Design, synthesis, and in vitro pharmacology of new radiolabeled γ-hydroxybutyric acid analogues including photolabile analogues with irreversible binding to the high-affinity γ-hydroxybutyric acid binding sites

Article abstract of DOI:10.1021/jm1006325

γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report the design and synthesis of the first ¹²⁵I-labeled radioligands in the field, one of which contains a photoaffinity label which enables it to bind irreversibly to the high-affinity GHB binding sites.

Full text of DOI:10.1021/jm1006325

6506 J. Med. Chem. 2010, 53, 6506–6510
DOI: 10.1021/jm1006325
Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues
Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid
Binding Sites
†
†
‡
†
‡
Paola Sabbatini,†,§ Petrine Wellendorph, Signe Høg, Martin H. F. Pedersen, Hans Brauner-Osborne, Lars Martiny,
€
Bente Frølund,† and Rasmus P. Clausen*,†
†Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, 2 Universitetsparken, Dk-2100
Copenhagen, Denmark, and ‡The Hevesy Laboratory, Radiation Research Division, Risø, The Technical University of Denmark, 399
Frederiksborgvej, Dk-4000 Roskilde, Denmark. §On leave from Dipartimento di Chimica e Tecnologia del Farmaco, Universitaꢀ di Perugia, 06123
Perugia, Italy.
Received May 25, 2010
γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential
physiologicalsignificance, the completemolecularmechanismsofaction remainunexplained. Tofacilitate
the isolation and identification of the high-affinity GHB binding site, we herein report the design and
synthesis of the first 125I-labeledradioligands inthe field, one of whichcontains a photoaffinity label which
enables it to bind irreversibly to the high-affinity GHB binding sites.
Introduction
actions of GHB as a neuromodulator and therapeutic drug
where concentrations of GHB in the CNS are in the lower
micromolar range.7,13
γ-Hydroxybutyric acid (GHBa) was originally synthesized
as a γ-aminobutyric acid (GABA) analogue able to readily
cross the blood-brain barrier.1 Subsequently, GHB was
found to occur naturally in the mammalian brain2 as a
metabolite and, under certain conditions, as a precursor of
GABA.3 During the years, GHB has been used for several
different scopes. GHB is a registered drug for the treatment of
catalepsy and excessive daytime sleepiness associated with
narcolepsy (Xyrem)4 and for ameliorating alcohol withdrawal
and reducing alcohol craving in recovered alcoholics
(Alcover).5 On the other hand, GHB is a widely popular drug
of abuse because of the euphoria, disinhibition, and heigh-
tened sexual awareness associated with its use.6
To date, the exact mechanism of action of GHB in the
mammalian central nervous system (CNS) is only partially
understood. When taken exogenously in high doses, reaching
millimolar concentrations in the CNS, many key effects of
GHB are mediated by the subtype B of GABA (GABAB)
receptors7 where GHB is a low-affinity weak partial agonist.8
Apart from the low-affinity binding to GABAB receptors,
GHB also binds to a unique population of high-affinity
binding sites in the CNS9 that are defined by a distinct
ontogenesis10 and distribution11 and hence suggested to re-
present a putative GHB receptor. In further support of this
view, brains from GABAB receptor knockout mice have been
shown to display intact [3H]GHB binding.12 Whereas exo-
genous GHB intake predominantly leads to GABAB receptor
effects, the specific high-affinity site appears critical for the
Despite substantial recent research and reports of two
different GHB receptors in rat14 and human,15 the identity
of the high-affinity site is debatable6,16,17 and has not been
convincingly linked to a specific protein. Uncovering the
identity of the high-affinity GHB binding site would be a
major breakthrough in terms of our understanding of the
therapeutic and recreational use of GHB and its action as an
endogenous signaling molecule.
In continuation of previous work17,18 and to obtain better
pharmacological tools for addressing the neurobiology and
pharmacology of GHB, we labeled a high-affinity GHB
ligand with a 125I isotope to obtain a higher specific radio-
activity than can be achieved with conventional 3H-labeling.19
Photolabeling is a method that has been widely used to study
the interaction of biologically relevant compounds with their
target macromolecules.20,21 An appropriate photoaffinity
probe is usually prepared by attachment of a photoreactive
group (arylazides, aryldiazirines, or benzophenones) to the
ligand molecule. UV irradiation triggers the photoactivating
group in the ligand and leads to the conversion of the
noncovalent binding into a covalent link between the ligand
and amino acidic residues present in the binding pocket in an
unspecific fashion.22
We here report the development of novel GHB analogues
containing a photoreactive azide group, one of whichhas been
labeled with the 125I isotope to generate the first reported
GHB photoaffinity radioligand with preference for the GHB
high-affinity binding site.
*To whom correspondence should be addressed. Phone: (þ45)
35336566. Fax: (þ45) 35336041. E-mail: rac@farma.ku.dk.
a Abbreviations: CNS, central nervous system; DMF, dimethylforma-
mide; GABA, γ-aminobutyric acid; GHB, γ-hydroxybutyric acid; HPLC,
high performance liquid chromatography; NCS-382, (E,RS)-(6,7,8,9-
tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid.
Results and Discussion
We recently reported the synthesis and binding charac-
teristics of a series of high-affinity and selective lipophilic
r
pubs.acs.org/jmc
Published on Web 08/17/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6507
Scheme 1a
Chart 1. Structures of Biarylic Selective GHB Ligands
a Reagents and conditions: (a) Pd(PPh3)4, (SnBu3)2, toluene, reflux,
22 h; (b) NaI, chloramine-T, 94% EtOAc, 4% DMF, 1% AcOH, 1%
H2O, room temp, 30 min, Na2S3O5; (c) NaOH, 1 h; (d) 3 mCi Na125I and
chloramine-T in a mixture of 94% EtOAc, 4% DMF, 1% AcOH, and
1% H2O, room temp, 30 min, Na2S3O5.
Chart 2. Novel Synthesized Photoaffinity-Labeled and Radi-
olabelled GHB Analogues
Scheme 2a
4-diarylic derivatives of GHB (Chart 1) notably devoid of
affinity for GABAA and GABAB receptors.18
For the purpose of developing labeled GHB analogues, we
considered the possibility of substituting functions on the
terminal aromatic ring of these biarylic GHB ligands.
Inparticular (R,S)-4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]-
butanoate (1) and (R,S)-4-hydroxy-4-[4-(3-iodobenzyloxy)-
phenyl]butanoate (2) (Chart 1) seemed like excellent templates
because of their high affinity binding in our [3H]NCS-382
binding assay (Ki of 60 and 75 nM, respectively)18 and their
already incorporated, well-tolerated iodine group.
a Reagents and conditions: (a) LiAlH4, Et2O, roomtemp, 2 h; (b) PPh3,
DEAD, THF, reflux, 18 h; (c) (SnBu3)2, tetrakis(PPh3)Pd(0), toluene,
reflux, 14 h; (d) NaNO2, NaN3, CF3CO2H, 0 °C, 10 min.
ligand 3 was generated in a similar fashion. Thus, radio-
iodination was carried out by treatment with Na125I using
the chloramine-T method23 followed by hydrolysis with
aqueous NaOH without isolation of the intermediate pro-
tected lactone. The radioligand 3 was purified by HPLC
and analyzed to have a radiochemical purity of 97% and a
total activity of 2.48 mCi (83% based on [125I]NaI).
Starting from these structures, we developed the 125I-radio-
labeled compound [125I]4-hydroxy-4-[4-(2-iodobenzyloxy)-
phenyl]butanoate (3) to introduce an azido group and the
strategies to generate the photoaffinity radioligand [125I]4-
hydroxy-4-[4-(2-azido-5-iodobenzyloxy)phenyl]butanoate
(6) (Chart 2). Herein we will describe in detail the chemistry
leading to3, 4, 5, and6, details on the radiochemistry to reach 3
and 6, and the initial in vitro pharmacological characterization
of the ligands. The detailed pharmacological analysis of 3 and
further characterization of 6 will be published elsewhere.
Chemistry. The synthesis of the stannyl precursor 8 for the
radioligand 3 was performed as depicted in Scheme 1. 5-(4-
(2-Bromobenzyloxy)phenyl)dihydrofuran-2(3H)-one (7) was
prepared by Mitsunobu reaction between 2-bromobenzyl
alcohol and 5-(4-hydroxyphenyl)tetrahydrofuran-2-one as
previously reported.18 The obtained aryl bromide 7 was
then treated with hexabutyldistannane in the presence of
tetrakis(triphenylphosphine)Pd(0) in refluxing toluene to
furnish the stannylated derivative 8. To test the efficiency of
the iodination reaction, 8 was initially converted into the
cold ligand 9 by reaction with NaI under oxidative condi-
tions with chloramine-T in a mixture of EtOAc, DMF,
AcOH, and H2O, followed by hydrolysis of the lactone
with NaOH. As this strategy proved functional, the radio-
Several routes were investigated in order to obtain the
photoaffinity-labeled derivatives 4 and 5 (Chart 2). Initially
we applied a synthetic strategy similar to the one reported for
the other substituted biarylic GHB analogues,18 as outlined in
Scheme 2. Commercially available 2-amino-5-bromobenzoic
acid 10 and 4-amino-3-bromobenzoic acid 11 were first re-
duced with LiAlH4 in 87% and 91% yield, respectively, to give
the corresponding benzyl alcohols 12 and 13. These were
refluxed overnight with 5-(4-hydroxyphenyl)tetrahydrofuran-
2-one 1418 in the presence of triphenylphosphine and diethyl-
azodicarboxylate to furnish the Mitsunobu products 15 and
16 in 36% and 32% yield, respectively, upon purification. The
following stannylation reaction proceeded very slowly, and
while 17 could be isolated only in 23% yield, no stannylated
product was obtained from 16. Compound 17 wasthentreated
with NaNO2 and NaN3 in trifluoroacetic acid with the aim of
converting the arylamine moiety into an arylazido group.
Unfortunately, noazide formation was detected and the acidic
condition used for the Sandmeyer reaction led to loss of the
stannyl group, to furnish the unstannylated derivative 18.

6508 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Sabbatini et al.
Scheme 3a
Figure 1. Concentration-dependent inhibition of [3H]NCS-382
binding to rat cerebrocortical membranes by GHB and the GHB
analogues 2, 4, and 5. Results are expressed as the mean ( SD of a
single representative experiment performed in triplicate. Average
Ki values for GHB (4.3 μM) and 2 (75 nM) have been reported
earlier.17 Novel 4 and 5 gave average Ki of 120 and 186 nM,
respectively (n = 2).
4-amino-3-iodobenzoate 26 (Scheme 3). After reduction with
LiAlH4, the resulting alcohols 27 and 28 were treated with
NaNO2 and NaN3 in trifluoroacetic acid to obtain the
desired arylazides 29 and 30 in 28% and 60% yield. The
transformation of the alcohols 29 and 30 into a mesyl group
gave 31 and 32 in 90% and 60% yield respectively. The
nucleophilic reaction between the tetrahydrofuran-2-one
1418 and the mesyl derivatives 31 and 32, respectively,
furnished the key intermediates 33 and 34 in 50% and 88%
yield. Treatment with 1.1 equiv of LiOH led to the photo-
affinity-labeled GHB analogues 4 and 5 in 41% and 25%
yield, respectively. Starting from 33, a stannylation reaction
was performed ((SnBu3)2, tetrakis(triphenylphosphine)Pd(0),
toluene, reflux, overnight) to obtain the desired stannyl deri-
vative 35 as a pure pale-yellow oil in 34% yield. The
tributylstannyl derivative 35 was then used as precursor for
the radioiodination step. Treatment with [125I]NaI using the
chloramine-T method23 followed by hydrolysis with aqueous
NaOH furnished the photoaffinity-labeled radioligand 6.
Compound 6 was purified by HPLC and had a radiochemi-
cal purity of 96% and a total activity of 1.66 mCi (83% based
on [125I]NaI).
In Vitro Pharmacological Evaluation. To evaluate the
affinity of our two novel photoaffinity-labeled GHB analo-
gues 4 and 5 (Scheme 3) for the high-affinity GHB binding
sites in mammalian brain tissue, we studied their ability to
compete for binding with the commercially available GHB
radioligand (E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-ben-
zocyclohept-6-ylidene)acetic acid, [3H]NCS-382,25 in our
previously reported binding assay using rat brain cortical
membranes.16 As shown in Figure 1, 4 and 5 are able to
inhibit [3H]NCS-382 binding in a concentration-dependent
manner. Both compounds bind with affinities several
orders of magnitude higher than GHB itself and with Ki
(120 nM for 4 and 186 nM for 5) only slightly below that of
the previously reported analogue 2 without the azido group
(Ki = 60 nM),18 thus rendering them both suitable GHB
high-affinity ligands.
a Reagents and conditions: (a) LiAlH4, THF, room temp, 4 h; (b)
NaNO2, NaN3, CF3CO2H, 0 °C, 10 min; (c) MsCl, Et3N, DMAP,
CH2Cl2, 0 °C, 1 h, room temp, 4 h; (d) 5-(4-hydroxyphenyl)tetrahydro-
furan-2-one (14), K2CO3, DMF, 70 °C, 14 h; (e) (SnBu3)2, tetrakis-
(PPh3)Pd(0), toluene, reflux, 14 h; (f) LiOH, THF, H2O, room temp, 24 h;
(g) 3 mCiNa125I and chloramine-T in a mixture of 94% EtOAc, 4%
DMF, 1% AcOH, and 1% H2O, room temp, 30 min, Na2S3O5; (h)
NaOH, 1 h.
Consequently, we decided to introduce the azido moiety
before the stannylation step in an alternative synthetic
strategy as shown in Scheme 3. With this strategy, 2-ami-
no-5-bromobenzyl alcohol 12 and 4-amino-3-bromobenzyl
alcohol 13 were first transformed into the corresponding
arylazide derivatives 19 and 20 in 88% and 82% yield,
respectively, by reaction with NaNO2 and NaN3 in trifluoro-
acetic acid at 0 °C. The benzyl alcohol was then transformed
into the mesyl derivatives 21 and 22 by treatment with mesyl
chloride and triethylamine in 90% and 92% yield, respec-
tively. The following reaction between 21 and 22 with the
tetrahydrofuran-2-one 1418 in DMF in the presence of 1.5
equiv of K2CO3 gave the desired derivatives 23 and 24 in 70%
and 77% yield, respectively. Unfortunately, the following
stannylation reaction did not work as expected. No transfor-
mation was observed when the reaction was performed
under mild conditions (bis-triphenylphosphinopalladium-
(II) chloride, dioxane, 50 °C), while stronger conditions
(bis-triphenylphosphinopalladium(II) chloride, dioxane,
reflux or (SnBu3)2, tetrakis(triphenylphosphine)Pd(0), to-
luene, reflux) led to the degradation of the azido group to
furnish 15 and 16. Since it was not possible to transform the
aryl bromides 23 and 24 into the corresponding tributyltin
derivatives without the reduction of the azido group, we
decided to replace the bromine atom with a more reactive
iodine atom that has been reported to allow transformation
into the corresponding arylstannane derivative in the pre-
sence of an azido group, in ortho and in para positions.24
Consequently, a similar synthetic strategy was used
starting from 2-amino-5-iodobenzoic acid 25 and methyl
To develop a protocol for studying the photolinking pro-
perties of 4 and 5 indirectly, we initially found that whereas
[3H]NCS-382could be dissociated frommembraneslabeledto
equilibrium with cold GHB or NCS-382, compounds 4 and 5
were not able to induce dissociation of the radioligand in the
same time frame. This excluded evaluation of the photo-
properties against [3H]NCS-382 within the same time period.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6509
GHB radioligand 6 with high affinity for the specific
GHB binding sites. The compounds are the first reported
125I-labeled analogues for the GHB binding site and will
serve as useful tools for the photolinking and identification
of the specific GHB high-affinity binding protein.
Experimental Section
Chemistry. The syntheses of selected compounds are de-
scribed below. The general chemistry, experimental informa-
tion, and syntheses of all other compounds are in Supporting
Information. Purity of tested compounds was established using
HPLC and is more than 95% pure.
Lithium 4-(4-(2-Azido-5-iodobenzyloxy)phenyl)-4-hydroxybu-
tanoate (4). To a solution of 33 (0.05 g, 0.11 mmol) in THF
(2 mL), a solution of LiOH (3 mg, 0.12 mmol) in H2O (0.2 mL)
was added all at once. The resulting orange solution was stirred
at room temperature for 24 h. Then the solvent was removed in
vacuo. The orange residue was recrystallized from EtOH/H2O
1
to furnish 21 mg (0.045 mmol, 41%) of 4 as a white solid. H
NMR (D2O) δ: 1.53-1.62 (2H, m), 1.88-1.97 (2H, m), 4.17-
4.19 (2H, m), 4.67 (2H, s), 6.33-6.40 (3H, m), 6.78-6.80
(2H, m), 7.14-7.16 (1H, m), 7.38 (1H, s).
Lithium 4-(4-(4-Azido-3-iodobenzyloxy)phenyl)-4-hydroxybu-
tanoate (5). Compound 5 was prepared according to the same
procedure as described for compound 4 starting from com-
pound 34 in 25% yield. 1H NMR (D2O) δ: 1.66-1.75 (2H, m),
1.92-1.99 (2H, m), 4.27 (1H, bs), 4.44-4.50 (1H, m), 4.67 (2H,
s), 6.54 (2H, d, J = 6.9 Hz), 6.67 (1H, d, J = 6.9 Hz), 6.90 (2H, d,
J = 6.9 Hz), 6.99 (1H, d, J = 6.9 Hz), 7.44 (1H, s).
Figure 2. (a) Dissociation of 100 nM 6 from rat cortical mem-
branes. After equilibrium binding, dissociation was time-depen-
dently initiated by the addition of 1 mM GHB. Data shown are the
mean ( SD of two independent experiments performed in triplicate.
(b) Photoincorporation dissociation experiments showing the effect
of UV irradiation on the ability of 1 mM GHB to initiate dissociation
of 100 nM 6. Samples (gray bars) were subjected to UV irradiation
(302 nm, 25 W) for 1 min and compared to samples with no irradia-
tion (white bars). Data shown are representative of two experiments
carried out in duplicate.
Radiochemistry. [125I]4-Hydroxy-4-[4-(2-iodobenzyloxy)-
phenyl]butanoic Acid (3). Radiolabeling was achieved without
isolation of the intermediate protected lactone. Chloramine-T
trihydrate (40.8 mg, 145 μmole) was suspended in a mixture
of EtOAc (4.75 mL), DMF (0.25 mL), acetic acid (50 μL), and
a small amount of water was added (50 μL) to dissolve the
compound. This solution (1000 μL) was added to the stannyl
precursor 8 (1.19 mg, 2.1 μmol) in a small HPLC vial. The vial
was capped and shaken to dissolve the compounds. [125I]NaI
(30 μL, 3 mCi) was added to the vial which was quickly reclosed.
The vial was shaken and the contents left to react for 30 min.
Sodium metabisulfite (9.70 mg) in water (350 μL) was added to
quench excess chloramine-T, and the reaction mixture was
washed with purified water (3 ꢀ 300 μL). The organic layer
was evaporated to dryness under an argon flow. Subsequently,
the reaction mixture was dissolved in acetonitrile (500 μL) and
NaOH (20 μL, 4 M), the latter to hydrolyze the lactone. The
sample was shaken and left overnight at 5 °C. Then it was
neutralized with HCl (80 μL, 1 M) and immediately subjected
to preparative HPLC for purification. The desired product 3 was
collected at 24.7min and analyzed to have a radiochemical purity
of 97% and a total activity of 2.48 mCi (83%). The product
was diluted 5 times with ultrapurified water and loaded onto
a preactivated C-18 Sep-PAK (160 mg). It was then washed
with ultrapurified water (5 mL), eluted with EtOH (5 mL),
and subsequently diluted to ∼5 MBq/mL with a dilute NaOH
solution (0.001 M).
Instead, for direct evaluation of their photolinking properties
of our compounds, we synthesized the photoaffinity radio-
ligand 6, the 125I-labeled analogue of 4 (Scheme 3). It was
chosen to synthesize the labeled analogue of 4 based on the
more feasible synthetic route of its stannyl precursor.
To set up the photoaffinity linking experiments using the
photoaffinity radioligand 6, we initially examined the bind-
ing kinetics of the radioligand without applying UV light.
This served to determine the time needed to achieve binding
and unbinding of the radioligand. Preliminary experiments
using 100 nM radioligand indicated a fast association time of
∼2 min and equilibrium binding after 10 min that was
constant for at least 1.5 h (data not shown). Consequently,
a 60 min incubation time was used for the assays. Dissocia-
tion was fast with a t1/2 of ∼1 min (Figure 2a). Thus, in
photoincorporation experiments we used an initial 60 min
preincubation time before UV radiation followed by a
secondary 30 min incubation time to potentially allow for
full dissociation of the radioligand.
Photoincorporation of 6 with the GHB binding protein
was then tested utilizing the demonstrated ability of the
radioligand to rapidly dissociate in the absence of photo-
linking. As shown in Figure 2b, the photoaffinity radioligand
6 covalently links to the high-affinity GHB binding sites, as
pre-equilibration of membranes with 6, irradiation by UV
light, and subsequent addition of a high concentration of
GHB did not lead to dissociation of the photolinker. By
contrast, non-UV-irradiated membrane samples were fully
displaced by GHB (Figure 2b). Taken together, this clearly
demonstrated that our novel photoaffinity radioligand 6 is
able to covalently link with the high-affinity GHB binding
site upon UV irradiation. Characterization of the linked
protein now awaits.
[
125I]4-Hydroxy-4-[4-(2-azido-5-iodobenzyloxy)phenyl]butanoic
Acid (6). Radiolabeling was achieved without isolation of the
intermediate protected lactone as described for 3. As the com-
pound is light sensitive, all experiments were performed protected
from light. The desired product 6 was collected at 26.7 min and
analyzed to have a radiochemical purity of >95% (Figure 2) and
a total activity of 1.75 mCi (88% based on Na125I). The product
was diluted 5 times with ultrapurified water and loaded onto a
preactivated C-18 Sep-PAK (160 mg). It was then washed with
ultrapurified water (5 mL), eluted with EtOH (5 mL), and
subsequently diluted to a concentration of ∼5 MBq/ml in a
NaOH solution (0.001 M).The use of slightly basic conditions
in the final product matrix eliminated the potential lactone
In conclusion, we have designed and developed two photo-
affinity-labeled GHB analogues 4 and 5 and a 125I-labeled

6510 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Sabbatini et al.
formation. The total yield of formulated and final product
was 1.66 mCi (83% based on [125I]NaI) with a radioactive
purity of 96% and a moderate specific activity of approximately
2 Ci/mmol. The moderate specific activity was due to traces of the
cold iodide compound used in the preparation of the stannyl
precursor.
(8) Mathivet, P.; Bernasconi, R.; De Barry, J.; Marescaux, C.; Bittiger,
H. Binding characteristics of γ-hydroxybutyric acid as a weak but
selective GABAB receptor agonist. Eur. J. Pharmacol. 1997, 321,
67–75.
(9) Wu, Y.; Ali, S.; Ahmadian, G. γ-Hydroxybutyric acid (GHB) and
γ-aminobutyric acidB receptor (GABABR) binding sites are dis-
tinctive from one another: molecular evidence. Neuropharmacology
2004, 47, 1146–1156.
Pharmacology. Rat brain (cerebral cortex) synaptic membrane
preparation, [3H]NCS-382 binding assay, and data analysis were
carried out exactly as previously described.16 Dissociation experi-
ments for 6 were carried out using a modified [3H]NCS-382 assay
protocol. In brief, well-washed membranes (10 μg of protein per
sample) were preincubated with 100 nM 6 in 50 mM phosphate
buffer, pH 6, for 1 h at room temperature in a total volume of
2 mL. To initiate dissociation, 20 μL of GHB (final concentration
of 1 mM) was added. At the indicated time points, the reactions
were terminated by rapid filtration through Whatman GF/C
filters (PerkinElmer Life and Analytical Sciences, Waltham, MA)
using a Brandel M-48R cell harvester, followed by washing with
3 ꢀ 3 mL of ice-cold assay buffer. The amount of filter-bound
radioactivity was quantified by adding 3 mL of scintillation fluid
(Opti-Fluor, PerkinElmer) to the dried filters and counting in
a Packard Tricarb 2100 liquid scintillation counter. Data were
fitted using the model for exponential decay in the program
Graphpad Prism 5.0, yielding the t1/2 as the time for half of the
radioligand to dissociate. Total bound radioligand accounted for
less than 10% of the total amount of radioactivity in the reaction.
(10) Snead, O. C. The ontogeny of [3H]γ-hydroxybutyrate and [3H]-
GABAB binding sites: relation to the development of experimental
absence seizures. Brain Res. 1994, 659, 147–156.
(11) Castelli, M. P.; Mocci, I.; Langlois, X.; Gommerendagger, W.;
Luyten, W. H.; Leysen, J. E.; Gessa, G. L. Quantitative autoradio-
graphic distribution of γ-hydroxybutyric acid binding sites in
human and monkey brain. Brain Res. 2000, 78, 91–99.
(12) Kaupmann, K.; Cryan, J. F.; Wellendorph, P.; Mombereau, C.;
Sansig, G.; Klebs, K.; Schmutz, M.; Froestl, W.; van der Putten,
€
H.; Mosbacher, J.; Brauner-Osborne, H.; Waldmeier, P.; Bettler,
B. Specific γ-hydroxybutyrate-binding sites but loss of pharmaco-
logical effects of γ-hydroxybutyrate in GABA(B)(1)-deficient mice.
Eur. J. Neurosci. 2003, 18, 2722–2730.
(13) Castelli, M. P. Multi-faceted aspects of gamma-hydroxybutyric
acid: a neurotransmitter, therapeutic agent and drug of abuse.
Mini-Rev. Med. Chem. 2008, 8, 1188–1202.
(14) Andriamampandry, C.; Taleb, O.; Viry, S.; Muller, C.; Humbert,
J. P.; Gobaille, S.; Aunis, D.; Maitre, M. Cloning and character-
ization of a rat brain receptor that binds the endogenous neuro-
modulator γ-hydroxybutyrate (GHB). FASEB J. 2003, 17, 1691–
1693.
(15) Andriamampandry, C.; Taleb, O.; Kemmel, V.; Humbert, J. P.;
Aunis, D.; Maitre, M. Cloning and functional characterization of a
γ-hydroxybutyrate receptor identified in the human brain. FASEB
J. 2007, 21, 885–895.
(16) Bettler, B.; Kaupmann, K.; Mosbacher, J.; Gassmann, M. Molec-
ular structure and physiological functions of GABAB receptors.
Physiol. Rev. 2004, 84, 835–867.
Acknowledgment. The authors thank Dr. Klemens
Kaupmann for protocols for the photoincorporation
experiments. This work was supported by the Danish
Medical Research Council, “Fonden til Lægevidenskabens
Fremme”, and “Carl and Ellen Hertz Legat for Dansk
Natur- og Lægevidenskab” (P.W.). P.W. was further sup-
ported by the Alfred Benzon Foundation and by the “For
(17) Wellendorph, P.; Høg, S.; Greenwood, J. D.; De Lichtenberg, A.;
€
Nielsen, B.; Frølund, B.; Brehm, L.; Clausen, R. P.; Brauner-
Osborne, H. Novel cyclic γ-hydroxybutyrate (GHB) analogues
with high affinity and stereoselectivity of binding to GHB sites in
rat brain. J. Pharmacol. Exp. Ther. 2005, 315, 346–351.
ꢁ
Women in Science” grant from L’Oreal, UNESCO, and
The Royal Danish Academy of Science and Letters.
(18) Høg, S.; Wellendorph, P.; Nielsen, B.; Frydenvang, K.; Dahl,
€
I. F.; Brauner-Osborne, H.; Brehm, L.; Frølund, B.; Clausen,
R. P. Novel high-affinity and selective biaromatic 4-substituted
γ-hydroxybutyric acid (GHB) analogues as GHB ligands: design,
synthesis and binding studies. J. Med. Chem. 2008, 51 (24), 8088–
8095.
Supporting Information Available: Zipped file containing
details for the synthesis of intermediates 8 and 12-35, radio-
labeling and photoincorporation, HPLC chromatograms, and
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
(19) Froestl, W.; Bettler, B.; Bittiger, H.; Heid, J.; Kaupmann, K.;
Mickel, S. J.; Strub, D. Ligands for expression cloning and isola-
tion of GABAB receptors. Farmaco 2007, 58, 173–183.
(20) Dorman, G.; Prestwich, G. D. Using photolabile ligands in drug
discovery and development. Trends Biotechnol. 2000, 18, 64–77.
(21) Colca, J. R.; Harrigan, G. G. Photo-affinity labeling strategies in
identifying the protein ligands of bioactive small molecules: exam-
ples of targeted synthesis of drug analog photoprobes. Comb.
Chem. High Throughput Screening 2004, 7, 699–704.
References
(1) Laborit, H.; Jouany, J.; Gerard, J.; Fabiani, F. Generalities con-
cerning the experimental study and clinical use of sodium gamma-
hydroxybutyrate. Agressologie 1960, 1, 397–406.
(2) Bessman, S. P.; Fishbein, W N. Gamma-hydroxybutyrate, a
normal brain metabolite. Nature 1963, 200, 1207–1208.
(3) Maitre, M. The gamma-hydroxybutyrate signalling system in
brain: organization and functional implications. Prog. Neurobiol.
1997, 51, 337–361.
(4) Robinson, D. M.; Keating, G. M. Sodium oxybate: a review of its
use in the management of narcolepsy. CNS Drugs 2007, 21, 337–
354.
(5) Caputo, F.; Vignoli, T.; Maremmani, I.; Bernardi, M.; Zoli, G.
Gamma hydroxybutyric acid (GHB) for the treatment of alcohol
dependence: a review. Int. J. Environ. Res. Public Health 2009, 6,
1917–1929.
(6) Drasbek, K. R.; Christensen, J.; Jensen, K. Gamma-hydroxybuty-
rate, a drug of abuse. Acta Neurol. Scand. 2006, 114, 145–156.
(7) Carter, L. P.; Koek, W.; France, C. P. Behavioral analyses of GHB:
receptor mechanisms. Pharmacol. Ther. 2009, 121, 100–114.
(22) Ruoho, A. E.; Kiefer, H.; Roeder, P. E.; Singer, S. J. The mechan-
ism of photoaffinity labeling. Proc. Nat. Acad. Sci. U.S.A. 1973, 70,
2567–2571.
(23) Lindegren, S.; Skarnemark, G.; Jacobsson, L.; Karlsson, B.
Chloramine-T in high-specific-activity radioiodination of anti-
bodies using N-succinimidyl-3-(trimethylstannyl)benzoate as an
intermediate. Nucl. Med. Biol. 1998, 25, 659–665.
(24) Horwell, D. C.; Hunter, J. C.; Kneen, C. O.; Pritchard, M. C.
Synthesis of novel iodinated radioligands with high affinity and
selectivity for CCK-B/Gastrin receptors. Bioorg. Med. Chem. Lett.
1995, 5 (21), 2501–2506.
(25) Mehta, A. K.; Muschaweck, N. M.; Maeda, D. Y.; Coop, A.;
Ticku, M. K. Binding characteristics of the γ-hydroxybutyric acid
receptor antagonist [3H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-
benzo[a][7]annulen-6-ylidene) ethanoic acid in the rat brain.
J. Pharmacol. Exp. Ther. 2001, 299, 1148–1153.