Bivalent dopamine D2 receptor ligands: Synthesis and binding properties

Article abstract of DOI:10.1021/jm2004859

Dopamine D₂ receptor homodimers might be of particular importance in the pathophysiology of schizophrenia and, thus, serve as promising target proteins for the discovery of atypical antipsychotics. A highly attractive approach to investigate and control GPCR dimerization may be provided by the exploration and characterization of bivalent ligands, which can act as molecular probes simultaneously binding two adjacent binding sites of a dimer. The synthesis of bivalent dopamine D₂ receptor ligands of type 1 is presented, incorporating the privileged structure of 1,4-disubstituted aromatic piperidines/piperazines (1,4-DAPs) and triazolyl-linked spacer elements. Radioligand binding studies provided diagnostic insights when Hill slopes close to two for bivalent ligands with particular spacer lengths and a comparative analysis with respective monovalent control ligands and unsymmetrically substituted analogues indicated a bivalent binding mode with a simultaneous occupancy of two neighboring binding sites.

Full text of DOI:10.1021/jm2004859

ARTICLE
pubs.acs.org/jmc
Bivalent Dopamine D2 Receptor Ligands: Synthesis and
Binding Properties
Julia K€uhhorn, Harald H€ubner, and Peter Gmeiner*
Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander University, Schuhstrasse 19, 91052 Erlangen,
Germany
S Supporting Information
b
ABSTRACT: Dopamine D2 receptor homodimers might be of
particular importance in the pathophysiology of schizophrenia
and, thus, serve as promising target proteins for the discovery of
atypical antipsychotics. A highly attractive approach to investigate
and control GPCR dimerization may be provided by the explora-
tion and characterization of bivalent ligands, which can act as
molecular probes simultaneously binding two adjacent binding sites of a dimer. The synthesis of bivalent dopamine D2 receptor ligands
of type 1 is presented, incorporating the privileged structure of 1,4-disubstituted aromatic piperidines/piperazines (1,4-DAPs) and
triazolyl-linked spacer elements. Radioligand binding studies provided diagnostic insights when Hill slopes close to two for bivalent
ligands with particular spacer lengths and a comparative analysis with respective monovalent control ligands and unsymmetrically
substituted analogues indicated a bivalent binding mode with a simultaneous occupancy of two neighboring binding sites.
Chart 1. General Structure of the Dopamine D2 Receptor
Bivalent Ligands
’ INTRODUCTION
G-protein-coupled receptors (GPCRs) have been tradition-
ally considered to exist as monomers. However, in recent years,
evidence has accumulated to suggest that GPCRs can cross-react
forming homo- or heterodimers or higher-order oligomers.1,2
Moreover, receptor dimerization is often essential for receptor
function and can also modulate ligand pharmacology, signal
transduction, and cellular trafficking.3 Previous investigations
indicate that dopamine D2 receptors, which are associated with
many central nervous system diseases including schizophrenia,
Parkinson’s disease, drug addiction, and erectile dysfunction,
exist as homomeric4ꢀ8 or heteromeric9,10 complexes. Using func-
tional complementation assays, communication between proto-
mers of the D2 receptors could be observed leading to a signi-
ficant modulation of GPCR activation in transfected cell lines.11
According to very recent findings,12 homodimers of dopamine
D2 receptors might be of particular importance in the pathophy-
siology of schizophrenia and, thus, serve as promising targets
for the discovery of atypical antipsychotics. Investigating post-
mortem striatal sections from schizophrenia patients and striatal
tissue of animal models of schizophrenia, the expression of D2
dimers proved to be significantly enhanced, while expression of
D2 monomers was decreased. Further studies in this exciting field
will request the invention of new methodologies and selective
pharmacological tools.
tools, a differential analysis of homo- and heteromers, tissue-
selective modulation, and a reversible control of monomer/
dimer equilibriums may be facilitated. In this paper, we describe
the synthesis of homodimeric dopamine D2 receptor ligands of
type 1 (Chart 1) incorporating the privileged structure of 1,4-
disubstituted aromatic piperidines/piperazines (1,4-DAPs) and
triazolyl-linked spacer elements. Radioligand binding studies are
also reported when Hill slopes close to two for homodimeric
ligands with particular spacer lengths and a comparative analysis
with respective monomers and unsymmetrically substituted ana-
logues indicated a bivalent binding mode with a simultaneous
occupancy of two adjacent binding sites.
A highly attractive approach to investigate and control GPCR
dimerization may be provided by the exploration and character-
ization of bivalent ligands, which can act as molecular probes
simultaneously binding two adjacent binding sites of a dimer.
Containing two identical or distinct pharmacophores linked by a
spacer, bivalent ligands can either address homomers13ꢀ18 or
heteromers.19ꢀ22 Employing bivalent ligands as pharmacological
’ RESULTS AND DISCUSSION
Design. The biological properties of 1,4-DAPs are encoded
by an aromatic headgroup, which controls intrinsic activity, and
Received: April 21, 2011
Published: May 22, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1a
a Reagents and conditions: (a) 3-bromo-1-propyne, 5-chloro-1-pentyne, or 6-chloro-1-hexyne, K2CO3, KI, CH3CN, reflux, 16ꢀ24 h (47ꢀ85%);
(b) 1,8-diazidooctane, 1,10-diazidododecane, 1,2-bis(2-azidoethoxy)ethane, or 1,10-oxybis[2-(2-azidoethoxy)ethane], sodium ascorbate, CuSO4, H2O/
tBuOH/CH2Cl2 1:2:2, RT, 20 h (65ꢀ97%); (c) 2-methoxyphenylpiperazine, Na(OAc)3BH, CH2Cl2, RT, 16 h (38ꢀ73%).
an amine moiety, which is responsible for the formation of a
reinforced hydrogen bond to the crucial residue Asp3.32 in the
transmembrane helix 3 (TM3). Thus, the phenylpiperidine/
phenylpiperazine scaffold simulates the endogenous biogenic
amine.23 Besides this, a lipophilic appendage is necessary enhanc-
ing ligand affinity. According to recent mutagenesis and mole-
cular docking studies, the lipophilic appendage, which is usually
represented by a heterocyclic system, directs to a hydrophobic
microdomain that is formed by the extracellular portion of TM2,
TM3, and TM7 and specific residues of the extracellular loop
2 (EL2).24ꢀ26 Because a formal elongation of this appendage is
expected to lead to the “entrance region” of the receptor and
from there to the binding pocket of a neighboring protomer, the
para-position of an aromatic moiety terminating the lipophilic
appendage was considered to be perfectly applicable as a point of
attachment for a linker unit. Instead of heterocyclic systems that
we used for our originally described monomeric molecular probes,
we chose a vanilline-derived carbocyclic moiety that could be easily
reacted in the para-position with a part of the linker sequence by a
Williamson ether synthesis and attached to the phenylpiperazine
moiety by reductive amination. Dimerization and construction of a
symmetric linker by click chemistry should result in spacer units of
approximately 25 Å, which is in agreement with structureꢀactivity
relationships of recently described bivalent GPCR ligands.21,27
Chemical Synthesis. A collection of homodimeric dopamine
receptor ligands with spacers built from 18ꢀ26 atoms was syn-
thesized. Because conformational flexibility and physicochemical
properties of the spacer seem to be essential for high specific
binding,18,28 flexible hydrophobic and hydrophilic spacer ele-
ments were implemented. The chemical composition of the
bridging moiety was varied using hydrophobic linkers composed
of methylene units as well as hydrophilic spacers incorporat-
ing oligo(ethylene glycol) units. As a dopamine surrogate, we
employed ortho-methoxyphenylpiperazine that was attached to a
benzylic CH2 position of a carbocyclic arene moiety.29 For com-
parative purposes, monovalent fragments containing one-half of the
linker and asymmetric “dummy ligands” incorporating one original
pharmacophore while the second pharmacophore is replaced by a
structurally similar nonbinding motif were synthesized.
in the presence of K2CO3 and KI.30 Employing copper(I)-
catalyzed azideꢀalkyne cycloaddition (CuAAC),31 the terminal
alkynes 2aꢀc were reacted with 1,8-diazidooctane,32 1,10-
diazidododecane,32 1,2-bis(2-azidoethoxy)ethane,33 or 1,10-oxybis-
[2-(2-azidoethoxy)ethane]33 to afford the synthetic intermediates
3aꢀg in good to excellent yields (65ꢀ97%). Reductive amination
of 3aꢀg with ortho-methoxyphenylpiperazine furnished the final
products 1aꢀg in 38ꢀ73% yield (Scheme 1).
For the preparation of the monovalent ligands 4aꢀc con-
taining capped spacers, the central building blocks 2aꢀc were
reacted with butylazide34 and reductively aminated by ortho-
methoxyphenylpiperazine (Scheme 2). To efficiently synthesize
the oxa-analogues 6a,b, the reaction sequence was inverted when
the final triazole formation was done using an excess of 1-azido-2-
ethoxyethane. The synthesis of the asymmetric molecular probes
of type 8 and 9 started from the terminal alkyne 5b, which was
subjected to CuAAC using a 5-fold excess of 1,8-diazidooctane to
afford the intermediate 7 in 52% yield. The synthetic intermedi-
ates 5c,d were obtained by reductive amination of the vanilline
derivative 2b with either methyl- or cyclohexylpiperazine. Cy-
cloaddition of the azide 7 and the alkynes 5c,d and pentyne
afforded the asymmetric ligands 9a,b and 8, respectively.
Receptor Binding. In vitro binding affinity of the molecular
probes 1aꢀg, along with their monovalent control agents 4aꢀc,
6a,b, 8, 9a,b, and the reference drug haloperidol was measured by
displacement of the radioligand [3H]spiperone from human
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. Our initial investigations were di-
rected to a careful comparison of the dimeric ligands 1a and 1b
displaying oxymethylene and oxypropylene units, respectively,
between the triazole and the benzene rings with their monova-
lent analogues 4a,b. The test compounds gave significant specific
binding for D2long, D2short, D3, and D4 (Table 1) and clearly re-
duced D1 affinity (Table 1). For both variants of the D2 subtype,
Ki values in the double-digit nanomolar range were observed with
slightly higher affinities for the dimeric test compounds. D3 affi-
nity was substantially lower with Ki values of 31ꢀ84 nM for the
dimers and 320ꢀ610 nM for the monomeric analogs. Ki values of
5.4ꢀ15 nM and 3.3ꢀ7.2 nM for 1a,b and 4a,b, respectively, indi-
cated that D4 binding was high for both dimeric and monomeric
ligands. Whereas 1a,b displayed only a 5-fold preference for D4
In detail, our central building block vanilline bearing a free
hydroxyl group in the para-position of the carbaldehyde was con-
verted to the corresponding alkynylethers 2aꢀc by alkylation with
3-bromo-1-propyne, 5-chloro-1-pentyne, or 6-chloro-1-hexyne
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Scheme 2a
a Reagents and conditions: (a) butyl azide, sodium ascorbate, CuSO4, H2O:tBuOH:CH2Cl2 1:2:2, RT, 16 h (60ꢀ94%); (b) 2-methoxyphenylpiper-
azine, Na(OAc)3BH, CH2Cl2, RT, 14 h (68ꢀ79%); (c) 2-methoxyphenylpiperazine or methylpiperazine or cyclohexylpiperazine, Na(OAc)3BH,
CH2Cl2, RT, 16 h (69ꢀ98%); (d) 5a or 5b, 1-azido-2-ethoxyethane, sodium ascorbate, CuSO4, CH2Cl2:MeOH 4:1 (73ꢀ83%); (e) 5b, 1,8-
diazidooctane, sodium ascorbate, CuSO4, CH2Cl2:MeOH 4:1, RT, 42 h (52%); (f) pentyne or 5c or 5d, sodium ascorbate, CuSO4, CH2Cl2:MeOH 4:1,
RT, 16 h (58ꢀ81%).
Table 1. Receptor Binding Data for the Bivalent Compounds and Their Monomeric Counterparts in Comparison to the
Reference Haloperidol Employing Porcine Dopamine D1 Receptor and the Human Subtypes D2long, D2short, D3, and D4.4
Ki valuesa (nM) ( SEM and Hill slopesb [absolute value of nH ( SEM]
[3H]SCH 23390
hD2long
[3H]spiperone
hD3
compd n spacer atoms
X
pD1
hD2short
hD4.4
1a
1
3
3
4
1
3
3
1
3
4
1
3
3
3
3
18
22
24
26
18
22
25
(CH2)8
(CH2)8
(CH2)10
(CH2)10
210 ( 28 16 ( 2.1 [1.6 ( 0.1]
220 ( 61 22 ( 2.4 [2.0 ( 0.1]
250 ( 77 23 ( 4.0 [1.6 ( 0.1]
25 ( 8.4 [1.7 ( 0.1]
41 ( 8.2 [2.0 ( 0.1]
17 ( 4.0 [1.7 ( 0.2]
31 ( 3.5 [1.5 ( 0.1] 5.4 ( 0.78 [1.5 ( 0.1]
84 ( 11 [1.6 ( 0.1] 15 ( 1.3 [1.6 ( 0.1]
96 ( 15 [1.6 ( 0.2] 19 ( 4.6 [1.7 ( 0.2]
74 ( 33 [1.2 ( 0.2] 11 ( 2.6 [1.2 ( 0.1]
36 ( 9.4 [0.9 ( 0.1] 6.8 ( 1.5 [0.8 ( 0.1]
61 ( 7.6 [1.1 ( 0.1] 12 ( 0.95 [1.1 ( 0.1]
72 ( 5.7 [1.1 ( 0.1] 19 ( 2.5 [0.9 ( 0.1]
320 ( 89 [0.9 ( 0.0] 3.3 ( 0.34 [0.9 ( 0.0]
610 ( 190 [1.2 ( 0.2] 7.2 ( 1.3 [1.0 ( 0.1]
590 ( 130 [1.0 ( 0.1] 13 ( 1.3 [0.9 ( 0.0]
440 ( 94 [1.0 ( 0.1] 8.4 ( 1.6 [0.9 ( 0.1]
250 ( 64 [1.0 ( 0.2] 6.4 ( 0.25 [1.2 ( 0.2]
230 ( 25 [1.1 ( 0.1] 14 ( 2.0 [1.2 ( 0.1]
1b
1c
1d
300 ( 100 17 ( 5.2 [1.0 ( 0.1] 8.2 ( 2.5 [1.1 ( 0.1]
1e
CH2(CH2OCH2)2CH2 390 ( 80 3.3 ( 0.92 [0.9 ( 0.0] 3.4 ( 0.55 [0.9 ( 0.1]
CH2(CH2OCH2)2CH2 220 ( 38 2.8 ( 0.26 [1.0 ( 0.1] 3.7 ( 1.1 [0.9 ( 0.1]
CH2(CH2OCH2)3CH2 270 ( 29 4.8 ( 0.68 [1.0 ( 0.0] 6.2 ( 1.4 [1.1 ( 0.1]
1f
1g
4a
2300 ( 410 63 ( 6.8 [1.0 ( 0.1]
1500 ( 140 67 ( 12 [1.1 ( 0.0]
2500 ( 540 52 ( 2.5 [0.9 ( 0.1]
70 ( 15 [1.0 ( 0.0]
53 ( 11 [1.0 ( 0.1]
67 ( 21 [0.9 ( 0.1]
4b
4c
6a
3800 ( 510 68 ( 13 [1.0 ( 0.0] 100 ( 17 [1.1 ( 0.1]
6b
870 ( 90 18 ( 2.6 [1.1 ( 0.1]
400 ( 45 36 ( 6.6 [1.1 ( 0.1]
16 ( 2.5 [1.1 ( 0.1]
32 ( 6.7 [1.1 ( 0.0]
8
9a
21
22
22
(CH2)8
(CH2)8
(CH2)8
980 ( 120 9.8 ( 4.0 [0.9 ( 0.1] 7.3 ( 0.55 [0.90 ( 0.0] 220 ( 34 [0.9 ( 0.1] 12 ( 2.9 [0.9 ( 0.1]
480 ( 130 19 ( 2.9 [1.2 ( 0.1] 17 ( 2.3 [1.0 ( 0.1] 100 ( 12 [1.1 ( 0.1] 19 ( 1.8 [1.3 ( 0.1]
93 ( 11 1.5 ( 0.44 [1.0 ( 0.1] 1.1 ( 0.16 [1.1 ( 0.1] 6.3 ( 1.6 [1.1 ( 0.1] 6.3 ( 1.3 [1.2 ( 0.1]
9b
haloperidol
a Ki values ( SEM derived from 4ꢀ10 experiments each done in triplicate. b Hill slopes ( SEM are derived from the same binding curves recorded for the
determination of Ki values; the original nH was negative but is displayed as absolute value.
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these probes gave Hill slopes close to one (nH = 0.9ꢀ1.1). Thus,
length and physicochemical properties of the spacer clearly affect the
binding mode when a hydrophobic molecular bridge and a linker
length of approximately 25 Å appear to be best for a bivalent binding
of two interacting receptor protomers, which is in accordance with
previous studies on bivalent GPCR ligands.21,27 To corroborate a
receptor-chelating binding mode, further control experiments were
performed using the asymmetric ligands 8 and 9a,b that are com-
posed of a pharmacophore with the complete spacer arm or one
pharmacophore, a spacer of 22 atom length, and a structurally
distorted second pharmacophore with a cyclohexyl or a methyl
unit displacing the methoxyphenyl headgroup. In fact, Hill
coefficients close to one (1.0ꢀ1.2) were observed, indicating
that the two D2 binding pharmacophores appear to be essential
for the cooperative binding mode. Ligand affinity of 8 and
9a,b for D2long and D2short (Ki = 7.3ꢀ36 nM) was similar to
the monovalent and bivalent parent compounds 1b and 4b.
To better characterize the biological properties and the deter-
minants for the unusual Hill slopes, the bivalent ligand 1b was
further investigated in comparison to the control ligand 4b. Since
the concentration of sodium ions was shown to significantly in-
fluence D2 affinity, maximal binding capacity, and ligand efficacy,38ꢀ40
the influence of Naþ on the ligand binding mode was studied.
While the monovalent ligand 4b showed very similar binding
properties in the absence (Ki = 53 ( 11 nM; nH = 1.0 ( 0.1) and
in the presence of 100 mM sodium chloride (Ki = 35 ( 6.0 nM;
nH = 0.8 ( 0.0), flattening of the binding curve could be observed
for the bivalent ligand 1b revealing a Hill slope close to one (nH =
1.1 ( 0.1 [þNaþ]; nH = 2.0 ( 0.1 [ꢀNaþ]) and Ki values of
12 ( 1.5 nM [þNaþ] and 41 ( 8.2 nM [ꢀNaþ]). Dopamine D2
receptor dimerization is described to be dependent on pH values
and temperature.41 According to our data, the ionic strength of the
system might also control the dimerization state. Thus, sodium
ions might perturb and destabilize receptor dimers, thus preclud-
ing a receptor-bridging binding mode of 1a.
Figure 1. Representative binding curves of 1b and 4b at the human
D2short receptor displaying different curve shapes resulting in Hill slopes
(absolute value of nH) of 2.0 (for 1b) and 1.0 (for 4b) indicating a bivalent
and a monovalent binding mode, respectively.
over D3, approximately 80ꢀ100-fold selectivity was observed for
4a,b. To gain diagnostic insights into the binding modes of our
bivalent test compounds, we analyzed the profiles of the compe-
tition binding curves in detail. Competitive binding curves that
follow the law of mass action have a slope of one. A Hill slope
(nH) less than one is considered a reflection of negative coopera-
tivity or the ability of a ligand to bind to G-protein precoupled
receptors with a higher affinity than uncoupled receptors. Thus,
agonists often show Hill slopes between 0.5 and 0.7. Antagonists
tend to have Hill slope values close to one since they fail to diff-
erentiate between the precoupled and uncoupled receptor popu-
lations, thereby following the general law of mass action for single
site competition.35 Examination of the binding curves of the
monomeric ligands 4a,b revealed Hill slopes close to one (nH =
0.9ꢀ1.2) indicating antagonist properties and a binding mode
that one receptor protomer binds one ligand. Interestingly, care-
ful analysis of the heterologous competition experiments that
we conducted with the dimers 1a,b revealed a steepening of
the competition curves leading to Hill slopes between 1.5 and
2.0. This is indicative of a positive cooperative binding. Positive
cooperativity is usually observed as a consequence of an allosteric
effect inducing conformational crosstalk within one receptor
protomer or modulation of the interaction between two proto-
mers of a receptor dimer.36,37 Bivalent ligands addressing two
adjacent binding sites of receptor dimers will also induce such
cooperativity because binding of the second pharmacophore is
significantly accelerated due to the vicinity of ligand and the thus
facilitated enrichment of local concentration. Thus, bivalent
binding leads to the liberation of two equivalents of radioligand
and substantial steepening of the competition curve. The highest
Hill slope of 2.0 was calculated for the interaction of D2long and
D2short with the dimeric probe 1b incorporating a 22-atom spacer
(Figure 1). To learn whether this effect depends on the length of
the spacer arm, the dimeric homologues 1c and 1d with 24- and
26-atom spacers, respectively, were investigated. The competi-
tive binding studies gave Hill coefficients between 1.6 and 1.7 for
1c and slopes close to one for the homologue 1d (1.0ꢀ1.2)
indicating that lengthening of the distance beyond 22 atoms leads
to a reduction of a bridging binding mode by increasing the con-
finement volume. The monovalent control ligand 4c showed Hill
slopes close to one (0.9ꢀ1.0). Subsequently, the more hydrophilic
oligo(ethylene glycol) derivatives 1eꢀgwere investigated. Although
significantly higher D2 affinity was observed (Ki = 2.8ꢀ6.2 nM),
To exclude that the unusual binding behavior of the bilvalent
ligand 1b is due to an allosteric modulation,42ꢀ45 we examined
the influence of 1b and 4b on the dissociation of the radioligand
[3H]spiperone from the D2 receptor. Accelerating dissociation of
[3H]spiperone, negative allosteric modulation of the dopamine
D2 receptor was observed for amiloride and for zinc ions.46,47
Positive allosteric cooperation should markedly decrease the dis-
sociation rate. After preincubation of the receptor with the
radioligand to reach equilibrium, dissociation was observed in a
time-dependent way after adding an excess of haloperidol as a
competitor, which resulted in a koff = 0.0054 minꢀ1 and a
corresponding dissociation half-life of t1/2 = 130 min. In fact,
these parameters were identical in presence of 1b (koff = 0.0054
minꢀ1, t1/2 = 130 min) and highly similar in presence of 4b (koff
=
0.0057 minꢀ1, t1/2 = 120 min) indicating that the atypical bind-
ing mode of the bivalent ligand 1b may not be due to an allosteric
modulation.
Agonist behavior of GPCR ligands is a consequence of a
binding preference for the precoupled receptor state leading to
Hill coefficients smaller than one. The bivalent ligand 1b and the
control agent 4b were tested for their functional behavior in a
mitogenesis assay measuring an agonist stimulated incorporation
of [3H]thymidine into cells expressing the human D2long recep-
tor. As we expected from the structure of the pharmacophore,
both ligands 1b and 4b displayed neutral antagonist properties in
the functional experiments revealing that the different binding
profiles are not a result of varying functional properties.
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’ CONCLUSION
HPLC system A (254 nm) purity >99%. (tR = 11.9 min); system B
(254 nm) purity >99% (tR = 13.7 min). HR-EIMS calcd m/z for
C13H14O3 218.0943, found 218.0942.
When bivalent ligands are employed as pharmacological tools,
a differential analysis of homo- and heteromers, tissue-selective
modulation, and a reversible control of monomer/dimer equili-
briums may be facilitated. We herein present bivalent ligands of
type 1, differing in length and structure of the spacer unit that
links two identical pharmacophores. Radioligand binding assays
revealed that the bivalent ligands exhibited a distinct binding
profile compared with monovalent analogues containing capped
spacer and asymmetric “dummy ligands” incorporating one ori-
ginal pharmacophore while the second pharmacophore is re-
placed by a structurally similar nonbinding motif. Some of the
bivalent ligands revealed a steepening of the competition curve
for the D2 receptors. The profiles of the test compounds were
dependent on the structure and on the length of the spacer arm
when the bivalent ligand 1b containing a 22-atom spacer gave the
steepest curve and a Hill slope of 2. Since homodimerization of
dopamine D2 receptors is assumed to be of particular importance
in the pathophysiology of schizophrenia, molecular probes of
type 1 will serve as promising tools for the discovery of atypical
antipsychotics.
4,40-[Octane-1,8-diylbis(1H-1,2,3-triazole-1,4-diylpropane-3,1-diyloxy)]-
bis(3-methoxybenzaldehyde) (3b). A suspension of 2b (334 mg, 1.5
mmol), 1,8-diazidooctane (100 mg, 0.5 mmol), sodium ascorbate (20
mg, 0.1 mmol), and copper sulfate pentahydrate (6.4 mg, 25 μmol) in
H2O (2.5 mL), tBuOH (5 mL), and CH2Cl2 (5 mL) was stirred at room
temperature for 16 h. The reaction was quenched using saturated
NaHCO3 and 0.1 N EDTA, extracted with CH2Cl2, dried (Na2SO4),
and evaporated. The residue was purified by flash chromatography
(hexane/EtOAc 1:1ꢀ2:98) to give 3b as a white solid (290 mg, 90%
yield); mp 96 °C. IR 2360, 1681, 1587, 1510, 1269, 1136 cmꢀ1. 1H NMR
(360 MHz, CDCl3) δ 1.24ꢀ1.32 (m, 8H), 1.85 (m, 4H), 2.28 (m, 4H),
2.94 (t, J = 7.2 Hz, 4H), 3.93 (s, 6H), 4.17 (t, J = 6.5 Hz, 4H), 4.29 (t, J =
7.2 Hz, 4H), 6.96 (d, J = 8.2 Hz, 2H), 7.26 (s, 2H), 7.41 (brs, 2H), 7.43
(d, J = 1.8 Hz, 2H), 9.84 (s, 2H). 13C NMR (90 MHz, CDCl3) δ 21.9,
26.1, 28.3, 28.5, 30.1, 49.9, 55.9, 68.1, 109.2, 111.5, 120.7, 126.7, 129.9,
146.6, 149.8, 153.9, 190.6. APCI-MS calcd m/z for C34H44N6O6 632.7,
found 634 (M þ H)þ. Anal. (C34H44N6O6) C, H, N.
1,10-[Octane-1,8-diylbis[1H-1,2,3-triazole-1,4-diylpropane-3,1-diyloxy-
(3-methoxy-4,1-phenylene)methylene]]bis[4-(2-methoxyphenyl)-
piperazine] (1b). A solution of 3b (50 mg, 0.08 mmol), 1-(2-methoxy-
phenyl)piperazine (45.6 mg, 0.24 mmol), and Na(OAc)3BH (53 mg, 0.24
mmol) in anhydrous CH2Cl2 (10 mL) was stirred under nitrogen atmo-
sphere at room temperature for 20 h. The reaction mixture was quenched
with saturated aqueous NaHCO3 and extracted with CH2Cl2. The com-
bined organic layers were dried (Na2SO4) and evaporated. The residue
was purified by flash chromatography (EtOAc/MeOH 99:1ꢀ98:2 þ 1%
TEA) to give 1b as a white solid (54.7 mg, 70% yield); mp 103 °C. IR
2933, 2813, 1590, 1502, 1452, 1238 1140, 1030, 748 cmꢀ1. 1H NMR (360
MHz, CDCl3) δ 1.23ꢀ1.37 (m, 8H), 1.85 (m, 4H), 2.21 (m, 4H), 2.64
(m, 8H), 2.93 (t, J = 7.6 Hz, 4H), 3.09 (m, 8H), 3.51 (s, 4H), 3.85 (s, 6H),
3.87 (s, 6H), 4.06 (t, J = 6.3 Hz, 4H), 4.28 (t, J = 7.2 Hz, 4H), 6.79ꢀ7.00
(m, 14H), 7.30 (s, 2H). 13C NMR (90 MHz, CDCl3) δ 22.2, 26.3, 28.7,
28.9, 30.2, 50.1, 50.7, 53.3, 56.1, 62.9, 68.2, 111.2, 113.0, 118.2, 120.8,
121.0, 121.5, 122.9, 131.0, 141.5, 147.3, 147.5, 149.4, 152.3. HPLC system
A (254 nm) purity 97% (tR = 11.9 min); system B (254 nm) purity 97%
(tR = 12.7 min). APCI-MS calcd m/z for C56H76N10O6 985.3, found 986
(M þ H)þ.
’ 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 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 value in hertz). Melting points were
determined with a MEL-TEMP II melting point apparatus (Laboratory
Devices, USA) in open capillaries and are 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% to 100% CH3OH in
15 min, 100% for 3 min, flow rate 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 1.0 mL/min,
λ 254 nm. The purity of all test compounds and key intermediates was
determined to be >95%. CHN elementary analyses were performed at
the chair of Organic Chemistry of the Friedrich-Alexander University
Erlangen-N€urnberg.
3-Methoxy-4-(pent-4-yn-1-yloxy)benzaldehyde (2b). A suspension
of 4-hydroxy-3-methoxybenzaldehyde (2.3 g, 15 mmol), 5-chloropent-
1-yne (3.2 g, 30 mmol), K2CO3 (2.1 g, 15 mmol), and KI (0.5 g, 3.0
mmol) in anhydrous CH3CN (50 mL) was stirred at reflux temperature
for 16 h. Then the reaction mixture was allowed to cool to room tem-
perature, 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 chro-
matography (hexaneꢀEtOAc 4:1) to give 2b as a white solid (1.55 g,
47% yield); mp 81 °C. IR 3747, 2360, 2337, 1676, 1587, 1279, 1134, 995,
658 cmꢀ1. 1H NMR (360 MHz, CDCl3) δ 1.98 (t, J = 2.6 Hz, 1H), 2.10
(m, 2H), 2.44 (dt, J = 6.9, 2.6 Hz, 2H), 3.92 (s, 3H), 4.22 (t, J = 6.3 Hz,
2H), 7.00 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.44 (dd, J = 8.2,
1.8 Hz, 1H), 9.85 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 15.1, 27.8,
56.0, 67.4, 69.2, 83.1, 109.3, 111.6, 126.6, 130.0, 149.8, 153.9, 190.8.
1-[[4-[3-(1-Butyl-1H-1,2,3-triazol-4-yl)propoxy]-3-methoxyphenyl]-
methyl]-4-(2-methoxyphenyl)piperazine (4b). Sodium azide (5.93 g,
91 mmol) was added to a solution of 1-bromobutane (3.94 mL, 36 mmol)
in anhydrous CH3CN (36 mL) and H2O (4 mL) and heated at reflux
temperature overnight. The reaction was quenched with saturated aqu-
eous NaHCO3, extracted with n-hexane, dried (MgSO4), and evaporated
to 3.83 mL to obtain a concentration of 1-azidobutane of 4.13 mmol/mL.
A suspension of 2b (344 mg, 1.58 mmol), a 4.13 M solution of
1-azidobutane in n-hexane (1.15 mL, 4.74 mmol), sodium ascorbate
(62.5 mg, 0.32 mmol), and copper sulfate pentahydrate (19.7 mg, 0.08
t
mmol) in H2O (5 mL), BuOH (10 mL), and CH2Cl2 (10 mL) was
stirred at room temperature overnight. The reaction was quenched with
saturated NaHCO3 and 0.1 N EDTA, extracted with CH2Cl2, dried
(Na2SO4), and evaporated. The residue was purified by flash chroma-
tography (hexane/EtOAc 1:3) to give 4-[1-butyl-1H-1,2,3-triazol-4-
yl)propoxy]-3-methoxybenzaldehyde as a white solid (354 mg, 71%
yield); mp 36 °C. IR 2958, 2935, 2873, 1682, 1589, 1512, 1466, 1423,
1269, 1134, 1030, 806 cmꢀ1. 1H NMR (360 MHz, CDCl3) δ 0.95 (t, J =
7.4 Hz, 3H), 1.34 (m, 2H), 1.86 (m, 2H), 2.29 (m, 2H), 2.94 (t, J = 7.5
Hz, 2H), 3.93 (s, 3H), 4.18 (t, J = 6.5 Hz, 2H), 4.32 (t, J = 7.3 Hz, 2H),
6.97 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.40ꢀ7.45 (m, 2H), 9.85 (s, 1H).
13C NMR (90 MHz, CDCl3) δ 13.4, 19.7, 22.0, 28.5, 32.3, 49.9, 56.0,
68.1, 109.3, 111.6, 120.8, 126.8, 130.1, 146.8, 149.9, 154.0, 190.8. HPLC
system A (254 nm) purity 97% (tR = 12.0 min); system B (254 nm)
4900
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Journal of Medicinal Chemistry
ARTICLE
purity 98% (tR = 12.9 min). HR-EIMS calcd m/z for C17H23N3O3
317.1739, found 317.1739.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ49 9131 85-29383. Fax: þ49 9131 85-22585. E-mail:
A solution of 4-[1-butyl-1H-1,2,3-triazol-4-yl)propoxy]-3-methoxy-
benzaldehyde (70 mg, 0.22 mmol), 1-(2-methoxyphenyl)piperazine
(63.6 mg, 0.33 mmol), and Na(OAc)3BH (98.0 mg, 0.44 mmol) in
anhydrous CH2Cl2 (10 mL) was stirred under nitrogen atmosphere at
room temperature for 14 h. The reaction mixture was quenched with
saturated aqueous NaHCO3 and extracted with CH2Cl2. The combined
organic layers were dried (Na2SO4) and evaporated. The residue was
purified by flash chromatography (hexane/EtOAc 1:1 þ 1% TEA) to
give 4b as a colorless oil (86 mg, 79% yield). IR 2935, 2873, 2816, 1592,
peter.gmeiner@medchem.uni-erlangen.de.
’ ACKNOWLEDGMENT
We thank S. L€ober for fruitful discussions on the design of the
project. L. Leeb and H. Koolman are acknowledged for their
contribution to the synthesis. This research was supported by a
grant of the Deutsche Forschungsgemeinschaft (DFG: Gm 13/8).
1
1501, 1452, 1260, 1240, 1141, 1031, 1011, 931 cmꢀ1. H NMR (600
MHz, CDCl3) δ 0.94 (t, J = 7.4 Hz, 3H), 1.34 (m, 2H), 1.86 (m, 2H),
2.22 (m, 2H), 2.64 (m, 4H), 2.93 (t, J = 7.4 Hz, 2H), 3.09 (m, 4H); 3.52
(s, 2H), 3.85 (s, 3H), 3.88 (s, 3H), 4.06 (t, J = 6.4 Hz, 2H), 4.31 (t, J = 7.2
Hz, 2H), 6.81 (d, J = 8.3 Hz, 1H), 6.83ꢀ6.87 (m, 2H), 6.89ꢀ6.96 (m,
3H), 6.99 (ddd, J = 7.7, 7.4, 1.7 Hz, 1H), 7.31 (s, 1H). 13C NMR (90
MHz, CDCl3) δ 13.4, 19.7, 22.2, 28.9, 32.3, 49.9, 50.7, 53.3, 55.3, 56.1,
62.9, 68.2, 111.2, 113.0, 118.2, 120.8, 121.0, 121.5, 122.8, 131.1, 141.5,
147.3, 147.5, 149.4, 152.3. HPLC system A (254 nm) purity >99% (tR =
11.5 min); system B (254 nm) purity >99% (tR = 11.8 min). HR-EIMS
calcd m/z for C28H39N5O3 493.3051, found 493.3053.
Radioligand Binding Studies. Receptor binding studies were
carried out as described previously.48 In brief, competition binding
experiments with the human D2long, D2short, D3, and D4.4 receptors were
run on the membrane preparations from CHO cells stably expressing
the corresponding receptor and [3H]spiperone (specific activity = 84
Ci/mmol, PerkinElmer, Rodgau, Germany) at final concentrations of
0.1ꢀ0.5 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
’ ABBREVIATIONS
GPCR, G-protein coupled receptor; 1,4-DAP, 1,4-disubstituted
aromatic piperidines/piperazines; TM, transmembrane domain; EL,
extracellular loop; CuAAC, copper(I)-catalyzed azideꢀalkyne
cycloaddition; CHO, Chinese hamster ovary; Ki, inhibition con-
stant; SEM, standard error of the mean; nH, Hill slope; HR-
EIMS, high-resolution electron ionization mass spectrometry;
APCI-MS, atmospheric-pressure chemical ionization mass spec-
trometry; CHN, combustion/elemental analysis
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