Synthesis of specific bivalent probes that functionally interact with 5-HT4 receptor dimers

Article abstract of DOI:10.1021/jm070552t

G-protein-coupled receptor dimerization directs the design of new drugs that specifically bind to receptor dimers. Here, we generated a targeted series of homobivalent ligands for serotonin 5-HT₄ receptor (5-HT ₄R) dimers composed of

Full text of DOI:10.1021/jm070552t

4482
J. Med. Chem. 2007, 50, 4482-4492
Synthesis of Specific Bivalent Probes That Functionally Interact with 5-HT4 Receptor Dimers
Olivier Russo,†,§,# Magali Berthouze,‡,§,# Mireille Giner,†,§ Jean-Louis Soulier,†,§ Lucie Rivail,†,§ Sames Sicsic,†,§
Frank Lezoualc’h,‡,§ Ralf Jockers,|, and Isabelle Berque-Bestel*,†,§
Mole´cules Fluore´es et Chimie Me´dicinale and Inserm, U769, Signalisation et Physiopathologie Cardiaque, Chaˆtenay-Malabry, F-92296,
UniVersite´ Paris-Sud, Faculte´ de Pharmacie, IFR141, UMR-S769, Chaˆtenay-Malabry, F-92296, Inserm, U567, Department of Cell Biology,
Paris, UniVersite´ Paris-Descartes, UMR-8104, Paris
ReceiVed May 11, 2007
G-protein-coupled receptor dimerization directs the design of new drugs that specifically bind to receptor
dimers. Here, we generated a targeted series of homobivalent ligands for serotonin 5-HT4 receptor (5-
HT4R) dimers composed of two 5-HT4R-specific ML10302 units linked by a spacer. The design of spacers
was assisted by molecular modeling using our previously described 5-HT4R dimer model. Their syntheses
were based on Sonogashira-Linstrumelle coupling methods. All compounds retained high-affinity binding
to 5-HT4R but lost the agonistic character of the monomeric ML10302 compound. Direct evidence for the
functional interaction of both pharmacophores of bivalent ligands with the 5-HT4R was obtained using a
bioluminescence resonance energy transfer (BRET) based assay that monitors conformational changes within
5-HT4R dimers. Whereas the monovalent ML10302 was inactive in this assay, several bivalent derivatives
dose-dependently increased the BRET signal, indicating that both pharmacophores functionally interact with
the 5-HT4R dimer. These bivalent ligands may serve as a new basis for the synthesis of potential drugs for
5-HT4R-associated disorders.
Introduction
protein metabolism,15,16 a key gene involved in Alzheimer’s
disease (AD). This observation and its well-known memory and
learning-enhancing effects17 indicate that 5-HT4 receptors are
promising pharmacological targets for the treatment of AD.18
However, the plethora of 5-HT4 ligands synthesized over the
past decades has only led to the commercialization of two drugs
used in the treatment of irritable bowel syndrome.14 Thus, it
clearly appears that additional studies are required to better
understand the molecular determinants of 5-HT4R activation.
Recently, the 5-HT4R has been shown to form constitutive
homodimers,19 opening a new avenue for the design of 5-HT4
probes, which could serve as a basis for the pharmacotherapy
of memory disorders such as AD. In a previous paper, we
reported the design and the synthesis of 5-HT4R-selective
bivalent ligands20 based on the structure of ML10302, a selective
partial 5-HT4 agonist.21 Bivalent molecules composed of two
ML10302 units and spacers ranging from 6 to 29 atoms were
prepared (Figure 1). All bivalent ligands conserved high-affinity
binding for 5-HT4R, and those with the longest spacers (18,
25, and 29 atoms) appeared to be functionally different in the
cAMP accumulation assay compared to the ML10302 reference
compound.20
In the past 2 decades, a large body of evidence has led to the
reconsideration of the classical dogma of monomeric G-protein-
coupled receptor (GPCR) entities. It is now well accepted that
GPCRs can form homo- and/or heterodimers in cell membranes
and that dimer formation may influence receptor function.1-7
Several studies have shown the role of dimerization in the
activation process of therapeutically relevant GPCRs, demon-
strating the crucial importance of elucidating this phenomenon
to develop novel pharmaceutical entities.8 For this purpose, one
of the obvious ways to create new tools is the design of specific
bivalent ligands. This strategy has been successfully used for
some GPCRs and has led to interesting results.9-11 For instance,
chimeric agonists comprising a somatostatin pharmacophore
targeting somatostatin SST5 receptors and a dopamine phar-
macophore targeting dopamine D2 receptors have been shown
to enhance the potency of each pharmacophore separately. The
physiological consequences are a suppression of growth hor-
mone and prolactin secretion in human pituitary somatotroph
adenoma cells.12 Furthermore, bivalent ligands composed of an
adenosine A1 and A3 receptor agonist linked by a spacer have
been shown to activate both receptors. This coactivation induces
a greater protection against myocardial ischemia than activation
of each receptor individually.13
We now extend this work by determining the optimal spacer
length for the ML10302-based bivalent ligands using molecular
modeling on our recently published 5-HT4R dimer model,20
which has been validated by site-directed mutagenesis.22 Model-
ing studies predicted an optimal spacer length of approximately
22 Å (20-24 atoms), which is in good agreement with recent
studies on µ-opioid receptor dimers and bivalent ligands.9,23
Since our previous series of bivalent ligands did not cover spacer
lengths between 18 and 25 atoms, we decided to synthesize
two new series of 5-HT4R-specific bivalent ligands containing
flexible and constrained spacers of 20-24 atoms. Position 4 of
the piperidin ring of ML10302 was conserved as the most
suitable attachment point for spacers based on molecular
modeling of 5-HT4R dimers and structure-activity relationship
studies.
Serotonin 5-HT4 receptors (5-HT4R) belong to the GPCR
superfamily and have been identified as a valuable target to
treat gastrointestinal diseases.14 Moreover, recent studies have
outlined the role of 5-HT4R activation in the amyloid precursor
* To whom correspondence should be addressed. Address: INSERM
U869, Universite´ Victor Segalen Bordeaux 2, Bordeaux F-33076, France.
Phone: +33 1 57 57 09 45. Fax: +33 1 57 57 10 15. E-mail:
isabelle.bestel@bordeaux.inserm.fr.
† CNRS UMR C 8076 (BioCIS), Universite´ Paris XI.
§ IFR-141, Universite´ Paris XI.
# These authors participated equally for this work.
‡ INSERM UMR S769, Universite´ Paris XI.
| Universite´ Paris Descartes.
INSERM U567.
10.1021/jm070552t CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/03/2007

Synthesis of BiValent Probes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4483
Figure 1. General structure of 5-HT4 bivalent ligands.
We first determined the pharmacological and functional
properties of these bivalent ligands and then addressed the
important issue of whether both or only one pharmacophore of
the bivalent compounds binds to 5-HT4R dimers by using an
innovative bioluminescence resonance energy transfer (BRET)
assay. BRET determines the ability of the monovalent ML10302
molecule and its bivalent derivates to promote ligand-induced
conformational changes within 5-HT4R dimers.19
Results
Molecular Modeling Studies. To estimate the optimal length
and structure of the spacer of bivalent ligands, we performed
molecular docking studies on our recently described 5-HT4R
dimer model. This model, which was initially generated by
GRAMM software calculations20 and subsequently confirmed
by site-directed mutagenesis,22 predicts the involvement of
helices II, III, and IV in the dimer interface. Initial positioning
of the two ML10302 pharmacophores into the 5-HT4R binding
site was based on experimental data and on a ML10302-5-
HT4R complex24,25 that has been previously optimized by large-
scale molecular dynamics in a virtual hydrated lipid bilayer.26
On the basis of these docking studies, position 4 of the piperidin
ring of ML10302 has been confirmed as the most suitable
attachment point for spacers. The evaluation of the minimal
length between these two spacer attachment points has to take
into account the bypassing of the two helices III, revealing a
minimal distance of approximately 22 Å. Modeling with short
spacers of 14 atoms was possible but required an unfavorable
highly extended conformation of the spacer. A more favorable
and extended conformation was obtained for spacers of 20-24
atoms. The final structure obtained for each bivalent-ligand-
5-HT4R dimer complex was then refined using molecular
dynamics and energy minimizations to allow a more favorable
positioning of the spacer with respect to essential ML10302-
5-HT4R interactions (Figure 2, compound 9c shown as a 22-
atom spacer). Optimal docking of spacers depends not only on
their lengths but also on their structures. Since the exact
chemical composition of optimal spacers is difficult to predict,
we decided to synthesize two series of bivalent ligands with
either flexible or constrained spacers ranging from 20 to 24
atoms.
Synthesis of Bivalent Ligands. In order to modulate the
spacer length by one-atom increments with homogeneous
chemical compositions, we designed spacers, which are pre-
sented in Table 1. By variation of the number of atoms n and
m and the positions of the substitutents on the central aromatic
ring (1,3 or 1,4), the desired repertoire of spacers could be
achieved. The synthesis of bivalent ligands was based on a
Sonogashira-Linstrumelle coupling reaction and is described
in Scheme 1. Introduction of a propargyl group on the nitrogen
atom of Boc-glycine or Boc-γ-aminobutyric acid brought three
supplementary carbons into the spacers. In this first step, Boc
protected amines 1a and 1b were alkylated using NaH as a base
in DMF to give the propargyl acids 2a and 2b in 58% and 77%
Figure 2. Molecular modeling studies: (a) side view of docked bivalent
ligand 9c in 5-HT4R dimer model, where both protomers including
helices and extra- and intracellular loops are represented in red and
blue; (b) top view of docked of bivalent ligand 9c in 5-HT4R dimer
model, where the loops are not displayed for a better visualization.
yields, respectively. Then a Sonogashira-Linstrumelle coupling
with either 1,3 or 1,4-diiodobenzene allowed introduction of
three or four atoms in the linker. This reaction was performed
at 80 °C, allowing the synthesis of the constrained spacers 3a,
3b, and 4 in moderate yields (46-62%). Finally, conformational
flexibility was introduced by reducing the triple bonds into single
bonds by classical hydrogenation on Pd/C, producing molecules
5a,b and 6. The synthesis of the bivalent ligands was then
performed by condensation between the already known amine
7a or 7b20 derived from ML10302 and previously synthesized
acids. A standard protocol using EDC, HOBt, and NEt3 in
anhydrous DMF was used to afford the constrained bivalent
ligands 8a-e (Scheme 2) or the flexible bivalent ligands 9a-e
(Scheme 3) in low to moderate yields (19-62%).
This straightforward synthesis is highly modular, avoiding
the use of protecting groups, thus leading to spacers bearing
terminal carboxylic functions ready for the coupling with amines
derived from ML10302. This strategy allowed us to prepare
both rigid and flexible bivalent molecules derived from ML10302
with spacer lengths ranging from 20 to 24 atoms.
Molecule 13, which is not a bivalent ligand because one of
the two ML10302 units of the bivalent ligands was replaced
by a cyclohexyl ring, was also prepared as a control compound.

4484 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Russo et al.
Table 1. General Structure of Flexible and Constrained 5-HT4R Bivalent Ligandsa
a The meta or para substitution of the aromatic central ring mixed with adequat aliphatic chain combinations (n or m) allow a one by one atom incremented
spacer length.
Scheme 1. Synthesis of Spacersa
a Reagents and conditions: (i) (a) NaH, DMF, 0 °C, 1 h; (b) propargyl bromide 80% in toluene, room temp, overnight; (ii) 1,4-diiodobenzene, PdCl2(PPh3)2
(5 mol %), CuI (10 mol %), NEt3, DMF, 80 °C, (iii) 1,3-diiodobenzene, PdCl2(PPh3)2 (5 mol %), CuI (10 mol %), NEt3, DMF, 80 °C; (iv) H2 (1 bar), Pd/C
(10%), MeOH, room temp.
A similar reaction as that used to produce the propargyl acid
2b was used again, except that dissymmetry was obtained using
a large excess of 1,4-diiodobenzene in a Sonogashira-Lin-
strumelle coupling reaction at room temperature, to give 10 in
49% yield (Scheme 4). The cyclohexylamine part of 13 was
obtained by coupling cyclohexylamine with 2b, using EDC,
HOBt, and NEt3 in DMF to give 11 in 61% yield. A second
Sonogashira-Linstrumelle coupling reaction between 10 and
11 at 80 °C yielded 12 in a moderate yield (53%). Compound
13 was then obtained with a good yield after hydrogenation
of the triple bonds of 12 and coupling with 7a, using HBTU
and NEt3 in anhydrous DMF. Bivalent molecules 14-18
(Figure 3), which possess other chemical composition spacers
and which were previously synthesized,20 were also used in this
study.
Pharmacological and Functional Properties of 5-HT4R-
Specific Bivalent Molecules. The pharmacological properties
of the newly synthesized series of ligands were determined in
C6 glial cells stably expressing the human 5-HT4R.27 [3H]-
GR113808 competition curves of bivalent ligands were monopha-
sic (see Table 2 for Ki values). All compounds retained high
affinity for 5-HT4R with Ki values that were very similar to
those obtained for recently published bivalent ligands and the
monovalent ML10302 reference compound.20 Similar results
were obtained in Chinese hamster ovary (CHO) cells transiently
expressing the 5-HT4R (data not shown). Next, the ability of
bivalent ligands to stimulate adenylyl cyclase activity was
analyzed by measuring ligand-induced cAMP production.
Compared to the monovalent ML10302, bivalent ligands either
conserved a ML10302-like partial agonistic character (8a) or

Synthesis of BiValent Probes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4485
Scheme 2. Synthesis of Constrained Bivalent Ligandsa
a Reagents and conditions: (i) EDC, HOBt, NEt3, anhydrous DMF, room temp.
Scheme 3. Synthesis of Flexible Bivalent Ligandsa
a Reagents and conditions: (i) EDC, HOBt, NEt3, anhydrous DMF, room temp.
lost these agonistic properties (8b,d,e, 9b-e) (Table 2). This
confirms our previous observation that bivalent ligands with
long spacers (20-29 atoms) are functionally different compared
to ML10302.20 To evaluate the respective impact of the spacer
and/or the second pharmacophore of the bivalent ligands on
these functional changes, we synthesized a ML10302 derivative
that possessed a spacer of 22 atoms but only one pharmacophore
(13). This compound retained high-affinity for 5-HT4R and
generated an intermediate cAMP production (17%), indicating
that both the spacer and the second pharmacophore participate
in the modification of the functional properties compared to
the ML10302 reference compound.
BRET Results. The functional differences observed between
ML10302 and the bivalent compounds suggest that ML10302

4486 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Scheme 4. Synthesis of a Nonbivalent Control Liganda
Russo et al.
a Reagents and conditions: (i) 1,4-diiodobenzene, PdCl2(PPh3)2 (5 mol %), CuI (10 mol %), NEt3, DMF, 0 °C to room temp, (ii) cyclohexylamine, EDC,
HOBt, NEt3, DMF/DCM (2/1), room temp; (iii) 11, PdCl2(PPh3)2 (5 mol %), CuI (10 mol %), NEt3, DMF, 80 °C; (iv) (a) H2 (1 bar), Pd/C (10%), MeOH,
room temp; (b) 7a, HBTU, NEt3, DMF, room temp.
Figure 3. Previously synthesized 5-HT4 bivalent ligands.20
Table 2. Pharmacological Evaluations of Synthesized 5-HT4R Bivalent
Ligands: Binding Affinities (Ki), cAMP Production (% of 5-HT), BRET
Signal Measurementsa
spacer
size
Ki
(nM)
cAMP
BRET
compd
(% of 5-HT)b
(% of control)c
GR113808
ML10302
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
13
14
15
16
17
0.5 ( 0.2
5 ( 2.5
44 ( 10
8 ( 3
1 ( 1
45 ( 1
49 ( 26
8 ( 8
99 ( 2
101 ( 5
107 ( 2
109 ( 2
106 ( 2
108 ( 1
115 ( 1*
114 ( 4*
111 ( 1
118 ( 1*
108 ( 2
115 ( 0*
105 ( 2
108 ( 1
110 ( 1
108 ( 4
114 ( 1*
114 ( 2*
20
21
22
23
24
20
21
22
23
24
18 ( 6
35 ( 10
6 ( 9
40 ( 11
25 ( 5
1 ( 2
19 ( 6
18 ( 1
10 ( 8
21 ( 3
5 ( 6
15 ( 10
12 ( 3
23 ( 8
15 ( 22
34 ( 11
7 ( 3.2
20 ( 12
9 ( 4
9 ( 1
Figure 4. BRET technique.
17 ( 3
46 ( 5
5 ( 1
9
14
18
25
29
ligand-induced conformational changes of ML10302 and its
bivalent derivatives, we used a recently developed BRET-based
assay.19 This assay relies on the observation that the degree of
physical proximity between two receptor molecules within a
dimer can be assessed in living cells by the level of energy
transfer occurring between fusion proteins of the receptor tagged
with the energy donor Renilla luciferase (RLuc) and another
receptor tagged with a fluorescent acceptor, the yellow fluo-
rescent protein (YFP) (Figure 4).31,32 Ligand-induced confor-
mational changes within preexisting dimers may be detected if
the relative position and orientation of the Rluc and the YFP
moieties within the dimer are altered.33-35 Carboxy-terminal
5-HT4R fusion proteins (5-HT4R-RLuc, 5-HT4R-YFP) were
transiently coexpressed in CHO cells. All receptor constructs
6 ( 4
10 ( 2
12 ( 3
50 ( 12
113 ( 32
18
a Very similar Ki values of 5.7 ( 1 and 3.5 ( 2 (ML10302), 0.5 ( 0.1
and 0.7 ( 0.1 (GR113808), and 10 ( 2 (compound 9c) were obtained for
5-HT4R-RLuc and 5-HT4R-YFP fusion proteins, respectively. 5-HT4R-
RLuc and 5-HT4R-YFP fusion proteins behaved as the wild type receptor
in the cAMP assay.19 b Results are the mean ( of two independent
experiments performed in triplicate. c Results are the mean ( of three
independent experiments performed in duplicate (/, p < 0.001).
interacts with 5-HT4R differently than its bivalent derivatives.
Recent studies indicate that functional differences (i.e., between
partial and full agonists) can be explained by the stabilization
of different conformational states of GPCRs.28-30 To detect

Synthesis of BiValent Probes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4487
Figure 5. BRET assays. Membrane preparations from CHO cells expressing 5-HT4R-RLuc and 5-HT4R-YFP at equimolar amounts corresponding
to nonsaturating conditions were incubated with different ligands: (a) effect of bivalent ligands 8e and 9c (1 µM) on the basal BRET signal in the
presence (W, black) or absence (W/O, gray) of 1 µM monovalent antagonist GR113808; dose response curves of bivalent ligands 9c (b), 17 (c),
and 18 (d). Data are the mean of at least three experiments performed in duplicate.
were functional because the coupling of 5-HT4R-RLuc and
5-HT4R-YFP to adenylyl cyclase19 and binding properties
(Table 2) were identical to those of the wild type receptor
expressed in C6 glial cells. BRET measurements were performed
using saturating concentrations (1 µM) of newly synthesized
and previously described ligands in cells expressing equimolar
amounts of 5-HT4R-RLuc and 5-HT4R-YFP (Figure 5).
Binding of ML10302 and its derivative 13, which lacks the
second pharmacophore but carries a long spacer, did not modify
the basal BRET signal. In contrast, the bivalent ligands, in
particular those with long spacers, induced a significant increase
of the basal BRET signal (Table 2, 8e, 9a,c,e, 17, 18). This
increase corresponds to a ligand-induced conformational change
in preexisting 5-HT4R dimers and not to a ligand-induced
dimerization because previous studies have shown that all
receptors exist already as covalent, disulfide-bridged dimers in
the absence of ligand.22
Importantly, the ligand-promoted BRET of flexible (9c) and
constrained (8e) ligands was abolished in the presence of the
reference monovalent antagonist GR113808 (Figure 5a). To
further confirm the specificity of the ligand-induced BRET,
dose-response curves were generated for ligands 9c, 17, and
18, which possess long flexible spacers with 22, 25, and 29
atoms, respectively. Half-maximal ligand-promoted BRET
values of 0.7, 150, and 10 nM for ligands 9c, 17, and 18,
respectively, were in good agreement with Ki values (Figure
5b-d). Bivalent ligands 14-16 with spacers containing less
than 18 atoms did not significantly modify the basal BRET
signal. Comparison of ligand-promoted BRET of bivalent
ligands with flexible and constrained spacers revealed interesting
differences. For the constrained molecules possessing a diyne
structure (8a-e), only compound 8e produced a significant
change of the basal BRET signal. In contrast, for bivalent ligands
with flexible spacers (9a-e, 15-18) five compounds (9a, 9c,
9e, 17, 18) significantly modified the basal BRET signal.
Intriguingly, in the series of bivalent ligands 9, compounds 9b
and 9d had no significant effect on basal BRET signals despite
the presence of an apparently sufficiently long spacer. A closer
look at the structure of compounds 9b and 9d revealed the
presence of a 1,3-meta-substituted central cyclic core compared
to a 1,4-para-substituted core present in 9a, 9c, and 9e. This
further highlights the crucial role not only of the length but
also of the flexibility and the chemical structure of the spacer
for the correct orientation of the two pharmacophores in bivalent
ligands.
Discussion
The concept of GPCR homo- and heterodimerization has an
important impact on GPCR drug design. An obvious way to
design new drug families for GPCRs is the synthesis of bivalent
compounds that are expected to bind simultaneously to the two
ligand binding sites present in GPCR dimers.12,13,36 Ideally,
bridging of the two protomers of the receptor dimer by bivalent
ligands would generate unique functional properties, which are
different from those of the monovalent pharmacophore. The
successful design and characterization of bivalent ligands rely
on the choice of the pharmacophore and the spacer linking both
pharmacophores as well as the availability of adequate assay
systems to determine their specific interaction on receptor dimers
and their functional properties.
In the present study, we conserved the ML10302 compound
as pharmacophore, as this compound has high affinity and
specificity for the 5-HT4R. Importantly, ML10302 is a partial
agonist (∼50% of maximal serotonin response) for 5-HT4R.27
Thus, differences in functional properties of the corresponding
bivalent derivatives should be easily revealed and an increase
and/or a decrease of the agonistic potential should be readily
detected. On the basis of the optimized docking of the
monovalent ML10302 molecule in our 5-HT4R dimer model,
position 4 of the piperidin ring of ML10302 was conserved as
the most suitable attachment point for spacers. We prepared
two series of bivalent ligands (8 and 9), one with constrained

4488 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Russo et al.
spacers and the other with flexible ones. Both series possessed
spacers ranging from 20 to 24 atoms of approximately 22 Å,
the predicted optimal spacer length according to our own
docking experiments and according to published results on
opioid receptor dimers.9,23
compound 8e possesses the longest spacer with 24 atoms,
suggesting that longer spacers are able to overcome the
conformational constraints due to this type of spacer.
In the flexible family (9a-e), which complements the
previously synthesized ligand family (14-18), most compounds
induced a significant ligand-promoted BRET (9a,c,e). The high
success rate for this ligand family in the BRET assay most likely
resides in the flexibility of the spacer favoring the convenient
orientation of the second pharmacophore.
We first determined the pharmacological properties of the
bivalent compounds. [3H]-GR113808 competition curves were
monophasic for all bivalent compounds with a single high-
affinity site for 5-HT4R. Conservation of high affinity of bivalent
compounds for 5-HT4R is consistent with the binding of at least
one pharmacophore of the bivalent ligands to 5-HT4R. However,
pharmacological studies did not provide any obvious evidence
for the binding of the second pharmacophore. Indeed, we did
not observe substantial changes in ligand affinity or the addition
of a second, pharmacologically distinct binding site that might
have indicated the existence of allosteric effects.37 Although
substantial differences in binding affinities have been reported
for some bivalent ligands,10,36,38-46 pharmacological evidence
for the occupation of both binding sites in a dimer is generally
difficult to obtain especially for the binding of two identical
pharmacophores to receptor homodimers.
The functional activity of bivalent ligands was tested in cAMP
accumulation assays. Despite the preservation of high affinity
for 5-HT4R, most bivalent compounds lost the agonistic
properties of the ML10302 reference compound. This observa-
tion is particularly true for ligands with long spacers, which
confirms our preliminary conclusion based on a limited set of
previously described bivalent ligands. These functional differ-
ences are compatible with the hypothesis that both pharma-
cophores of bivalent ligands interact with 5-HT4R dimers.
However, in this flexible family, two ligands, 9b and 9d, were
inactive in the BRET assay despite apparently adequate spacer
length. The functional properties of these two ligands may be
explained by a different type of position in the central cyclic
core of the spacer that changes the relative orientation of the
two pharmacophores. Whereas compounds 9b and 9d have a
1,3-meta-substituted central cyclic core, the other compounds
have a 1,4-para0substituted core (Scheme 3). The absence of
ligand-induced BRET for these 1,3-meta-substituted compounds
further highlights the importance not only of spacer length but
also of the orientation of the two pharmacophores, which is
determined by the chemical nature of the spacer. The inability
of compounds 9b and 9d to promote ligand-induced BRET
changes further confirms the sensitivity and the specificity of
the BRET signals. Altogether, the BRET results confirmed the
prediction of our molecular docking studies. A spacer length
of 20-24 atoms appears to be optimal for binding of the two
pharmacophores of bivalent ligands to receptor dimers. Fur-
thermore, additional structural constraints introduced by triple
bonds (8a-c) or 1,3-meta-substituted aromatic rings (9b and
9d) are equally important parameters to be considered for
optimal docking of bivalent ligands. Interestingly, overlay of
extended conformations of compounds with constrained (8c)
and flexible (9c) linkers of the same size revealed a similar
distance between the two ML10302 moieties. In contrast, the
structure of the 5-HT4R dimer plays a key role in the
conformation adopted by the spacer because major obstacles
such as helices III have to be bypassed.
The docking studies of bivalent ligands on the 5-HT4R dimer
model may provide a possible explanation for the loss of
receptor activation in the presence of these bivalent ligands
compared to the ML10302 reference compound. Since spacers
of bivalent ligands most likely have to bypass the two helices
III of the dimer, this may hinder the movement of this helix,
which has been shown to be crucial for the activation process
of GPCRs.47
To consolidate data obtained with mono- and bivalent
5-HT4R-specific compounds, we propose the following model,
where two monovalent ligands occupy both ligand binding sites
of 5-HT4R dimers with similar affinities (Figure 6a). Depending
on the functional properties of each ligand, different confor-
mational changes are induced leading to receptor activation or
inactivation. None of these conformational changes are detect-
able in the BRET 5-HT4R-dimerization assay. For bivalent
ligands that promote significant ligand-induced BRET changes,
interaction of both pharmacophores with the same receptor dimer
occurs. These ligands bind to 5-HT4R in a similar manner as
monovalent ligands; however, the spacer imposes additional
conformational constraints between the two protomers of the
dimer (Figure 6b). This has two important consequences, a
change of functional properties compared to the monovalent
reference compound and the detection of ligand-promoted
conformational change with the BRET assay. For bivalent
ligands without detectable ligand-induced BRET changes, two
alternative possibilities may be envisioned (Figure 6c,d). For
ligands with short spacers and functional properties similar to
those of the monovalent compound, binding of two single
molecules to the receptor dimer appears likely (Figure 6d). For
those with adequate spacers and modified functional properties,
simultaneous binding of both pharmacophores to the same
receptor dimer may be hypothesized (Figure 6c). However, the
conformational constraints imposed by this type of spacer are
not detectable in the BRET assay. Indeed, ligand binding to
In an effort to obtain evidence for the specific interaction of
the second pharmacophore with 5-HT4R, we tested bivalent
ligands in a BRET assay (Figure 4). This assay has recently
emerged as a sensitive approach to monitor conformational
changes of a wide range of proteins in living cells including
membrane receptors.31,34 Whereas the ML10302 reference
molecule was inactive in the BRET assay, several bivalent
compounds significantly increased the basal BRET signal. The
effect of compounds 9c, 17, and 18 was dose-dependent with
half-maximal doses in agreement with the respective Ki values.
These ligand-induced BRET changes were inhibited in the
presence of the 5-HT4R-specific monovalent antagonist
GR113808, further confirming the specificity of the signals.
Importantly, the monovalent ML10302 derivate 13 was inactive
in the BRET assay. This defines the specificity of the ligand-
promoted BRET signal that exclusively relies on the properties
of the second pharmacophore, which qualifies the BRET assay
as an excellent tool to study the interaction of the second
pharmacophore with 5-HT4R.
Testing of the constrained family of bivalent ligands (8a-e)
in the BRET assay revealed that only ligand 8e significantly
enhanced the basal BRET signal. The fact that only one 1 of 5
compounds was able to induce a significant BRET change may
be explained by the rigidity introduced by the diyne central core
in the spacer of this family preventing the binding of the second
pharmacophore of these bivalent ligands. Interestingly, the active

Synthesis of BiValent Probes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4489
International (Fontenay-sous-Bois, France). Liquid chromatography
was performed on Merck silica gel 60 (70/30 mesh), and TLC was
performed on silica gel 60F-254 (0.26 mm thickness) plates.
Visualization was achieved with UV light and Dragendorff reagent
unless otherwise stated.
General Procedure for the Preparation of Compounds 2a and
2b: Preparation of 2-(tert-Butoxycarbonyl(prop-2-ynyl)amino)-
acetic Acid (2a). To a stirred solution of 1a (3.0 g, 17.12 mmol,
1 equiv) in 40 mL of dry DMF at 0 °C was added NaH (60% in
mineral oil, 2.12 g, 53.07 mmol, 3.5 equiv). After 1 h at this
temperature, propargyl bromide (80% in toluene, 3.24 mL, 29.11
mmol, 1.7 equiv) was added, and the reaction mixture was allowed
to reach room temperature and was stirred for 18 h. Water was
added (20 mL), and the pH was adjusted to 3 with 1 N HCl. This
aqueous solution was extracted with 3 × 20 mL of CH2Cl2, and
the combined organic layers were dried with Na2SO4 and concen-
trated. The oil obtained was purified by flash chromatography using
cHex/AcOEt (1/1) containing 0.1% AcOH to yield 2.20 g (66%)
of 2a as an amber solid. Rf(cHex/AcOEt (1/1) + 0.5% AcOH) )
1
0.67. H NMR (200 MHz) δ 10.84 (bs, 1H, OH), 4.18-4.07 (m,
4H), 2.25 (t, J ) 2.6 Hz, 1H), 1.41 (s, 9H). 13C NMR (50 MHz) δ
174.4, 154.6, 81.4, 78.2, 72.9, 47.0, 36.5, 28.0. F ) 118 °C.
General Procedure for the Preparation of Compounds 3a,
3b, and 4: Preparation of 2,2′-(3,3′-(1,4-Phenylene)bis(prop-2-
yne-3,1-diyl))bis(tert-butoxycarbonylazanediyl)diacetic Acid (3a).
A 30 mL solution of freshly distilled NEt3 containing also 1,4-
diiodobenzene (1.95 g, 5.92 mmol, 1 equiv), PdCl2(PPh3)2 (0.165
g, 0.24 mmol, 0.05 equiv), and CuI (0.090 g, 0.47 mmol, 0.1 equiv)
was stirred at room temperature. Then 30 mL of anhydrous DMF
containing a solution of 2a (3.0 g, 14.20 mmol, 2.4 equiv) was
added dropwise. After the mixture was heated at 80 °C overnight,
the solvents were removed. The crude product was taken up in 20
mL of water, and the mixture was acidified to pH 3 with 1 N HCl.
This aqueous solution was extracted with 3 × 20 mL of CH2Cl2,
and the combined organic layers were dried with Na2SO4 and
concentrated. The resulting oil was purified by flash chromatog-
raphy using cHex/AcOEt (6/4) containing 0.1% AcOH to yield 1.72
g (58%) of 3a as an amber oil. Rf(cHex/AcOEt (6/4) + 0.5% AcOH)
) 0.13. 1H NMR (200 MHz) δ 10.03 (bs, 2H), 7.30 (s, 4H), 4.40-
4.03 (m, 8H), 1.41 (s, 18H). 13C NMR (50 MHz) δ 174.5, 154.6,
131.5, 122.5, 85.6, 84.1, 81.4, 47.2, 37.3, 28.1.
Figure 6. Proposed model: (a) binding of two monovalent ligands
on both 5-HT4R dimer binding sites; (b) binding of bivalent ligands
inducing BRET modifications; (c) binding of bivalent ligands without
detectable BRET modifications; (d) binding of short spacers bivalent
ligands without detectable BRET modifications.
GPCRs does not systematically lead to detectable BRET changes
because the conformational modification may not necessarily
change the relative position of the energy donor (Rluc) and the
energy acceptor (YFP).8
Taken together, we have synthesized two targeted series of
bivalent ligands for 5-HT4R dimers possessing spacers of 20-
24 atoms corresponding to an optimized spacer length of
approximately 22 Å. We applied the recently developed BRET
GPCR dimerization assay for the first time to the study of
bivalent ligands on GPCR dimers. Whereas classical competition
binding experiments were unable to reveal differences in the
binding properties of mono- and bivalent ligands, the BRET
assay provided new insights in the interaction of bivalent ligands
with 5-HT4R dimers. We were able to provide evidence for the
interaction of the two pharmacophores of several bivalent
compounds with 5-HT4R dimers. Having a validated molecular
model of 5-HT4R dimers, optimized spacers, and specific BRET
assays at one’s disposal, the development of heterobivalent
ligands for 5-HT4R homodimers or 5-HT4R heterodimers can
now be envisaged. Furthermore, our knowledge about the
dimerization interface of 5-HT4R dimers may lead in the future
to the design of 5-HT4R dimerization inhibitors. In conclusion,
5-HT4R molecules able to stabilize or inhibit receptor dimer-
ization may serve as a new basis for the synthesis of new drugs
for 5-HT4R-associated disorders.
General Procedure for the Preparation of Compounds 5a,
5b, and 6: Preparation of 2,2′-(3,3′-(1,4-Phenylene)bis(propane-
3,1-diyl))bis(tert-butoxycarbonylazanediyl)diacetic Acid (5a). To
a stirring solution of 3a (300 mg, 0.60 mmol) in 10 mL of MeOH
at room temperature was added 30 mg of 10% Pd/C. The mixture
was stirred under 1 bar of H2 overnight and filtered through a pad
of Celite. Evaporation of the solvent yielded 278 mg (91%) of 5a
1
as an amber oil. H NMR (200 MHz) δ ppm 10.17 (bs, 2H), 7.05
(s, 4H), 3.88 (bd, J ) 20.9 Hz, 4H), 3.54-3.09 (m, 4H), 2.55 (t,
J ) 7.4 Hz, 4H), 1.99-1.62 (m, 4H), 1.32 (s, 18H). 13C NMR (50
MHz) δ 174.3 and 174.1 (two signals due to rotamers), 156.1 and
155.3 (two signals due to rotamers), 139.1, 128.4, 80.5, 49.1 and
48.8 (two signals due to rotamers), 48.3 and 48.1 (two signals due
to rotamers), 32.7, 29. 9, 28.3.
General Procedure for the Preparation of Dimers 8 and 9:
Preparation of Dimer 8a. To a solution of 3a (595 mg, 1.19 mmol,
1 equiv), 7b (986 mg, 2.38 mmol, 2 equiv),20 HOBt‚H2O (321 mg,
2.38 mmol, 2 equiv), and NEt3 (1.5 mL, 10.7 mmol, 9 equiv) in 30
mL of anhydrous DMF at room temperature was added EDC‚HCl
(456 mg, 2.38 mmol, 2 equiv). The mixture was stirred overnight,
and the solvent was removed. The crude product was dissolved in
30 mL of CH2Cl2 and washed with 20 mL of saturated Na2CO3
and 20 mL of brine. The organic layer was dried with Na2SO4 and
concentrated in vacuo. Chromatography on silica gel using AcOEt/
MeOH (90/10) followed by AcOEt/MeOH/NH4OHaq20% (87/10/3)
afforded 330 mg (24% yield) of 8a as white hygroscopic foam. 1H
NMR (200 MHz) δ 7.79 (s, 2H), 7.32 (s, 4H), 6.28 (s, 2H), 4.49
(bs, 4H), 4.33 (m, 8H), 3.98 (s, 4H), 3.82 (s, 6H), 3.13 (t, J ) 6,0
Hz, 4H), 2.92 (m, 4H), 2.70 (t, J ) 6,0 Hz, 4H), 2.05-1.94 (m,
8H), 1.65-1.26 (m, 24H). 13C NMR (50 MHz) δ 171.7, 169.2,
Experimental Section
Chemistry. Melting points were determined on a Kofler melting
point apparatus. NMR spectra were performed on a Bruker AMX
200 (1H, 200 MHz; 13C, 50 MHz) or Bruker AVANCE 400 (1H,
400 MHz; 13C, 100 MHz). Unless otherwise stated, CDCl3 was
used as solvent. Chemical shifts δ are in ppm, and the following
abbreviations are used: singlet (s), broad singlet (bs), doublet (d),
triplet (t), and multiplet (m). Elemental analyses (C, H, N) were
performed at the Microanalyses Service of the Faculty of Pharmacy
at Chaˆtenay-Malabry (France) and were within 0.4% of the theorical
values otherwise stated. Mass spectra were obtained using a Bruker
Esquire electrospray ionization apparatus.
Materials. DMF distilled from CaSO4, CH2Cl2 distilled from
calcium hydride, and the usual solvents were purchased from VWR

4490 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Russo et al.
164.4, 160.2, 147.9, 155.4, 133.1, 131.6, 122.5, 109.8, 98.2, 85.9,
81.5, 80.4, 61.8, 56.7, 55.9, 53.4, 50.9, 44.7, 38.7, 35.6, 29.6, 28.1.
MS (ESI) m/z ) 1147 [M+H]+. Anal. (C58H76N8O12Cl2‚2.5H2O),
C, H, N.
DMF, and to this solution at room temperature was added 7a (144
mg, 0.36 mmol, 1 equiv),20 HBTU (145 mg, 0.38 mmol, 1.05
equiv), and NEt3 (200 µL, 1.44 mmol, 4 equiv). After 5 h at room
temperature, the solvents were removed, and the crude product was
dissolved in 20 mL of AcOEt and washed with 10 mL of saturated
Na2CO3 and 10 mL of brine. The organic layer was dried with
Na2SO4 and concentrated in vacuum. Purification by chromatog-
raphy on silica gel, using AcOEt/MeOH (95/5) followed by AcOEt/
MeOH/NH4OHaq20% (92/5/3) afforded 280 mg (82% yield) of 13
as light-yellow foam. Rf(AcOEt/MeOH/NH4OHaq20%, 92/5/3) )
4-(tert-Butoxycarbonyl(3-(4-iodophenyl)prop-2-ynyl)amino)-
butanoic Acid (10). To a stirring solution of 1,4-diiodobenzene
(5.469 g, 16.59 mmol, 5 equiv), PdCl2(PPh3)2 (117 mg, 0.17 mmol,
0.05 equiv), and CuI (63 mg, 0.33 mmol, 0.1 equiv) in 10 mL of
freshly distilled NEt3 at 0 °C was added dropwise a solution of 2b
(800 mg, 3.32 mmol, 1 equiv) in 20 mL of anhydrous DMF. The
solution was stirred at 0 °C for 1 h and allowed to warm to room
temperature overnight. After removal of the solvents, the crude
mixture was treated with 70 mL of 5% K2CO3 and extracted with
150 mL of AcOEt. The aqueous layer was acidified to pH 3 with
1 M HCl and extracted with 100, 70, and 35 mL of CH2Cl2. The
combined organic layers were washed with 100 mL of saturated
NaCl and dried over Na2SO4. After concentration, purification by
chromatography on silica gel using AcOEt/cHex (3/7) containing
0.5% AcOH afforded 719 mg (49% yield) of 10 as a brown oil.
Rf(cHex/AcOEt (4/6) + 0.5% AcOH) ) 0.53. 1H NMR (200 MHz)
δ 7.62 (d, J ) 8.4 Hz, 2H), 7.12 (d, J ) 8.4 Hz, 2H), 4.23 (bs,
2H), 3.43 (t, J ) 6.9 Hz, 2H), 2.40 (t, J ) 7.2 Hz, 2H), 1.96 (m,
2H), 1.47 (s, 9H). 13C NMR (50 MHz) δ 178.7, 157.8, 155.3, 137.6,
133.3, 122.5, 86.7, 82.6, 80.7, 46.0, 37.3, 31.3, 28.5, 23.3.
tert-Butyl 4-(Cyclohexylamino)-4-oxobutyl(prop-2-ynyl)car-
bamate (11). To a solution of 2b (795 mg, 3.32 mmol, 1 equiv) in
30 mL of a 2/1 mixture of dry DMF and dry CH2Cl2 was added
NEt3 (1.39 mL, 9.95 mmol, 3 equiv), cyclohexylamine (270 µL,
3.98 mmol, 1.2 equiv), HOBt‚H2O (539 mg, 3.98 mmol, 1.2 equiv),
and EDC‚HCl (764 mg, 3.98 mmol, 1.2 equiv). The mixture was
stirred overnight at room temperature, and the solvents were
removed under vacuum. The crude product was dissolved in 50
mL of AcOEt and washed with 25 mL of 1 M KHSO4, 25 mL of
saturated Na2CO3, and 25 mL of brine. The organic layer was dried
over Na2SO4 and concentrated. Purification on silica gel, using
AcOEt/cHex (4/6) as eluent, afforded 646 mg (61% yield) of 11
1
0.21. H NMR (200 MHz) δ 7.76 (s, 1H), 7.05 (s, 4H), 6.72 (bs,
1H), 6.48 (bs, 1H), 6.27 (s, 1H), 4.62 (bs, 2H), 4.32 (t, J ) 5.9 Hz,
2H), 3.72 (m, 5H), 3.19 (m, 8H), 2.89 (m, 2H), 2.70 (t, J ) 5.9
Hz, 2H), 2.52 (m, 4H), 2.23 (m, 2H), 2.08 (m, 4H), 1.79 (m, 12H),
1.29-0.99 (m, 28H). 13C NMR (50 MHz) δ 172.0, 171.6, 164.6,
160.3, 156.2, 156.1, 148.1, 139.2, 133.3, 128.3, 109.9, 109.6, 98.3,
79.6, 62.3, 56.8, 56.1, 52.6, 48.2, 46.9, 46.3, 45.9, 33.8, 33.7, 33.1,
32.9, 32.2, 30.2, 28.5, 25.6, 24.9, 24.7. MS (ESI) m/z ) 978 [M +
H]+. Anal. (C51H79N6O9Cl‚1.5H2O), C, H, N.
Biological Methods. Cell Culture and Transient Transfection.
Chinese hamster ovary (CHO) cells were grown at 37 °C and 5%
CO2 in HamsF12 medium and Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% (v/v) fetal calf serum,
10 mM HEPES (pH 7.4), and antibiotics. Transient transfection
experiments were performed using the transfection reagent Jet-PEI
(Polyplus-Transfection, Illkirch, France) according to the manu-
facturer’s instructions. Final concentration of DNA was adjusted
to 8 µg per 100 mm Petri dish.
Membrane Preparation and Radioligand Binding Assays. C6
glial cells stably transfected with h5-HT4(e) receptors, grown to
confluence, were incubated with serum-free medium for 4 h, washed
twice with phosphate buffered saline (PBS), and centrifugated at
300g for 5 min. The pellet was used immediately or stored at -80
°C. The pellet was resuspended in 10 volumes of ice-cold HEPES
buffer (50 mM, pH 7.4) and centrifugated at 40000g for 20 min at
4 °C. The resulting pellet was resuspended in 15 volumes of HEPES
(50 mM, pH 7.4). The protein concentration was determined by
the method of Bradford using bovine serum albumin as the standard.
Radioligand binding studies were performed in 250 µL of HEPES
buffer (50 mM, pH 7.4), 20 µL of the studied ligand (seven
concentrations), 20 µL of [3H]-GR113808 at a concentration of 0.2
nM, and 50 µL of membranes preparation (100-200 µg of protein).
Nonspecific binding was determined with 10 µM GR113808. Tubes
were incubated at 25 °C for 30 min, and the reaction was terminated
by filtration through Watman GF/B filter paper using the Brandel
48R cell harvester. Filters were presoaked in a 0.1% solution of
polyethylenimine. Filters were subsequently washed with ice-cold
buffer (50 mM Tris-HCl, pH 7.4) and placed overnight in 4 mL of
ready-safe scintillation cocktail. Radioactivity was measured using
a Beckman model LS6500C liquid scintillation counter. Binding
data (Ki) were analyzed by computer-assisted nonlinear regression
analysis (Prism, Graphpad Software, San Diego, CA). The data are
the results of two or three determinations in triplicate.
cAMP Accumulation. C6 glial cells stably transfected with h5-
HT4(e) receptors were grown to confluence and incubated with
serum-free medium for 4 h before the beginning of the assay. Then
the cells were preincubated for 15 min with serum-free medium
supplemented with 5 mM theophylline and 10 µM pargyline. 5-HT
(1 µM) and/or compounds were added and incubated for an
additional 15 min at 37 °C in 5% CO2. The reaction was stopped
by aspiration of the medium and addition of 50 µL of ice-cold
perchloric acid (20%). After a 30 min period, neutralization buffer
was added (25 mM HEPES, 2 N KOH), supernatant was extracted
after 5 min of centrifugation at 2000g, and cAMP was quantified
using a radioimmunoassay kit (cAMP competitive radioimmunoas-
say, Beckman, France). The 5-HT concentration-effect curve was
calculated using seven concentrations (10-10-10-6) alone or in the
presence of compounds. The ligand concentration-effect curves
were calculated using seven concentrations (10-10-10-5).
1
as a pale-yellow oil. Rf(cHex/AcOEt, 6/4) ) 0.34. H NMR (200
MHz) δ 3.98 (bs, 2H), 3.74 (m, 1H), 3.35 (t, J ) 6.7 Hz, 2H),
2.21-2.06 (m, 3H), 1.98-1.81 (m, 4H), 1.78-1.51 (m, 2H), 1.46
(s, 9H), 1.37-1.01 (m, 4H). 13C NMR (50 MHz) δ 177.3, 175.8,
161.6, 159.0, 155.8, 155.4, 131.6, 87.1, 83.0, 80.7, 48.4, 46.0, 37.3,
33.9, 33.1, 31.3, 28.3, 25.7, 24.9, 23.4.
4-(tert-Butoxycarbonyl(3-(4-(3-(tert-butoxycarbonyl(4-(cyclo-
hexylamino)-4-oxobutyl)amino)prop-1-ynyl)phenyl)prop-2-yny-
l)amino)butanoic Acid (12). To a stirring solution of 10 (656 mg,
1.48 mmol, 1 equiv), PdCl2(PPh3)2 (53 mg, 0.074 mmol, 0.05
equiv), and CuI (30 mg, 0.15 mmol, 0.1 equiv) in 20 mL of a 1/1
mixture of freshly distilled NEt3 and anhydrous DMF at room
temperature was added dropwise a solution of 11 (497 mg, 1.55
mmol, 1.05 equiv) in 10 mL of anhydrous DMF. The solution was
stirred at 80 °C for 3.5 h and cooled to room temperature. After
removal of the solvents, the crude mixture was purified by
chromatography on silica gel, using AcOEt/cHex (4/6) containing
0.5% AcOH, to give 503 mg (53% yield) of 12 as a yellow oil.
Rf(cHex/AcOEt (1/1) + 0.5% AcOH) ) 0.39. 1H NMR (200 MHz)
δ 7.32 (s, 4H), 4.41 (bs, 2H), 4.23 (bs, 2H),3.70 (m, 1H), 3.41 (dd,
J ) 11.8 and 6.7 Hz, 4H), 2.38 (t, J ) 7.2 Hz, 2H), 2.17 (t, J )
7.2 Hz, 2H), 1.90 (m, 4H), 1.64 (m, 4H), 1.56-1.39 (m, 22H),
1.17 (m, 2H). 13C NMR (50 MHz) δ 177.3, 175.8, 161.6, 159.0,
155.8, 155.4, 131.6, 87.1, 83.0, 80.7, 48.4, 46.0, 37.3, 33.9 and
33.1 (two signals due to rotamers), 28.5, 28.3, 25.7 and 24.9 (two
signals due to rotamers), 23.4.
2-(4-(4-(tert-Butoxycarbonyl(3-(4-(3-(tert-butoxycarbonyl(4-
(cyclohexylamino)-4-oxobutyl)amino)propyl)phenyl)propyl)-
amino)butanamido)piperidin-1-yl)ethyl 4-Amino-5-chloro-2-
methoxybenzoate (13). To a stirring solution of 12 (343 mg, 0.54
mmol) in 30 mL of MeOH at room temperature was added 36 mg
of 10% Pd/C. The mixture was stirred under 1 bar of H2 overnight
and filtered through a pad of Celite. Evaporation of the solvent
yielded 327 mg (94%) as a colorless oil. An amount of 230 mg
(0.36 mmol, 1 equiv) of this oil was dissolved in 30 mL of dry
Bioluminescence Resonance Energy Transfert (BRET) Assay.
The BRET experiment and the 5-HT4R fusion proteins used for

Synthesis of BiValent Probes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4491
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BRET studies (5-HT4R-RLuc, 5-HT4R-YFP) have been previ-
ously described.19 Two days after transfection, CHO cells trans-
fected with 5-HT4R fusion proteins were detached and washed with
PBS. Membrane preparations were distributed in a 96-well optiplate
(Packard) in the presence or absence of ligand at 25 °C. Coelentera-
zine h substrate (Molecular Probes, Eugene, OR) was added at a
final concentration of 5 µM, and readings were performed with a
lumino/fluorometer Fusion (Packard), which allows the sequential
integration of luminescence signals detected with two filter settings
(RLuc filter, 485 ( 10 nm; YFP filter, 530 ( 12.5 nm). Emission
signals at 530 nm were divided by emission signals at 485 nm.
The difference between this emission ratio obtained with cotrans-
fected RLuc and YFP fusions proteins and that obtained with the
RLuc fusion protein alone was defined as the BRET ratio. Results
were expressed in milli-BRET units (mBU, with 1 mBU corre-
sponding to the BRET ratio value multiplied by 1000).
Molecular Modeling. Manual docking of the ML10302 part is
derived from our previously refined [5-HT4R-ML10302] complex
model.26 After the different spacers were built, molecular dynamics
at 1000 K followed by annealing to 300 K were carried out, the
seven-transmembrane domain being frozen. The final structure was
then energy-minimized using 10 000 steps of a conjugated gradient
procedure.
Modeling was performed on an “Octane” Silicon Graphics
computer, using software from Molecular Simulation Inc. (InsightII,
Builder, and Discover). For all these calculations, a distance-
dependent dielectric constant was used to avoid overevaluation of
electrostatic interactions between charged groups. A cutoff of 2
nm was used to truncate the list of interactive atoms to a reasonable
size in relation to the available central processing unit (CPU) time.
Acknowledgment. This work was supported by grants from
INSERM and CNRS. We thank Patty Chen (Institut Cochin)
for critically reading the manuscript and Monique Gastineau
for her excellent technical assistance.
Supporting Information Available: Chemistry experimental,
spectroscopic data, and results from elemental analyses of all the
listed compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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