Synthesis of the nanomolar photoaffinity GABA(B) receptor ligand CGP 71872 reveals diversity in the tissue distribution of GABA(B) receptor forms

Article abstract of DOI:10.1016/S0968-0896(99)00214-X

A radioiodinated probe, [¹²⁵I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain reveale

Full text of DOI:10.1016/S0968-0896(99)00214-X

Bioorganic & Medicinal Chemistry 7 (1999) 2697±2704
Synthesis of the Nanomolar Photoanity GABAB Receptor
Ligand CGP 71872 Reveals Diversity in the Tissue
Distribution of GABAB Receptor Forms
Michel Belley, a,*,y Richard Sullivan, a,y Austin Reeves, b
Jilly Evans, a Gary O'Neill a and Gordon Y. K. Ng a,*
aDepartments of Chemistry, Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research,
16711 TransCanada Highway, Kirkland, Quebec, H9H 3L1, Canada
bMerck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Harlow,
Essex CM20 2QR, UK
Received 18 March 1999; accepted 21 June 1999
AbstractÐA radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and
used to characterize GABAB receptors. Photoanity labeling experiments using crude membranes prepared from rat brain revealed
two predominant ligand binding species at ꢀ130 and ꢀ100 kDa believed to represent the long (GABABR1a) and short
(GABABR1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABAB receptor-
speci®c antibody con®rming the receptor speci®city of the photoanity probe. Most convincingly, [125I]-CGP 71872 binding was
competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, ( )-baclofen, (+)-baclofen and (l)-
glutamic acid with a rank order and stereospeci®city characteristic of the GABAB receptor. Photoanity labeling experiments
revealed that the recombinant GABABR2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that
CGP 71872 is a GABABR1 selective antagonist. Photoanity labeling experiments using rat tissues showed that both GABABR1a
and GABABR1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABABR1b receptor form was
detected in kidney and liver whereas the long GABABR1a form was selectively expressed in the adrenal gland, pituitary, spleen and
prostate. We report herein the synthesis and biochemical characterization of the nanomolar anity [125I]-CGP 71872 and CGP
71872 GABABR1 ligands, and di€erential tissue expression of the long GABABR1a and short GABABR1b receptor forms in rat
and dog. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
pattern of gene expression, agonist and antagonist
mediated spectrum of physiological activities.
Amino acids such as glutamic acid, g-aminobutyric acid
(GABA) and glycine are neurotransmitters that bind to
speci®c receptors in the vertebrate nervous system and
mediate synaptic transmission. Of these amino acids,
GABA is the most widely distributed amino acid inhibi-
tory neurotransmitter in the vertebrate central nervous
system. The biological activities of GABA are mediated
by three types of GABA receptors: ionotropic GABAA
receptors, metabotropic GABAB G protein-coupled
receptors, and ionotropic GABAC receptors. Each type of
receptor has its own characteristic molecular structure,
Biology
GABAB receptors were ®rst distinguished by Hill and
Bowery.1 Based on their anatomical location and physio-
logical functions, GABAB receptors act presynaptically as
autoreceptors and heteroreceptors and also act post-
synaptically. Functionally, GABAB receptors couple
through G proteins to neuronal K+ or Ca2+ channels.
Receptor activation leading to increased K+ conductance
is responsible for late inhibitory postsynaptic potentials
(IPSPs), whereas receptor mediated decrease in Ca2+
conductance results in presynaptic inhibition of transmit-
ter release. There is also evidence that GABAB receptor
activation leads to inhibition or potentiates adenylyl
cyclase activity, although the functional signi®cance of
Key words: CGP 71872; GABAB receptor; GABA; photoanity
labeling.
* Corresponding authors. Fax:+1-514-428-4900; e-mail: belley@
merck.com (chemistry); gordon_ng@merck.com (biology)
y
Co-®rst authors.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00214-X

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M. Belley et al. / Bioorg. Med. Chem. 7 (1999) 2697±2704
these second messenger changes remains unclear. The
molecular cloning of two members of the rat GABAB
receptor family, GABABR1a and GABABR1b, was
®nally achieved by expression cloning by Kaupmann et
al.2 using high-anity GABAB CGP 64213 and CGP
71872 receptor antagonists. This pivotal paper, how-
ever, did not report the synthesis of these powerful
GABAB receptor probes. In studying the recombinant
GABABR1 receptors and a GABAB-like orphan GPR
51 receptor, now identi®ed as GABABR2, we and others
observed that both receptors are functionally inactive
when expressed individually in multiple heterologous
expression systems, and that the functional GABAB
receptor is a heterodimer of GABABR1 and GABABR2
subtypes.3±7 However, our understanding of the mole-
cular basis of GABAB heterodimer activation and ligand
binding is still rudimentary. With the aim of clarifying
the pharmacological properties and therapeutic potential
of the functional GABAB receptor, we report herein the
detailed synthesis of [125I]-CGP 71872, the characteriza-
tion of its pharmacological speci®city and the tissue dis-
tribution of the GABAB receptors in rat and dog as
determined by photoanity labeling studies.
Chemistry
[
125I]-CGP 71872 was derived from structure±activity rela-
tionship studies starting from the GABAB receptor agonist
3-aminopropylphosphinic acid, a close analogue of g-ami-
nobutyric acid.2,8 It was designed to be a photoanity
probe: when bound to the receptor, the aryl azide function
can be activated by photolysis (360 nm) to generate a
highly reactive nitrene intermediate which can then react to
create a covalent bond with the surrounding protein.
[
125I]-CGP 71872 was prepared in a manner similar to
its synthesis summarized in a patent,9±11 with some
modi®cations (Scheme 1). The preparation of ethyl (1,1-
diethoxyethyl)phosphinate 1 from phosphinic acid and
Scheme 1. Synthesis of CGP 71872.

M. Belley et al. / Bioorg. Med. Chem. 7 (1999) 2697±2704
2699
triethyl orthoacetate under Lewis acid catalysis using the
reported procedures10,12 gave low and non-reproducible
yields of 1, along with varying amounts of diethyl (1,1-
diethoxyethyl)phosphonate (EtO)2CMePO(OEt)2. Redu-
cing the temperature and the time of the reaction greatly
improved the preparation. Deprotonation of 1 with
sodium hydride and reaction with N-(5-bromo-
pentyl)phthalimide a€orded 2, which was deprotected with
chlorotrimethylsilane to give the phosphinic acid 3. Treat-
ment of 3 with chlorotrimethylsilane and triethylamine
yielded a trivalent silylated phosphonite that is very sensi-
tive to oxidation and should be ®ltered under argon and
concentrated with a high vacuum pump. Reaction with
fused zinc chloride and epichlorohydrin at 80ꢁC (in the
absence of solvent) and hydrolysis of the silyl ether with
acetic acid yielded the intermediate 4. The (cyanophenyl)-
ethylamine 6 was prepared via a new route in three steps by
a chiral reduction with ( )-B-chlorodiisopinocampheyl-
borane13,14 followed by a Mitsunobu reaction with
phthalimide and deprotection to the amine.15±17 Substitu-
tion of the chlorine of 4 with the primary amine 6 a€orded
the secondary amine 7. The nitrile was hydrolyzed with
NaOH and the phthalimide removed with concentrated
HCl to yield the amine 8. This is reacted with the commer-
cially available N-hydroxysuccinimidyl-4-azidosalicylic
acid 9 to give the intermediate 10 which is iodinated to
CGP 71872 with chloramine T and sodium iodide.
Figure 1. [125I]-CGP 71872 labels GABABR1a and GABABR1b
receptors. (A) Membranes (25 mg) derived from rat brain were photo-
labeled with [125I]-CGP 71872 in the absence ( ) and presence of 1 mM
CGP 71872 (+) and visualized by autoradiography. (B) GABABR1a
and GABABR1b receptors were immunoprecipitated using GABAB
receptor-speci®c antibodies 1713.1±1713.2 ( ) but not preimmune
serum (+). (C) [125I]-CGP 71872 labeled dog brain GABAB receptor
forms in the absence ( ) and presence (+) of 1 mM CGP 71872.
label the GABABR2 receptor. Photoanity labeling
experiments using 100 nM ®nal concentration of [125I]-
CGP 71872 revealed that the recombinant GABABR2
receptor3±7 (when expressed in COS-7 cells) does not bind
the ligand, even though its expression was con®rmed by
immunoblot analysis (data not shown). This suggests that
[
125I]-CGP 71872 is a GABABR1 receptor selective ligand
and that the GABABR1 subunit is primarily responsible for
ligand binding to the GABABR1±GABABR2 heterodimer.
Results and Discussion
The abundant brain expression of GABABR1a and
GABABR1b receptors suggests that both the long and
short GABAB receptors contribute to the reported
GABA and baclofen activated G protein-gated inwardly
rectifying K+ channels and/or Ca2+ channels which
have been implicated in hippocampal long-term poten-
tial, absence epilepsy, slow-wave sleep, muscle relaxa-
tion and antinociception activities.8,20
Since the ligand binding properties of CGP 71872 have not
been reported in detail previously, its characterization is
given herein. With the high speci®c activity of iodine
(2200 Ci/mmol) attached to the ligand, the tissue distribu-
tion and the therapeutic potential of GABAB receptors has
also been de®ned.
[
125I]-CGP 71872 labels GABABR1a and GABABR1b
receptors, but not GABABR2 receptors
CGP 71872 exhibits nanomolar anity for GABABR1a
and GABABR1b receptors
Photolabeling experiments were carried out to character-
ize the substrate speci®city of [125I]-CGP 71872. Upon
exposure to UV light, [125I]-CGP 71872 photolabeled
major bands at ꢀ130 and ꢀ100 kDa in crude membranes
prepared from rat brain at the predicted molecular mass
reported for GABABR1a and GABABR1b receptors2
(Fig. 1A). Indeed, GABAB receptor GB-1713.1 and GB-
1713.2 polyclonal antibodies but not preimmune sera were
able to immunoprecipitate these ligand binding species
(Fig. 1B) con®rming the receptor speci®city of the ligand.
Similar ꢀ130 and ꢀ100 kDa ligand binding species were
observed in dog brain membranes (Fig. 1C). Faintly
labeled species at approximately twice the molecular mass
of the receptor monomers in rat and dog brain mem-
branes suggest that GABABR1 receptors may undergo
homodimerization,18 also reported for the structurally
related metabotropic glutamate receptors.19
Displacement of [125I]-CGP 71872 binding to mem-
branes prepared from whole brain in ®ltration-binding
experiments was in the appropriate rank order for
GABAergic ligands: CGP 71872 (Ki 0.48‹0.08 nM
(n=4) (mean‹standard deviation)) > GABA (2.50
‹0.06 mM (n=3))
> saclofen (156.13‹45.88 mM
(n=3)) > ( )-baclofen (0.52‹0.04 mM (n=3)) > (+)-
baclofen (1.14‹0.31 mM (n=3)) > l-glutamic acid
(4.91‹0.30 mM (n=3)) (Fig. 2(a)). The anity of
baclofen in whole brain tissue is lower compared to
previous studies using brain cortex tissues.2 One expla-
nation might be that the baclofen anity in whole brain
re¯ects binding to multiple and pharmacologically dif-
ferent GABAB receptor subtypes that are not expressed
in brain cortex. GABABR1 and GABABR2 receptors
are di€erentially expressed in brain, especially in the
basal ganglia, where GABABR1 RNA expression is
high whereas GABABR2 RNA expression is low.3±7
Since the GABABR1 receptor requires the coexpression
of the GABABR2 receptor for membrane targeting and
the formation of the functional GABAB receptor,3±7 this
With the identi®cation of the functional GABAB receptor
which requires the coexpression and heterodimerization of
GABABR1 and GABABR2 receptors,3±7 it seemed impor-
tant to test whether the potent CGP 71872 antagonist can

2700
M. Belley et al. / Bioorg. Med. Chem. 7 (1999) 2697±2704
suggests the existence of other GABAB receptors in the
basal ganglia that would enable the formation of a
functional GABAB receptor heterodimer. Further,
GABABR1 receptor isoforms exhibit di€erential cellular
and extrasynaptic localization in brain, suggesting di€er-
ent pharmacology and functions.21,22 Photoanity
labeling experiments showed that the long GABABR1a
and short GABABR1b receptor forms di€er slightly in
their intensity of labeling, suggesting that the short form
may have a higher anity for CGP 71872 or is more
highly expressed compared to the long GABABR1a
receptor (Fig. 2(b)). Taken together, CGP 71872 is the
highest anity ligand so far described with a Ki of
ꢀ0.5 nM at GABABR1a and GABABR1b receptors.
Peripheral tissue expression of GABABR1a and
GABABR1b receptors
There is clear evidence of the functional importance of
GABAB receptors in the brain, but the lack of high-a-
nity GABAB receptor-speci®c ligands and low peripheral
expression have made it dicult to establish a clear role
for this receptor in GABAergic mechanisms outside the
CNS.23,24 The potential importance of GABAB receptors
in peripheral GABAergic functions is now supported by
photoanity labeling studies using the nanomolar CGP
71872 ligand in tissues prepared from rat and dog.
CGP 71872 labeling studies revealed long and short
GABAB receptor proteins in stomach and testis tissues
(Table 1, Fig. 3). The known involvement of GABAB
receptors in stomach physiology has been to mediate
gut relaxation by inhibition of postganglionic choliner-
gic nerves.23 The role(s) of GABAB receptors outside a
nervous based mechanism of action is less clear. In the
isolated stomach, GABA has been localized to secretory
granules of gastric mucosal epithelium and shown to
modulate gastric acid secretion likely via GABAA
receptors.24,25 The morphological feature of GABAB
receptor-positive cells in the gastrointestinal mucosa26
Figure 2. Ligand binding properties of rat brain GABABR1a and
GABABR1b receptors. (a) Pharmacological characterization of GABAB
receptors using ®ltration-competition binding experiments. (b) CGP
71872 competition of [125I]-CGP 71872 binding in photoanity assays.
Table 1. Summary of di€erential expression of long and short GABAB receptor forms in rat and dog tissuesa
Rat Dog
130 kDa long
isoform
100 kDa short
isoform
130 kDa long
isoform
100 kDa short
isoform
Rat/dog ®lm
exposure time
Spinal cord
Brain
Heart
Lung
Muscle
Liver
Spleen
Small intestine
PBL
Pituitary
Adrenal gland
Prostate
Testis
++
+++
ND
ND
ND
ND
+
ND
ND
+
++
+++
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
+
ND
ND
ND
ND
++
+
ND
ND
ND
ND
++
+++
Ð
ND
ND
ND
+
ND
Ð
+
+
+
Ð
Ð
Ð
ND
Ð
+
++
+++
Ð
ND
ND
ND
ND
ND
Ð
ND
ND
ND
Ð
Ð
Ð
ND
Ð
+
ND
Ð
Ð
Ð
h±1 day
h±1 day
4 weeks
4 weeks
4 weeks
2 weeks
4 weeks/2 weeks
4 weeks
4 weeks
4 weeks/2 weeks
4 weeks
4 weeks/2 weeks
1 week
4 weeks
+
+
+
Ovary
ND
ND
ND
ND
ND
+
ND
ND
ND
ND
Trachea
Pancreas
Colon
4 weeks
4 weeks
4 weeks
1/3 weeks
1 week
4 weeks
4 weeks
4 weeks
4 weeks
Kidney
Stomach
Thyroid
Thymus
Bone marrow
Lymph node
ND
Ð
Ð
Ð
Ð
Ð
a
ND: not detected; +++: high expression; ++: intermediate expression; +: low expression; Ð: tissue not tested.

M. Belley et al. / Bioorg. Med. Chem. 7 (1999) 2697±2704
2701
The biological actions of GABA in these regions are likely
mediated via GABAA receptors,31 although baclofen
binding studies have indicated expression of GABAB
receptors in the kidney as well.32 The demonstration of
high-anity GABA uptake sites on hepatocytes, localiza-
tion of GABA and GAD on these cells, and demonstra-
tion of bicuculline-sensitive (GABAA receptor) GABA
binding support a GABAergic component to bile secre-
tion.24,33 The localization of GABABR1b to this exocrine
tissue may suggest an in¯uence on bile secretion as well.
Figure 3. Di€erential expression of GABABR1a and GABABR1b
receptors in rat tissues. Photoanity [125I]-CGP 71872 labeling of
GABAB receptor forms in multiple tissues. The long GABABR1a and
short GABABR1b receptors are indicated. Autoradiogram exposure
times are as follows: brain, 1 week; kidney, 3 weeks; stomach, 3 weeks;
liver, 10 weeks; spleen, 10 weeks; pituitary, 10 weeks; prostate, 10
weeks; testis, 9 weeks; spinal cord, 1 week; adrenal gland, 12 weeks.
We showed for the ®rst time di€erential peripheral tissue
expression of the long GABABR1a and short
GABABR1b receptor forms. Because the functional
GABAB receptor is a GABABR1±GABABR2 hetero-
dimer, this suggests that possibly GABABR1a±
GABABR2 and GABABR1b±GABABR2 heterodimers
mediate di€erent physiological functions, thus accounting
for the diverse biological activities of GABA. Clearly
more detailed in situ hybridization studies are needed to
con®rm that the expression of GABAB receptors is not
con®ned to nerve tissue within the periphery. GABAB
receptors were not detected in a number of tissues: tra-
chea, heart, lymph node, peripheral blood leukocytes,
bone marrow, skeletal muscle, small intestine, colon,
ovary, pancreas, thyroid, thymus, and lung. One expla-
nation for the e€ects of the GABAB receptor agonists
such as baclofen on the physiology of some of these
organs may be that it involves central GABAB receptors
or that other GABAB receptor forms not labeled by CGP
71872 remain to be discovered at the molecular level.
would, however, support the expression of GABAB
receptors in the stomach such that both GABAA and
GABAB receptors may be involved in mediating the
local hormone actions of GABA on the secretion of
gastrin and somatostatin.27,28
A role for GABAB receptors in the testis has not been
described and is unknown. GABA has been detected in
spermatozoa and presumably acts via GABAA recep-
tors to regulate the motility of the spermatozoon.24
Recent biochemical ®ndings have shown GABA mod-
ulation of androgen secretion in testicular tissue, prob-
ably by a direct action on endocrine cells of the testis,29
but the receptors involved remain to be determined.
The long form of the receptor, GABABR1a, was
expressed absent of the short GABABR1b receptor in
restricted peripheral tissues (Table 1, Fig. 3). [125I]-CGP
71872 labeled GABABR1a in endocrine tissues such as
pituitary, adrenal gland, prostate, and epithelial tissues
such as spleen. GABA and glutamic acid decarboxylase
have been shown to be localized to both anterior and
intermediate lobes of the pituitary,24 suggesting that
GABABR1a receptor activation may contribute to
GABA mediated decrease in corticotrophin-releasing
hormone, prolactin-releasing factor, luteinizing hor-
mone levels including increasing androgen produc-
tion.30 Indeed, baclofen, a selective GABAB receptor
agonist, exhibits robust inhibition of pituitary secre-
tions.30 We observed GABABR1a expression in the adre-
nal gland. Although its role in local hormone actions is
not known, GABAB receptors have been reported to
modulate acetylcholine-evoked catecholamine release.24
A role for GABABR1a receptors in prostate and spleen
physiology is less clear. Much of the evidence suggests a
role for the GABAA receptor in the immune modulatory
role of GABA on the proliferation of thymic cells and of
spleen lymphocytes.24 The localization of GABABR1a in
spleen suggests that this GABAB receptor form may be
important as well. No information is available about
GABAB receptors in the prostate although its expression
was detected by [125I]-CGP 71872 binding.
Experimental
Synthesis of [125I]-CGP 71872; general comments
N-Hydroxysuccinimidyl-4-azidosalicylic acid was pur-
chased from Pierce. Many intermediates in the synthesis
are hard to reveal on TLC (silica gel): they have to be
spotted heavily (a whole drop of reaction mixture from a
pipette), checked by UV and revealed with iodine followed
by a cerium/molybdate dip to give, sometimes, only faint
and di€use spots. The chemical shifts of the protons for
compounds 6, 7, 9 and CGP 71872 also vary from one
sample to another due to pH variation and/or contamina-
.
tion by solvents or impurities such as i-Pr2NEt AcOH or
NH4OAc. Mass spectrometry was used often to follow
reactions and to identify the product distribution.
Ethyl (1,1-diethoxyethyl)phosphinate 1. Hypophosphor-
ous acid was concentrated down, at 40ꢁC under high
vacuum overnight, to a yellow syrup which crystallized
ꢁ
in the fridge.34 At 5 C, BF3 Et2O (10 mL) was added to
triethyl orthoacetate (450 mL) and the mixture was
cooled down to 20ꢁC. A solution of concentrated
hypophorous acid (39 g, 417 mmol) in THF (200 mL)
was then added slowly and the mixture was warmed to
0ꢁC for 2 h (until all the solid H3PO2 was dissolved; the
reaction could be monitored by TLC using acetone:toluene
2:3 as eluent). 0.4 M NaH2PO4 was then added slowly and
the product was extracted into i-PrOAc, washed with brine,
dried over Na2SO4 and concentrated. Distillation at 61ꢁC/
0.1 mm Hg yielded 74.35 g of 1 with a purity of 87% (69%
.
[
125I]-CGP 71872 revealed selective expression of the
short GABABR1b receptor form in exocrine and epithelial
tissues such as kidney and liver (Table 1, Fig. 3). GABA
is believed to have a possible role in renal ammonia-
genesis and in the function of proximal tubular cells.23

2702
M. Belley et al. / Bioorg. Med. Chem. 7 (1999) 2697±2704
yield). The major impurity co-distilling with 1 was diethyl
1
washed with 1 N HCl and brine, dried over Na2SO4 and
concentrated to dryness. Flash chromatography on silica
with EtOAc:toluene 1:5 a€orded 3.25g (65% yield) of 10
with an enantiomeric excess of 97% (determined from the
(1,1-diethoxyethyl)phosphonate. H NMR (acetone-d6) d
1.17 (6H, t), 1.32 (3H, t), 1.38 (3H, d), 3.68 (4H, m), 4.17
(2H, m), 6.85 (1H, d, J=540Hz, PH).
1
1H NMR of its Mosher esters). H NMR (acetone-d6) d
N-(5-((1,1-Diethoxyethyl)ethoxyphosphoryl)pentyl)phthali-
mide 2. At 0ꢁC, 80% NaH in oil (502 mg, 16.7 mmol) was
added to 1 (4.11g, 80% purity, 15.6 mmol) in THF
(80 mL) and the mixture was stirred at room temperature
for 4 h. At 0ꢁC, N-(5-bromopentyl)phthalimide (4.12 g,
13.9 mmol) was added and the mixture was stirred again at
room temperature for 4 h and was quenched by the addi-
tion of half saturated NH4Cl. The product was extracted
into i-PrOAc, dried over Na2SO4 and puri®ed by ¯ash
chromatography on silica with acetone:toluene 1:4 to give
2.08g (35% yield) of 2. 1H NMR (acetone-d6) d 1.14 (6H,
2t), 1.25 (2H, t), 1.40 (2H, d), 1.46 (2H, m), 1.58±1.77 (6H,
m), 3.60 (1H, m), 3.65 (4H, 2q), 3.74 (1H, m), 4.08 (2H, m),
7.83 (4H, s). MS (APCI, pos.) 380.2 (M OEt).
1.42 (3H, d), 4.46 (1H, d, OH), 4.93 (1H, m), 7.52 (1H, dd),
7.62 (1H, d), 7.72 (1H, d), 7.78 (1H, s).
N-(1-(R)-(3-Cyanophenyl)ethyl)phthalimide. At 0ꢁC, di-
isopropyl azodicarboxylate (4.2 mL) was added dropwise
to a solution of 5 (2.17 g, 14.7 mmol), triphenylphosphine
(5.60 g) and phthalimide (3.13 g) in THF (100 mL) and the
mixture was warmed to room temperature slowly and stir-
red at that temperature overnight.15±17 Half-saturated
NH4Cl was added and the product was extracted into i-
PrOAc, washed with 2 N HCl and brine and dried over
Na2SO4. Puri®cation by ¯ash chromatography on silica
with EtOAc:toluene 1:19 and trituration of the solid in
ether:hexane 1:1 (50 mL) a€orded 2.83 g (70% yield) of a
white solid. 1H NMR (acetone-d6) d 1.93 (3H, d), 5.60 (1H,
q), 7.58 (1H, dd), 7.68 (1H, d), 7.80 (1H, d), 7.83(4H, s), 7.90
(1H, s).
N-(5-(Ethoxyphosphoryl)pentyl)phthalimide 3. Trimethyl-
chlorosilane (6.6 mL) was added slowly to a solution of 2
(6.79 g, 16.0 mmol) in CH2Cl2:MeOH 9:1 (100mL) at 0ꢁC
and the mixture was stirred at room temperature over-
night. After addition of half saturated NH4Cl, the product
was extracted into i-PrOAc, dried over Na2SO4 and pur-
i®ed by ¯ash chromatography on silica with acet-
one:toluene:AcOH 50:50:1 to give4.40g (90%yield) of 3 as
a yellow oil. 1H NMR (acetone-d6) d 1.28 (3H, t), 1.47 (2H,
m), 1.59 (2H, m), 1.71 (4H, m), 3.65 (2H, t), 3.97±4.09 (2H,
m), 6.98 (1H, d, J=526Hz, PH), 7.85 (4H, s). MS (APCI,
pos.) 310.9, 309.9 (M+1), 281.9, 264.0 (M OEt).
(R)-1-(3-Cyanophenyl)ethylamine 6. N-(1-(R)-(3-Cyano-
phenyl)ethyl)phthalimide (2.83 g, 10.24 mmol) and hydra-
zine hydrate (1.03 g, 2 equiv) were heated to re¯ux in
ethanol (50 mL) for 1.6 h. 2 M Na2CO3 and i-PrOAc were
added and the solid dissolved by heating. The products
were extracted into i-PrOAc, dried over Na2SO4 and con-
centrated. The solid was triturated with ether:hexane 1:1
and ®ltered. The ®ltrate was concentrated and distilled on
a Kugelrohr apparatus under 15mm Hg to give 1.41g
25
d
1
(94% yield) of 6. ‰ꢀŠ 28.0 (c 1.3, EtOH). H NMR
N-(5-((3-Chloro-2-(R)-hydroxypropyl)ethoxyphosphoryl)-
pentyl)phthalimide 4. A solution of 3 (4.92 g, 15.9 mmol)
in ether (50 mL) was cooled to 0ꢁC under argon. Et3N
(5.0 mL, 2.3 equiv) was added, followed by TMSCl
(4.0 mL, 2 equiv) dropwise and the mixture was stirred at
room temperature for 3.5 h. THF (30 mL) was added and
the triethylamine hydrochloride was removed by ®ltration
under argon. The oxygen sensitive phosphine intermediate
was concentrated to dryness under high vacuum and then
fused ZnCl2 (990mg, 0.5 equiv) and (R)-( )-epi-
chlorohydrin (2.0 g, 1.4 equiv) were added quickly while
maintaining a positive pressure of argon. After heating to
80ꢁC for 3 h under argon, the reaction mixture was dis-
solved in MeOH:AcOH 35:1 (70 mL) and was stirred at
room temperature for 3 days to cleave the silyl ether. Eva-
poration and ¯ash chromatography of the residue on silica
with MeOH:CH2Cl2 1:19 a€orded 5.03g of 4 (79% yield).
1H NMR (acetone-d6) d 1.27 (3H, t), 1.45 (2H, m), 1.62
(2H, m), 1.70 (2H, m), 1.80 (2H, m), 1.90±2.13 (2H, m),
3.57±3.70 (4H, m), 4.02 (2H, 2q), 4.13±4.25 (1H, m), 4.77
(1H, s, OH), 7.84 (4H, s). MS (APCI, pos.) 401.9 (M+1).
(CDCl3) d 1.37 (3H, d), 4.17 (1H, q), 7.40 (1H, dd), 7.50
(1H, d), 7.59 (1H, d), 7.65 (1H, s).
N-(5-((3-(1-(R)-(3-Cyanophenyl)ethylamino)-2-(S)-hy-
droxypropyl)hydroxyphosphoryl)pentyl)phthalimide 7. A
mixture of 4 (160mg, 0.4 mmol), 6 (64 mL) and diisopro-
pylethylamine (83 mL) in EtOH (500mL) was heated to
80ꢁC for 1 day and 100ꢁC for 2 days in a glass bomb. Satu-
rated NaHCO3 was added and the product was extracted
into i-PrOAc, washed with brine, dried over Na2SO4 and
puri®ed by ¯ash chromatography on silica with
MeOH:CH2Cl2 3:97 and NH4OH:MeOH:CH2Cl2 1:10:75
to give 114 mg (56% yield) of 7. 1H NMR (methanol-d4) d
1.27 (3H, 2t), 1.37 (3H, d), 1.41 (2H, m), 1.60 (2H, m), 1.68
(2H,m), 1.82(2H,m), 1.86±2.05(2H,m), 2.42(1H,m), 2.52
(1H, m), 3.67 (2H, t), 3.83 (1H, q), 3.88±4.05 (3H, m), 7.49
(1H, dd), 7.58 (1H, d), 7.66 (1H, d), 7.73 (1H, s), 7.78 (2H,
m), 7.84 (2H, m). MS (APCI, pos.) 512.2 (M+1).
N-(5-((3-(1-(R)-(3-Carboxyphenyl)ethylamino)-2-(S)-hy-
droxypropyl)hydroxyphosphoryl)pentyl)phthalimide.
A
mixture of 7 (2.24 g, 4.37 mmol), 10 N NaOH (2.6 mL),
ethanol (5 mL) and water (8 mL) was heated to 80ꢁC
overnight (the reaction could be followed by TLC using
MeOH:CH2Cl2 1:1 as eluent). 2 N HCl (13 mL) was then
added at 0ꢁC and the solvents were coevaporated with
toluene. The product was dissolved in MeOH, ®ltered,
concentrated, stripped again with toluene and used as such
(S)-1-(3-Cyanophenyl)ethanol 5.13,14 To a solution of
( )-B-chlorodiisopinocampheylborane (20 g, 2 equiv) in
THF (100 mL) at 25ꢁC was added slowly a solution of 3-
acetylbenzonitrile (5.00g, 34.4mmol) in THF (60mL) and
the mixture was warmed slowly to 4ꢁC over 1 h. Acetal-
dehyde (7.7 mL) was then added and the mixture was stir-
red at room temperature overnight. 10% Diethanolamine
was added and the product was extracted into i-PrOAc,
1
in the next step. H NMR (methanol-d4) d 1.50 (2H, m),
1.58±1.76 (9H, m), 1.85±2.03 (2H, m), 3.00 (2H, m), 3.35

M. Belley et al. / Bioorg. Med. Chem. 7 (1999) 2697±2704
2703
(2H, t), 4.23 (1H, br s), 4.50 (1H, m), 7.42 (1H, d), 7.52 (1H,
dd), 7.58 (2H, m), 7.75 (1H, d), 7.94 (1H, d), 8.07 (1H, d),
8.18 (1H, s). MS (APCI, neg.) 501.2 (M 1), 371.0, 164.3.
[
125I]-3-(1-(R)-(3-((5-(4-Azido-2-hydroxy-5-iodobenzoyl-
amino)pentyl)hydroxyphosphoryl)-2-(S)-hydroxypropyl-
amino)ethyl)benzoic acid [125I]-CGP 71872. To a mixture
of 10 (4.5 nmol) in 0.1 M NH4OAc (37.5 mL) and Na125
I
3-(1-(R)-((3-((5-Aminopentyl)hydroxyphosphoryl)-2-(S)-hy-
droxypropyl)amino)ethyl)benzoic acid, dihydrochloride 8.
N-(5-((3-(1-(R)-(3-Carboxyphenyl)ethylamino)-2-(S)-
hydroxypropyl)hydroxyphosphoryl)pentyl)phthalimide
(4.37 mmol) was heated in concentrated HCl (15 mL) at
100ꢁC for 2 days (the reaction was monitored by TLC
using H2O:MeOH 1:3 as eluent). The mixture was diluted
with H2O (15 mL) and washed with THF:i-PrOAc 1:1
twice to remove the phthalimide. The water was evapo-
rated o€ and the product dissolved in MeOH, ®ltered and
evaporated to give 1.908 g of 8 as a hygroscopic foam
(98% yield for two steps). 1H NMR (methanol-d4) d 1.50
(2H, m), 1.58±1.70 (4H, m), 1.73 (3H, d), 1.80 (2H, m),
1.93±2.08 (2H, m), 2.93 (2H, t), 3.05 (2H, d), 4.24 (1H, m),
4.53 (1H, q), 7.60 (1H, dd), 7.77 (1H, d), 8.09 (1H, d), 8.20
(1H, s). MS (APCI, pos.) 373.4 (M+1).
(5 mCi, 2200 Ci/mmol; 2.3 nmol) in water (100 mL) was
added a solution of chloramine T (9 nmol) in H2O (75 mL)
and the mixture was stirred at room temperature for 10min
before being quenched by 0.1 M Na2SO3 (250 mL), diluted
with MeOH:H2O 1:1 (250mL) and puri®ed quickly by
HPLC (Nova-Pak C18 column 3.9Â150mm; ¯ow rate
1 mL/min, detection 235 nm, solvent 47% MeOH, 53%
NH4OAc, 2 g/L, containing 0.002% mercaptoethanol).
The fractions containing [125I]-CGP 71872 (1.24 mCi; 25%
radiochemical yield) were concentrated to a ®nal volume of
2 mL.
Preparation of membrane fractions
P2 membrane fractions were prepared at 4ꢁC. Tissues
were washed twice with cold PBS, collected by cen-
trifugation at 100Âg for 7 min, and resuspended in
10 mL of bu€er A: 5 mM Tris±HCl, 2 mM EDTA con-
taining (1X) protease inhibitor cocktail Complete2
tablets (Boehringer Mannheim), pH 7.4 at 4ꢁC. Tissues
were disrupted by polytron homogenization, centrifuged
at 100Âg for 7 min to pellet unbroken cells and nuclei,
and the supernatant collected. The resulting pellet was
homogenized a second time in 10 mL of bu€er A, cen-
trifuged as described above and supernatant fractions
saved. The pooled S1 supernatant was centrifuged at
high speed (40,000Âg for 20 min) and the pellet was
washed once with bu€er A, centrifuged (40,000Âg for
20 min) and resuspended in bu€er A to make the P2
membrane fraction, and stored at 80ꢁC. Protein con-
tent was determined using the Bio-Rad Protein Assay
Kit according to manufacturer instructions.
3-(1-(R)-(3-((5-(4-Azido-2-hydroxybenzoylamino)pentyl)-
hydroxyphosphoryl)-2-(S)-hydroxypropylamino)ethyl)ben-
zoic acid 10. A mixture containing 8 (29 mg, 65mmol), N-
hydroxysuccinimidyl-4-azidosalicylic acid 9 (22 mg, 1.2
equiv) and diisopropylethylamine (45 mL) in MeOH (2 mL)
was stirred at room temperature overnight in the dark. It
was then diluted with CH2Cl2 (2 mL), poured on top of a
¯ash chromatography column and eluted with
MeOH:CH2Cl2:AcOH 50:50:0, then 50:50:1 and 100:0:1 to
give 45mg of a solid containing 50% 10 (yield 64%), along
.
with some N-hydroxysuccinimide, i-Pr2NEt AcOH and
1
solvents. It was used as such in the next step. H NMR
(methanol-d4:D2O) (1.42 (2H, m), 1.52 (4H, m), 1.62 (2H,
m), 1.70 (3H, d), 1.70 (1H, m), 1.82 (1H, m), 2.94±3.04 (2H,
m), 3.38 (2H, t), 4.18(1H, m), 4.46(1H, q), 6.59(1H, s), 6.68
(1H, d), 7.49 (1H, dd), 7.58 (1H, d), 7.78 (1H, d), 7.95 (1H,
d), 7.98 (1H, s). MS (APCI, neg.) 532.7 (M 1), 504.7
(M N2).
Receptor ®lter-binding assays
Competition of [125I]-CGP 71872 binding experiments
were performed with ꢀ7 mg P2 membrane protein and
3-(1-(R)-(3-((5-(4-Azido-2-hydroxy-5-iodobenzoylamino)pen-
tyl)hydroxyphosphoryl)-2 -(S)-hydroxypropylamino)ethyl)-
benzoic acid, acetic acid salt CGP 71872. A solution of 10
(112mmol) in MeOH:H2O 2:1 (3 mL) was added to NaI
(35 mg, 2 equiv) and chloramine T (64 mg, 2 equiv) at 0ꢁC
and the mixture was stirred at that temperature for 3 h.
Sodium sul®te (2 equiv) in water was then added and the
mixture was injected directly onto an HPLC system for
puri®cation (Nova-Pak C18 cartridge 25Â10cm, ¯ow rate
20mL/min, detection 300 nm, solvent 53% MeOH, 47%
NH4OAc, 2 g/L). Four peaks could be detected by HPLC:
the ®rst peak corresponds to unreacted 10, the third to the
diiodination product and the fourth to CGP 71872. The
fractions containing CGP 71872 were concentrated to a
small volume and the product was freeze-dried to give
56mg of a light brown oil containing 84% CGP 71872 as
3
increasing concentrations of cold ligand (10 12±10
M). The concentration of radioligand used in the com-
petition assays was 1 nM (®nal). Each concentration
was examined in duplicate and incubated for 2 h at 22ꢁC
in the dark in a total volume of 250 mL binding bu€er:
50 mM Tris±HCl, 2.5 mM CaCl2 (pH 7.4) with (1X)
protease inhibitor cocktail Complete2 tablets. Bound
ligand was isolated by rapid ®ltration through a Brandel
96-well cell harvester using Whatman GF/B ®lters. Data
were analyzed by non-linear least-squares regression
using the computer-®tting program GraphPad Prism
version 2.01 (San Diego).
Photoanity labeling
1
an acetic acid salt (58% yield) and 16% NH4OAc. H
P2 membranes were resuspended in binding bu€er, and
incubated in the dark with 1 nM ®nal concentration
NMR (methanol-d4:D2O) d 1.42 (2H, m), 1.53 (4H, br s),
1.62 (2H, m), 1.70 (3H, d), 1.72 (1H, m), 1.84 (1H, m), 2.02
(3H, s, AcO), 2.93 (1H, dd), 3.01 (1H, dd), 3.36 (2H, t),
4.19 (1H, m), 4.47 (1H, q), 6.80 (1H, s), 7.55 (1H, dd),
7.68 (1H, d), 8.00 (1H, d), 8.05 (1H, s), 8.19 (1H, s). MS
(APCI, neg.) 658.6 (M 1), 630.5 (M±N2 1), 502.5.
[
125I]-CGP 71872 (2200 Ci/mmol) in a ®nal volume of
1 mL for 2 h at 22ꢁC. The membranes were centrifuged
at 40,000Âg for 10 min and the pellet was washed in ice-
cold binding bu€er, centrifuged at 40,000Âg for 20 min
and resuspended in 1 mL of ice-cold binding bu€er and

2704
M. Belley et al. / Bioorg. Med. Chem. 7 (1999) 2697±2704
exposed on ice 2 inches from 360 nm ultraviolet light for
10 min. Photolabeled membranes were washed, pelleted
by centrifugation and solubilized in sample bu€er
(50 mM Tris±HCl pH 6.5, 10% SDS, 10% glycerol, and
0.003% bromophenol blue with 10% 2-mercaptoetha-
nol). Samples were electrophoresed on precast NOVEX
10% Tris±glycine gels, ®xed, dried and exposed to
Kodak XAR ®lm with an intensifying screen at 70ꢁC.
Huang, L.; Tang, C.; Shen, Q.; Salon, J. A.; Morse, K.; Laz,
T.; Smith, K. E.; Nagarathnam, D.; Noble, S. A.; Branchek,
T. A.; Gerald, C. Nature 1998, 396, 674.
5. White, J. H.; Wise, A.; Main, M. J.; Green, A.; Fraser, N.
J.; Disney, G. H.; Barnes, A. A.; Emson, P.; Foord, S. M.;
Marshall, F. H. Nature 1998, 396, 679.
6. Kuner, R.; Kohr, G.; Grunewald, S.; Eisenhardt, G.; Bach,
A.; Kornau, H.-C. Science 1999, 283, 74.
7. Ng, G. Y. K.; Clark, J.; Coulombe, N.; Sullivan, R.; Ethier,
N.; Hebert, T.; Belley, M.; Kargman, S.; Chateauneuf, A.;
Tsukamoto, N.; MacDonald, T.; Whiting, P.; Mezey, E.;
Johnson, M. P.; Liu, J. Q.; Kolakowski, F. L., Jr.; Evans, J.;
Bonner, T.; O'Neill, G. P. J. Biol. Chem. 1999, 274, 7607.
8. Kerr, D. I. B.; Ong, J. Pharmac. Ther. 1995, 67, 187.
9. Stampf, P.; Frostl, W.; Mickel, S. J.; Zimmermann, K. WO
97/11953 (3 April 1997). For chemistry on closely related
compounds, see refs 10 and 11.
10. Frostl, W.; Mickel, S. J.; Hall, R. G.; von Sprecher, G.;
Strub, D.; Baumann, P. A.; Brugger, F.; Gentsh, C.; Jaekel, J.;
Olpe, H.-R.; Rihs, G.; Vassout, A.; Waldmeier, P. C.; Bittiger,
H. J. Med. Chem. 1995, 38, 3297.
11. Frostl, W.; Mickel, S. J.; von Sprecher, G.; Diel, P. J.;
Hall, R. G.; Maier, L.; Strub, D.; Melillo, V.; Baumann, P. A.;
Bernasconi, R.; Gentsh, C.; Hauser, K.; Jaekel, J.; Karlsson,
G.; Klebs, K.; Maitre, L.; Marescaux, C.; Pozza, M. F.;
Schmutz, M.; Steinmann, M. W.; van Riezen, H.; Vassout, A.;
Mondadori, C.; Olpe, H.-R.; Waldmeier, P. C.; Bittiger, H. J.
Med. Chem. 1995, 38, 3313.
GABAB receptor antibodies
Amino acids 212±227 (DVNSRRDILPDYELKLC)
and 333±346 (CATLHNPTRVKLFEK) within the N-
terminus of the rat GABABR1a were chosen as immu-
nogenic peptides. Identical sequences are shared with
the GABABR1b receptor, but no signi®cant homology
to other G protein-coupled receptors was found after
querying the Genbank database. Anity puri®ed rabbit
GABAB receptor polyclonal antibody 1713.1 (acetyl-
DVNSRRDILPDYELKLC-amide) and 1713.2 (acetyl-
CATLHNPTRVKLFEK-amide) were custom prepared
by Quality Control Biochemicals (Hopkinton, MA).
Brie¯y, immunogenic peptides were incubated with thiol
coupling gel (1 mg of peptide for 1 mL of gel) and non-
speci®c sites on the gel blocked. The peptide-coupled gel
was incubated with serum, and bound antibody eluted
with glycine bu€er and dialyzed.
12. Wadleworth, P. S.; Baylla, E. K. EP 307362 (5 September
1988).
13. Dhar, R. K. Aldrichimica Acta 1994, 27, 43. See also ref 14.
14. Ramachandran, P. V.; Teodorovic, A. V.; Brown, H. C.
Tetrahedron 1993, 49, 1725.
Solubilization and immunoprecipitation of receptors
Membranes were prepared by sonication in bu€er A as
described. The pellet was resuspended and stirred at 4ꢁC
overnight in 2 mL of freshly prepared solubilization bu€er
consisting of 100 mM NaCl, 10mM Tris±HCl, pH 7.4,
2% digitonin, and 5 mM EDTA with (1X) protease inhi-
bitor cocktail Complete2 tablets. The homogenate was
centrifuged at 40,000Âg for 20min and the solubilized
fraction were washed with 10mL cold bu€er C: 100 mM
NaCl, 10mM Tris±HCl, pH 7.4, with protease inhibitors
and concentrated in Centriprep 30 cartridges (Amicon).
Solubilized receptors were immunoprecipitated during
agitation with rabbit preimmune serum or GABAB
receptor antibodies 1713.1 and 1713.2 (1:1000 dilution
each) and agarose ®xed goat anti-primary IgG (1:40 dilu-
tion) (Sigma, MO). The immunoprecipitate was washed
six times with 5 volumes of cold bu€er C for 20min,
solubilized in SDS sample bu€er, sonicated and electro-
phoresed on 10% Tris±glycine gels as described.
15. Mitsunobu, O. Synthesis 1981, 1.
16. Rao, A. V. R.; Gurjar, M. K.; Kaiwar, V. Tetrahedron:
Asymmetry 1992, 3, 859.
17. Iida, H.; Yamazaki, N.; Kibayashi, C. Tetrahedron Lett.
1985, 26, 3255.
18. Ng, G. Y. K.; O'Dowd, B. F.; Chung, H. T.; Lee, S. P.;
Brann, M. R.; Seeman, P.; George, S. R. Biochem. Biophys.
Res. Commun. 1996, 227, 200.
19. Romano, C.; Yang, W.; O'Malley, K. L. J. Biol. Chem.
1996, 271, 28612.
20. Malcangio, M.; Bowery, N. G. Clinical Neurochemistry
1995, 18, 285.
21. Billinton, A.; Upton, N.; Bowery, N. G. Br. J. Pharmacol.
1999, 126, 1387.
22. Fritschy, J. M.; Meskenaite, V.; Weinmann, O.; Honer,
M.; Benke, D.; Mohler, H. Eur. J. Neurosci. 1999, 11, 143.
23. Erdo, S. L. TIPS 1985, 6, 205.
24. Erdo, S. L.; Wol€, J. R. J. Neurochem. 1990, 54, 363.
25. Tsai, L. H.; Taniyama, K.; Tanaka, C. Am. J. Physiol.
1987, 253, G601.
26. Nakajima, K.; Tooyama, I.; Kuriyama, K.; Kimura, H.
Neurochem. Res. 1996, 21, 211.
References
27. Harty, R. F.; Franklin, P. A. Gastroenterology 1986, 91, 1221.
28. Koop, H.; Arnold, R. Horm. Metab. Res. 1986, 18, 94.
29. Ritta, M. N.; Calandra, R. S. GABA and Endocrine Func-
tion; Racagni; G., Donoso, A. O., Eds.; Raven Press: New
York, 1986; pp 291±297.
1. Bowery, N. G.; Doble, A.; Hill, D. R.; Hudson, A. L.;
Shaw, J. S.; Turnbull, M. J.; Warrington, R. Eur. J. Pharma-
col. 1981, 71, 53.
2. Kaupmann, K.; Huggle, K.; Heid, J.; Flor, P. J.; Bischo€,
S.; Mickel, S. J.; McMaster, G.; Angst, C.; Bittiger, H.;
Froestl, W.; Bettler, B. Nature 1997, 386, 239.
3. Kaupmann, K.; Malitschek, B.; Schuler, V.; Heid, J.; Froestl,
W.; Beck, P.; Mosbacher, J.; Bischo€, S.; Kulik, A.; Shigemoto,
R.; Karschin, A.; Bettler, B. Nature 1998, 396, 683.
30. Rey-Roldan, E.; Lux-Lantos, V.; Gonzalez-Iglesias, A.;
Becu-Villalobos, D.; Libertun, C. Life Sciences 1996, 58, 1059.
31. Amenta, F.; Cavollotti, C.; Ferrante, F.; Erdo, S. L.
Pharmacology 1988, 36, 390.
32. Erdo, S. L. Eur. J. Pharmacol. 1990, 184, 405.
33. Minuk, G. Y.; Bear, C. E.; Sarjeant, E. J. Am. J. Physiol.
1987, 352, G642.
4. Jones, K. A.; Borowsky, B.; Tamm, J. A.; Craig, D. A.; Dur-
kin, M. M.; Yao, W.-J.; Dai, M.; Johnson, M.; Gunwaldsen, C.;
34. Fitch, S. J. J. Am. Chem. Soc. 1964, 86, 61.