
3277, 2951, 1714, 1601, 1515, 1435, 1295, 1272, 1222, 1134, 1033,
1
762 cm−1. H NMR (360 MHz, CDCl3) δ 1.98 (t, J = 2.7 Hz, 1H),
2.08 (m, 2H), 2.43 (dt, J = 6.9, 2.7 Hz, 2H), 3.89 (s, 3H), 3.91 (s, 3H),
4.18 (t, J = 6.4 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 2.0 Hz,
1H), 7.65 (dd, J = 8.4, 2.0 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ
15.1, 27.9, 51.9, 56.0, 67.2, 69.0, 83.2, 111.7, 112.4, 122.7, 123.5, 148.9,
152.4, 166.9. HPLC system A (254 nm) purity 97% (tR = 13.6 min);
system B (254 nm) purity 98% (tR = 15.2 min). HR-EIMS calcd m/z
for C14H16O4, 248.1049; found 248.1049.
D
2long, 675−2595 fmol/mg for D2short, 1450−11080 fmol/mg for D3,
and 565−1810 fmol/mg for D4 receptor. Nonspecific binding was
determined in the presence of 10 μM haloperidol. Specific binding
represented about 85% of the total binding. The D1 receptor binding
experiments were performed with homogenates prepared from porcine
striatum as described.46 Assays were run with membranes at protein
concentrations per well of 20−40 μg/mL and radioligand concen-
trations of 0.3−0.5 nM [3H]SCH 23390 (specific activity of 60 Ci/
4,4′-[Octane-1,8-diylbis(1H-1,2,3-triazol-1,4-diylpropan-3,1-
diyloxy)]bis(3-methoxybenzoic acid) (5). A suspension of 4 (500
mg, 2.0 mmol), 1,8-diazidooctane45 (200 mg, 1.0 mmol), sodium
ascorbate (60 mg, 0.3 mmol), and copper sulfate pentahydrate (25 mg,
0.1 mmol) in MeOH (3 mL) and CH2Cl2 (12 mL) was stirred at
room temperature overnight. The reaction was quenched with
saturated aqueous NaHCO3 and 0.1 M EDTA, extracted with
CH2Cl2, dried (Na2SO4), and evaporated. The residue was dissolved
in a methanolic solution of KOH (10 mL, 2M). The resulting solution
was stirred at 60 °C for 2 h. Then MeOH was evaporated. The residue
was adjusted to pH 1 with 2 M HCl and extracted with a mixture of
CH2Cl2 and MeOH (3:1). The organic layer was dried (Na2SO4) and
evaporated. The residue was purified by flash chromatography
(CH2Cl2/MeOH, 95:5 to 92:8 + 0.5% TFA) to give 5 as a white
solid (652.4 mg, 97% yield), mp 187 °C. IR 2929, 1670, 1581, 1516,
mmol, Biotrend, Koln, Germany) with K values of 0.51−0.67 nM.
̈
D
Protein concentration was established by the method of Lowry using
bovine serum albumin as a standard.47
Adenylyl Cyclase Inhibition Assay. Bioluminescence based
cAMP-Glo assay (Promega, Mannheim, Germany) was performed
according to the manufacturer's instructions. Briefly, CHO cells
expressing D2long receptor were seeded into a white half-area 96-well
plate (5000 cells/well) 24 h prior to the assay. On the days of the
assays, cells were washed with phosphate buffered saline (PBS, pH
7.4) to remove traces of serum and incubated with various
concentrations of compounds in the presence of 20 μM forskolin in
serum-free medium that contained 500 μM IBMX and 100 μM Ro 20-
1724, pH 7.4. After 15 min of incubation at 25 °C, cells were lysed
with cAMP-Glo lysis buffer, kinase reaction was performed with a
reaction buffer containing PKA, and finally an equal volume of Kinase-
Glo reagent was added. Bioluminescence was read on a microplate
reader Victor3V (Perkin-Elmer, Rodgau, Germany). The experiments
were performed two to nine times per compound with each
concentration in triplicate.
1467, 1423, 1271, 1226, 1028, 766 cm−1 1H NMR (360 MHz,
.
DMSO-d6) δ 1.13−1.25 (m, 8H), 1.75 (m, 4H), 2.06 (m, 4H), 2.77 (t,
J = 7.6 Hz, 4H), 3.81 (s, 6H), 4.07 (t, J = 6.4 Hz, 4H), 4.27 (t, J = 7.2
Hz, 4H), 7.01 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 1.9 Hz, 2H), 7.53 (dd,
J = 8.3, 1.9 Hz, 2H), 7.88 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ
21.6, 25.7, 28.2, 28.4, 29.6, 49.1, 55.5, 67.5, 111.9, 112.2, 121.8, 123.06,
123.14, 146.0, 148.4, 151.9, 167.1. APCI-MS calcd m/z for
C34H44N6O8, 664.7; found 665 (M + H)+.
4,4′-[Octane-1,8-diylbis(1H-1,2,3-triazol-1,4-diylpropan-3,1-
diyloxy)]bis[N-[4-[indan-2-yl(propyl)amino]butyl]-3-methoxy-
benzamide] (2). A solution of 5 (30 mg, 45 μmol) in anhydrous
DMF (2 mL) was cooled to 0 °C before a solution of HATU (69 mg,
Data Analysis. The resulting competition curves of the receptor
binding experiments and activity assay were analyzed by nonlinear
regression using the algorithms in PRISM 5.0 (GraphPad Software,
San Diego, CA). Competition curves were fitted to a sigmoid curve by
nonlinear regression analysis in which the log EC50 and the Hill
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Journal of Medicinal Chemistry
Article
coefficient were free parameters. EC50 values were transformed to Ki
values according to the equation of Cheng and Prusoff.48
ABBREVIATIONS USED
■
GPCR, G-protein-coupled receptor; 1,4-DAP, 1,4-disubstituted
aromatic piperidine/piperazine; TM, transmembrane domain;
EL, extracellular loop; CuAAC, copper(I)-catalyzed azide−
alkyne cycloaddition; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate; HATU, 2-(7-aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate; CHO, Chinese hamster ovary; HEK, human
embryonic kidney; Ki, inhibition constant; SEM, standard
error of the mean; nH, Hill slope; HR-EIMS, high resolution
electron ionization mass spectrometry; APCI-MS, atmospheric
pressure chemical ionization mass spectrometry
Immunocytochemistry. The antibody-feeding immunocyto-
chemistry experiments were performed as described.49 Briefly, cells
stably expressing FLAG-tagged D2long receptor were grown on
coverslips pretreated with gelatin to ∼50% confluency. Live cells
were fed M1 antibody (Sigma) directed against the FLAG epitope
(1:1000, 30 min) and then incubated with ligands (10 μM, 30 min) or
left untreated. Cells were then fixed with 4% formaldehyde in PBS, pH
7.4. After fixation, cells were permeabilized in Blotto (3% milk, 0.1%
Triton X, 1 mM CaCl2, 50mM Tris·HCl, pH 7.5) and stained with
Alexa Fluor 594 or Alexa Fluor 488 goat anti-mouse IgG2b antibody
(Invitrogen, 1:500, 45 min). The coverslips were mounted using
mounting medium from Vectashield. Images were acquired at 63×
with a LSM 510 laser confocal microscope (Zeiss) or a TCS SP5
confocal microscope (Leica Microsystems). Confocal images were
exported as TIFF files and processed using GIMP 2.6.
Construction of the Dopamine D2 Receptor Dimer. The
previously published homology model of the dopamine D2 receptor24
was used to construct a dopamine D2 dimer. The model was built
using the crystal structure of the β2-adrenergic receptor32 as a
template. The dimeric arrangement of two monomers was
accomplished according to Guo et al.9 The dimer was constructed
by means of VMD50 and afterward submitted to energy minimization
in order to avoid repulsive interactions between proximate amino acid
side chains. Therefore, the SANDER classic module of AMBER 1051
was used by applying 500 cycles of steepest descent minimization,
followed by 4500 cycles of conjugate gradient minimization. The
calculation was carried out in a water box with periodic boundary
conditions and a nonbonded cutoff of 8.0 Å. The all atom force field
ff99SB was applied.52 The minimized receptor dimer was used for the
insertion of a bivalent ligand.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed reaction scheme for the synthesis of the mono- and
heterobivalent target compounds (6−9), experimental and
spectroscopic data of the nonkey target compounds 3 and 6−9
and the respective precursors thereof, receptor binding data
employing porcine 5-HT1, 5-HT2, and α1 receptors, confocal
images of the antibody-feeding immunocytochemistry experi-
ments after stimulation with quinpirole, with the compounds 1
and 8−10, or with no ligand added. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 9131 85-29383. Fax: +49 9131 85-22585. E-mail:
peter.gmeiner@medchem.uni-erlangen.de.
■
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ACKNOWLEDGMENTS
■
We thank Prof. Thomas Stamminger (Institute for Clinical and
Molecular Virology, University of Erlangen-Nuremberg,
Germany) for providing the confocal microscope and Dr.
Nuska Tschammer for conducting microscopy experiments.
This research was funded by a grant of the Deutsche
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