Ligands for expression cloning and isolation of GABAB receptors

Article abstract of DOI:10.1016/S0014-827X(03)00018-1

The scope of the plenary lecture at the occasion of the Xth Meeting on Heterocyclic Structures in Medicinal Chemistry, Palermo 2002, is considerably larger than that of the main lecture at the XVIth International Symposium on Medicinal Chemistry, Bologna 2000, described by Froestl et al. in Farmaco 56 (2001) 101. Additional information is presented, in particular, on the reaction conditions for the 31 step synthesis of the combined affinity chromatography and photoaffinity radioligand [¹²⁵I]CGP84963 and on the recent developments of the molecular biology of GABA_B receptors.

Full text of DOI:10.1016/S0014-827X(03)00018-1

Il Farmaco 58 (2003) 173ꢀ183
/
www.elsevier.com/locate/farmac
Ligands for expression cloning and isolation of GABA receptors
B
Wolfgang Froestl *, Bernhard Bettler, Helmut Bittiger, Jakob Heid,
Klemens Kaupmann, Stuart J. Mickel, Dietrich Strub
Nervous System Research, Novartis Pharma AG, WKL-136.5.25, CH-4002 Basel, Switzerland
Accepted 4 July 2002
Abstract
The scope of the plenary lecture at the occasion of the Xth Meeting on Heterocyclic Structures in Medicinal Chemistry, Palermo
002, is considerably larger than that of the main lecture at the XVIth International Symposium on Medicinal Chemistry, Bologna
000, described by Froestl et al. in Farmaco 56 (2001) 101. Additional information is presented, in particular, on the reaction
2
2
1
25
conditions for the 31 step synthesis of the combined affinity chromatography and photoaffinity radioligand [ I]CGP84963 and on
the recent developments of the molecular biology of GABAB receptors.
´
2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
#
125
B B
Keywords: GABA receptors 1a to 1g; GABA receptor 2; Interplay between GB1 and GB2; Synthesis of [ I]CGP84963
1
. Historical Background
In 1950 Roberts and Frankel and the group of
bicuculline first introduced by Curtis et al. in 1970 [7].
Hence, the bicuculline insensitive receptor was desig-
nated as GABAB receptor, the bicuculline sensitive as
GABA receptor [8].
Awapara independently found that g-aminobutyric
acid (GABA, Fig. 1) is present in brain tissue [1,2]. In
1
A
In 1987 Barnard et al. at Cambridge succeeded to
clone the GABA receptor, an ion channel coupled
A
966 Krnjevic and Schwartz could show via electro-
physiological studies that GABA acts as an inhibitory
neurotransmitter [3]. GABA is synthesized in the brain
receptor, which on activation modulates fast synaptic
activity via chloride ion influx into the cell [9]. Several
groups of highly competent scientists attempted to
elucidate the molecular structure of the G-protein
via decarboxylation of
glutamic acid decarboxylase (GAD), whereas, given
orally, it is not capable to penetrate the bloodꢀbrain
L-glutamic acid by the enzyme
/
coupled, or metabotropic, GABA receptor. Two
B
barrier. In 1962 the CIBA chemist Heinrich Keberle
synthesized a lipophilic derivative of GABA, b-p-
chlorophenyl-GABA, baclofen, which enters the CNS
to a small extent sufficient to exert valuable muscle
relaxant and analgesic properties [4]. This drug was
marketed, as a racemate, in 1972 and is still widely
prescribed for the treatment of spasticity in patients
suffering from multiple sclerosis or hemi- and tetra-
plegia [5]. In 1980 Norman G. Bowery recognized that
groups tried to isolate the receptor via protein purifica-
tion from bovine (Kuriyama et al. [10]) or pig brain
(Facklam and Bowery [11]). Much work was devoted to
attempts of expression cloning in Xenopus oocytes, by
Barnard in England, by the groups of Tanaka and
Sekiguchi in Japan [12,13], by American (Woodward
and Miledi [14], Lester, Olson) and German researchers
(Seeburg). However, all attempts pursuing either ap-
proach ultimately failed.
the (R)-(ꢁ)-enantiomer of baclofen (Fig. 1) stereoselec-
/
tively interacts with a GABA receptor [6]. This interac-
tion could not be blocked by the GABA antagonist
2. A second class of selective GABA receptor agonists
B
In 1984 chemists at the Central Research Labora-
*
Corresponding author.
E-mail address: wolfgang.froestl@pharma.novartis.com (W.
Froestl).
tories of Cibaꢀ/Geigy in Manchester discovered a novel
class of GABAB receptor ligands. They replaced the
´
0
014-827X/03/$ - see front matter # 2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00018-1

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W. Froestl et al. / Il Farmaco 58 (2003) 173ꢀ183
/
3
6 742 with IC50 values of 2, 27 and 38 mM, respectively,
3
for the inhibition of binding of [ H]CGP27492 to
GABAB receptors on rat cortex membranes; Fig. 2)
[
21]. Compounds with substituents in size between
methyl and ethyl, such as difluoromethyl (CGP47656,
IC ꢂ89 nM), display properties of partial GABAB
/
5
0
receptor agonists [17]. Also the phosphonic acid analo-
gue of baclofen, phaclofen, synthesized simultaneously
in the laboratories of Rolf H. Prager in Adelaide and at
Fig. 1.
Cibaꢀ
/
Geigy in Manchester, is a low affinity GABAB
130 mM;
receptor antagonist (racemic phaclofen: IC ꢂ
/
5
0
carboxylic acid group of all natural aminoacids by the
bioisosteric phosphinic acid moiety [15,16]. None of the
phosphinic acid analogues of a-aminoacids showed any
interesting biological activity. On the contrary, the
phosphinic acid analogue of GABA, CGP27492 (Fig.
Fig. 2) [22].
Four years of optimization work of four chemistry
laboratories were required to significantly improve the
potency of this class of GABA receptor antagonists in
B
order to obtain compounds with single digit nanomolar
affinity. The decisive breakthrough was achieved by
Stuart J. Mickel, when he substituted the amino-group
of the g-aminopropyl-phosphinic acids with branched
benzyl-substituents [23,24]. Most potent compounds are
the 3,4-dichlorobenzyl derivative with an a-methyl
2), showed a very high affinity towards GABAB
receptors exceeding significantly the affinities of both
GABA and baclofen (IC50 values of 2, 25 and 35 nM,
3
respectively, for the inhibition of binding of [ H]baclo-
fen to GABA receptors on cat cerebellum membranes)
B
[
17]. CGP27492 interacted with GABA receptors with
A
substituent in (S)-configuration CGP54626A (IC ꢂ
/
5
0
4
the most potent GABAB receptor agonist. [ H]CGP-
500 times lower affinity. Even today this compound is
3
125
.2 nM; inhibition of binding of [ I]CGP64213 to
2
GABA receptors on rat cortex membranes, Fig. 3) and
3
7492 has replaced [ H]baclofen as radioligand due to
B
2
the meta carboxy-substituted benzyl derivative with an
a-methyl substituent in (R)-configuration CGP62349
its higher potency, its high specific binding and due to
the possibility of carrying out filtration assays [18,19].
Also the methyl homologue CGP35024 (Fig. 2), iden-
tical with SK&F97541, synthesized simultaneously by
Smith Kline & French chemists, is a potent GABAB
(
IC ꢂ0.7 nM; Fig. 3). It is not understood, why the a-
/
5
0
methyl substituents are oriented in opposite stereoche-
mical alignments in the 3,4-dichlorobenzyl and the 3-
carboxybenzyl-substituted ligands for the high affinity
diastereoisomers, respectively.
receptor agonist (IC ꢂ7 nM) [17,20].
/
5
0
Both compounds are also available as tritiated radi-
3
oligands [25,26]. In particular, [ H]CGP62349 with a
3
. Discovery of GABA receptor antagonists
B
specific radioactivity of 85 Ci/mmol is used frequently
1
1
for autoradiographic studies [27]. [ C]CGP62349 was
evaluated as a potential PET ligand at the Ospedale San
Raffaele in Milan in the laboratories of Ferruccio Fazio
[28,29].
In 1987, 25 years after the synthesis of baclofen, it was
found that phosphinic acid analogues with substituents
larger than methyl display properties of low affinity
GABA receptor antagonists (CGP’s 36 216, 35 348 and
B
Fig. 2.
Fig. 3.

W. Froestl et al. / Il Farmaco 58 (2003) 173ꢀ
/183
175
4
. Ligands for expression cloning of GABA receptors
B
A ligand for expression cloning must fulfil three
requirements. We need:
.
an antagonist labeling all affinity states of the
receptor
.
.
with very high affinity to the receptor (IC B
/
5 nM),
which contains a radioisotope with very high specific
5
0
1
25
32
radioactivity as I or P. In 1991 we synthesized
CGP54748A (IC ꢂ5 nM; Fig. 4), which we thought
would fulfill the above mentioned criteria. However,
Fig. 5.
/
5
0
1
25
during the exchange reaction with Na I Amersham
chemists reported complete decomposition of the
product.
We first formulated a radical cleavage reaction for
this decomposition (Fig. 5). However, as CGP57076A
(
IC ꢂ19 nM; Fig. 4), without the 2-hydroxy substi-
/
5
0
tuent, also decomposed under same reaction conditions,
fragmentation according to the mechanism outlined in
Fig. 5 appears unlikely. Fragmentation of an intermedi-
ate benzylic radical seems to be more probable (Fig. 6).
However, no further efforts were made to elucidate the
precise mechanism of this reaction.
Fig. 6.
At this point we abandoned the attempts to prepare
1
25
I radioligands (maximal specific radioactivity: 2000
Ci/mmol) and we turned to other radioligands with less
3
specific radioactivity, such as S (maximal specific
5
3
5
radioactivity: 1300 Ci/mmol). S-labelled methionine
derivatives are commercially available. We prepared
novel phosphinic acid analogues containing a primary
amino-group, to which the radiolabelled aminoacids
were attached via peptide bonds.
Fig. 7.
The o-aminopentyl phosphinic acid CGP57604A (Fig.
) proved to be useful. It shows only moderate affinity
7
after deprotection, to CGP61857 with IC50 values of 31
and 26 nM, respectively (Fig. 8), which is not sufficient
to be useful for expression cloning.
to GABAB receptors (IC ꢂ
/
110 nM). However, after
acylation, high affinity GABAB receptor antagonists
such as CGP’s 80 936 or 63 776 were obtained (IC ꢂ13
50
/
After all these negative results we decided to com-
pletely abandon radioactive ligands and to prepare
fluorescent ligands instead, with the help of which one
may also be capable to visualize transfected GABAB
receptors. We condensed the o-aminopentyl phosphinic
5
0
nM for both; Fig. 7). The condensation of CGP57604A
with Boc-protected methionine led to CGP61302, and
acid CGP57604A (Fig. 7) with fluoresceinꢀ
/
isothiocya-
nate to obtain CGP63626 (IC ꢂ123 nM; Fig. 9), a
/
5
0
compound with unacceptable affinity.
Fig. 4.
Fig. 8.

176
W. Froestl et al. / Il Farmaco 58 (2003) 173ꢀ
/183
Fig. 9.
At this point we were very close to give up. In a last
attempt we returned to fragmentation scheme outlined
time, the determination of the molecular weights of two
GABA receptor splice variants [30,31].
B
in Fig. 6 and tried to differentiate between inter- and
intra-molecular fragmentation processes. If the strong
This was crucial information, as it showed that the
cDNA clone for the larger GABAB receptor splice
variant must have an insert size of more than 4 kb,
whereas the insert sizes of the clones of the commercial
cDNA library were, at best, 3 kb. Therefore, a new
cDNA library of two million clones was prepared from
rat brain of postnatal day 7 and after screening of
another 620 000 clones, the clones encoding GABAB
receptor 1a, a protein of 960 amino acids and of GABAB
receptor 1b, a protein of 844 amino acids, both with the
typical seven transmembrane spanning domains of G-
protein coupled receptors, were identified. These results
were published in Nature in March 1997 [32], more than
1
25
radiation of the
I isotope cleaves the benzylic
carbonꢀhydrogen bond of a neighboring molecule in
/
an inter-molecular process, we cannot influence the
decomposition. But, if the fragmentation occurs due to
1
25
the proximity of the
I isotope to the fragile tertiary
benzylic carbonꢀhydrogen bond in an intra-molecular
/
process, we can place the radioactive iodine isotope as
far away from this bond as possible. This reasoning
1
25
finally led to the synthesis of [ I]CGP64213 (Fig. 10) in
July 1994, a compound with very high affinity and
specific radioactivity, which was stable for one half-life
17 years after the first pharmacological characterization
of GABA receptors by Bowery [6].
1
25
of the I isotope, i.e. 60 days [30].
In late 1994 the molecular biologists Bettler and
Kaupmann began to set up a screening procedure for
B
1
25
[
I]CGP64213 binding at transfected COS cells. They
5. Ligands for the isolation of extracellular domains of
GABA receptors
started with a commercial rat brain cDNA library
containing 1.5 million clones, which were divided into
pools of 2000 cDNA clones each and transfected into
COS cells. Within 9 months they screened a total of
B
In 1999 Malitschek et al. succeeded to express the
soluble N-terminal extracellular domains (ECDs) of
GABAB receptors 1a and 1b in insect cells using
recombinant baculovirus. They showed that the photo-
7
20 000 clones.
By introducing the photolabile azide group we
1
25
1
25
obtained the photoaffinity radioligand [ I]CGP71872
Fig. 11) in July 1995, which on irradiation with a 24 W
lamp of wavelength 365 nm for 3 min bound irreversibly
affinity ligand [ I]CGP71872 binds to both receptor
fragments and this binding can be blocked by prior
exposure of the proteins to the potent GABA receptor
(
B
to native GABA receptors in rat cortex, cerebellum and
B
antagonist CGP54626A indicating that the N-terminal
extracellular domains of both GABA receptors 1a and
1b can fold correctly even when they are dissociated
spinal cord membranes revealing two bands of 130 and
1
B
00 K in SDS gel electrophoresis allowing, for the first
Fig. 10.

W. Froestl et al. / Il Farmaco 58 (2003) 173ꢀ
/183
177
Fig. 11.
from the transmembrane domains [33]. Now we can
and that the binding pocket of biotin in avidin is located
˚
attempt to isolate the soluble N-terminal extracellular
domains of GABAB receptor 1a or 1b, purify these
proteins and crystallize them. The X-ray structure will,
finally, provide precise data on the three dimensional
at a depth of 9 A. One example for such a ligand is
CGP73415 (IC ꢂ33 nM; Fig. 12), with 22 bonds
between the GABA receptor binding part and biotin,
/
50
B
3
5
for which experiments using [ S]streptavidin proved
that the ligand interacted simultaneously with native
GABAB receptors from rat cerebral membranes and
streptavidin [34].
environment of the binding site of GABA receptor 1.
B
A very efficient method of protein purification is
affinity chromatography using ligands to which biotin is
attached with its extremely high affinity to avidin (K ꢂ
/
CGP73415 may retain the ECD of GABA receptor 1
B
D
ꢁ
15
1
0
between the GABA receptor binding part of the ligand
M). We estimated that we would need a spacer
on avidin columns. Desorption of the receptor fragment
after purification may be achieved by competition with
potent antagonists, such as CGP54626A (Fig. 3).
But, due to the equilibrium between association and
dissociation of the ligand to the receptor binding site we
B
˚
and biotin of a length of approximately 24 A. We
calculated that the binding region of a ‘classical’ G-
˚
protein coupled receptor is located at a depth of 15 A
Fig. 12.
Fig. 13.

178
W. Froestl et al. / Il Farmaco 58 (2003) 173ꢀ183
/
1
25
will obtain, in any case, a mixture of three species, the
GABA receptor fragment with and without the ligand
moiety of the photoaffinity radioligand [ I]CGP71872
(Fig. 11).
B
plus unbound ligand. We thought that we may have a
higher chance to induce crystallization in a solution of a
single species than from a mixture of three components.
We designed a ligand combining in one molecule a
photoaffinity and an affinity chromatography moiety.
The most straightforward approach is to attach a spacer
plus biotin as fifth substituent on the azidosalicylic acid
Theoretically we can envisage four chemically feasible
permutations of the penta-substituted iodo azido sal-
icylic acid as the OH-group directs the iodine substituent
into para (A and B) or ortho positions (C and D),
respectively (Fig. 13). Reactive groups at the receptor
fragment bind to the nitrene intermediate generated by
the photoreaction from the azide. In structures A, B or
Fig. 14.
Scheme 1. Reagents and conditions: (1) NaH, THF, r.t., 3 h; 5-bromovaleronitril, r.t., 16 h; (2) Raney nickel, 4% NH
ethoxycarbonyl-phtalimid, Na CO , H O, CH Cl , r.t., 5 h; (4) Me SiCl, EtOH, CH Cl (1:9), r.t., 17 h; (5) Me SiCl, Et
epichlorohydrin, 10 mol.% ZnCl , THF, 80 8C, 17 h; HOAc, MeOH, r.t., 17 h; (6) i-Pr EtN, EtOH, 80 8C, 7 days; (7) LiOH, EtOH, H
00 8C, 17 h; MeOH, H PO ; (8) conc. HCl, 100 8C, 17 h.
3
in EtOH, 45 8C, 16 h; (3) N-
N, THF, r.t., 17 h; (R)-
O (1:1),
2
3
2
2
2
3
2
2
3
3
2
2
2
1
3
4

W. Froestl et al. / Il Farmaco 58 (2003) 173ꢀ
/183
179
D both ortho positions adjacent to the azido group are
substituted, only in structure C one ortho position is free
causing less steric hindrance for the reaction with the
protein than structures A, B, or D.
from the avidin columns. A viable solution to this
problem is the use of 2-iminoÃbiotin, whose affinity to
avidin is pH-dependent. Protonated 2-iminiumÃbiotin
binds to avidin with eight orders of magnitude weaker
/
/
ꢁ
3
However, by attaching the ligand of structure C
irreversibly to the GABA receptor fragment we have
affinity (K ꢂ
/
10
M) than 2-iminoÃ
/
biotin (K ꢂ
/
D
D
ꢁ
11
B
3.5ꢃ/10
M). Recovery of the proteinꢀ/ligand com-
to face another serious problem, i.e. to detach the
plex from avidin columns can be achieved by washing
with ammonium acetate solutions at pH 4 [35].
receptorꢀ
/
ligand complex after the purification step
Scheme 2. Reagents and conditions: (1) NaNO
tolyl) , CH CN, Et N, 70 8C, 96 h; (3) MeOH, H
6) 100% HNO , 0ꢀ5 8C, 3 h, r.t., 16 h; (7) 1 N NaOH, 80 8C, 4 h; (8) N
bar, 90 min; (10) N-hydroxysuccinimidyl-benzylcarbonate, NaHCO , acetone, H
2
, H
2
SO
4
, 0ꢀ
/
5 8C, I
2
, NaI, H
, Pd/C, EtOAc, r.t., 1 bar, 30 min; (5) TosCl, K
O, EtOH, reflux, 16 h, 0.1 N HCl; (9) H
O, r.t., 16 h; (11) N-hydroxysuccinimide, DCC, dioxane, r.t., 16 h.
2
O, 0ꢀ
/
5 8C, 6 h, r.t., 18 h; (2) N-3-butenyl-phtalimide, Pd(OAc)
CO , acetone, reflux, 16 h;
, PtO , MeOH, HCl, r.t., 1
2
, P(o-
3
3
3
2
SO
4
, reflux, 16 h; (4) H
2
2
3
(
3
/
2
H
4
×
/
H
2
2
2
3
2
2 2
Scheme 3. Reagents and conditions: (1) i-Pr EtN, DMF, r.t., 94 h; (2) H , Pd/C, MeOH, HCl, r.t., 1 bar, 2 h.

180
W. Froestl et al. / Il Farmaco 58 (2003) 173ꢀ183
/
125
It required 31 steps to synthesize [ I]CGP84963 (Fig.
4), for which a dissociation constant KD of 2 nM was
their cytoplasmic C-termini. This was the first example of
a heterodimeric G-protein coupled receptor. Jean-Phi-
lippe Pin and Roberto Pellicciari showed independently
1
calculated from best-fit analyses of saturation curves.
Thirty percent of the total radioactivity was bound
irreversibly to native GABAB receptors of rat cortex
after the photoreaction [34].
that the ligands bind via a Venus Flytrap module [40ꢀ43].
/
GB1 binds GABA and all known GABAB receptor
ligands. GB2 does not bind GABA or another endo-
genous ligand. According to current knowledge GB2 has
three tasks: first to move GB1 to the cell surface by
The synthesis of combined photoaffinity and affinity
1
25
chromatography radioligand [ I]CGP84963 is outlined
in the following five reaction Schemes starting from
cheap inorganic hypophosphorus acid, which is con-
densed with triethylorthoacetate and boron trifluoride
etherate to give ethyl(1,1-diethoxyethyl)-phosphinate (1)
masking an intracellular retention signal [44ꢀ46], second,
/
to increase the affinity of agonist binding to GB1 [36,47]
and third, to effect G-protein activation via a distinct
hydrophobic residue in its third intracellular loop [47ꢀ
/
(
Scheme 1) in 60% yield [17]. Scheme 1 shows the eight
50]. This makes the GABA receptor the only GPCR, in
B
step synthesis of the o-aminopentyl phosphinic acid
which the agonist binding site and the G-protein coupling
domain are located in two different subunits. The
question is, of course, how the signal is translocated
from one subunit to the other.
CGP57604A, which contains the GABA receptor
B
binding part. Scheme 2 shows the 11 step synthesis of
the photoaffinity moiety of the ligand, Scheme 3 the
condensation of the GABA receptor binding part and
B
This is, however, not the only unresolved question in
the photoaffinity part (two steps), Scheme 4 the synth-
esis of the spacer consisting of three molecules of GABA
GABA receptor research. The most important one*
/
B
from the viewpoint of drug research*is the issue of
/
(
[
five steps). Scheme 5 illustrates the last five steps to
25
GABA receptor subtypes, which were proposed after
B
1
I]CGP84963.
Experiments are currently underway in the labora-
numerous pharmacological [51,52] and electrophysiolo-
gical studies [53,54]. One of the major functions of
GABA receptors is the modulation of the release of a
tories of Professor Markus Gr u¨ tter at the University of
Zurich to purify the extracellular domain of GABAB
receptor 1a expressed in large quantities in Escherichia
coli using this ligand with the aim of obtaining a
B
multitude of neurotransmitters, such as glutamate,
dopamine, noradrenaline, serotonin, acetylcholine sub-
stance P, cholecystokinin and somatostatin via presy-
crystalline receptor fragmentꢀ
/
ligand complex suitable
naptic GABA heteroreceptors, and of GABA via the
B
for X-ray structure determination.
proposed presynaptic autoreceptor. It would be highly
attractive to selectively target each individual subtype.
But, it appears that the GB1 plus GB2 heterodimer is
6
. Update on most recent developments and outlook
the only GABA
out of the pan GB1a & 1b subunit is sufficient to
suppress all GABA mediated effects in vivo [55,56].
We do not know about the physiological importance
of different combinations of GABA receptor subunits
to form distinct heterodimers. So far at least seven
isoforms of GB1, 1aꢀ1g (1c [57], 1c and 1d [58], 1e [59],
1f [60], 1g [61]) have been identified. Three isoforms of
B
receptor in the brain, since the knock-
In late 1998 the GABA receptor 2, a protein of 941
B
B
aminoacids, was cloned simultaneously by four groups,
at Novartis, Glaxo, Synaptic Pharmaceuticals and BASF
[36ꢀ39]. In order to get functionally active receptors
GABA receptors 1 and 2 have to form heterodimers via
B
/
/
B
a coiled-coil interaction, a stretch of 23 aminoacids at
3 2 2 3 2 2
Scheme 4. Reagents and conditions: (1) DCC, Et N, CH Cl , r.t., 16 h; (2) 2 N NaOH, MeOH, r.t., 16 h; (3) DCC, Et N, CH Cl , r.t., 16 h; (4) 2 N
NaOH, MeOH, r.t., 16 h; (5) N-hydroxy-succinimide, DCC, dioxane, r.t., 65 h.

Scheme 5. Reagents and conditions: (1) i-Pr
2
EtN, DMF, r.t., 65 h; (2) H
2
, Pd/C, MeOH, HCl, r.t., 1 bar, 3.5 h; (3) N-hydroxysuccinimido-2-iminobiotin hydrobromide (Pierce), i-Pr
2
EtN, DMF, r.t.,
125
6
5 h; (4) NaNO , 80% HOAc, ꢁ15 8C, 10 min, NaN
2
/
3
, H O, ꢁ15 8C to 0ꢀ
2
/
/5 8C, 72 h; (5) Na I, phosphate buffer, pH 7.4, H
2
O
2
, catalytic amounts lactoperoxidase, 0ꢀ/5 8C, 6 h.

182
W. Froestl et al. / Il Farmaco 58 (2003) 173ꢀ183
/
b- and g-aminoalkylphosphonous acids, Tetrahedron 45 (1989)
787ꢀ3808.
17] W. Froestl, S.J. Mickel, R.G. Hall, G. von Sprecher, D. Strub,
P.A. Baumann, F. Brugger, C. Gentsch, J. Jaekel, H.-R. Olpe, G.
Rihs, A. Vassout, P.C. Waldmeier, H. Bittiger, Phosphinic acid
GB2, 2aꢀ2c have been described [62], but their existence
was recently questioned [63].
/
3
/
[
Likewise, we do not know about the differences of
GABAB receptor subunit expression in normal and in
pathological tissues in diseases such as spasticity,
epilepsy, chronic pain, esophageal reflux, and the
various forms of drug abuse, for all of which GABAB
receptor ligands may provide valuable medications.
analogues of GABA. 1. New potent and selective GABA
agonists, J. Med. Chem. 38 (1995) 3297ꢀ3312.
18] H. Bittiger, N. Reymann, R. Hall, P. Kane, CGP27492, a new
B
/
[
[
B
potent and selective radioligand for GABA receptors, Eur. J.
Neurosci. (1988) Abstr. 16.10.
19] R.G. Hall, P.D. Kane, H. Bittiger, W. Froestl, Phosphinic acid
analogues of g-aminobutyric acid (GABA). Synthesis of a new
radioligand, J. Labelled Compounds Radiopharm. 36 (1995)
1
[20] W. Howson, J. Mistry, M. Broekman, J.M. Hills, Biological
29ꢀ135.
/
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selective GABA
B
agonist with CNS activity, Bioorg. Med. Chem.
518.
[
[
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