Noncompetitive antagonist-binding sites of rat and housefly γ-aminobutyric acid receptors display different enantiospecificities for tert-butyl(isopropyl)bicyclophosphorothionate

Article abstract of DOI:10.1016/S0968-0896(00)00162-0

The enantiomers of 4-tert-butyl-3-isopropyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfide (TBIPPS) were prepared in nine steps from diethyl tert-butylmalonate, and their abilities to compete with [³H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane (EBOB), a noncompetitive antagonist of ionotropic γ-aminobutyric acid (GABA) receptors, at their binding site were investigated using rat brain and housefly head membranes. The (S)-(-)-isomer of TBIPPS (IC₅₀=398 nM) was more potent than was the (R)-(+)-isomer of TBIPPS (IC₅₀=1220 nM) in rat receptors, while the potencies of (S)-TBIPPS (IC₅₀=104 nM) and (R)-TBIPPS (IC₅₀=94.4 nM) in housefly receptors were almost the same. The different enantiospecificities of rat and housefly receptors indicate that the three-dimensional structure of the binding site might be different between these receptors. In a region of the rat binding site there might be a steric bulk that interacts less favorably with (R)-TBIPPS than with (S)-TBIPPS, while in the corresponding region of the housefly binding site there might not be such a steric bulk that leads to specificity for these compounds. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00162-0

Bioorganic & Medicinal Chemistry 8 (2000) 2337±2341
Noncompetitive Antagonist-Binding Sites of Rat and House¯y
ꢀ-Aminobutyric Acid Receptors Display Di€erent
Enantiospeci®cities for
tert-Butyl(isopropyl)bicyclophosphorothionate
Xiu-Lian Ju and Yoshihisa Ozoe*
Department of Life Science and Biotechnology, Shimane University, Matsue, Shimane 690-8504, Japan
Received 8 May 2000; accepted 8 June 2000
AbstractÐThe enantiomers of 4-tert-butyl-3-isopropyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane 1-sul®de (TBIPPS) were pre-
pared in nine steps from diethyl tert-butylmalonate, and their abilities to compete with [3H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-
trioxabicyclo[2.2.2]octane (EBOB), a noncompetitive antagonist of ionotropic g-aminobutyric acid (GABA) receptors, at their
binding site were investigated using rat brain and house¯y head membranes. The (S)-( )-isomer of TBIPPS (IC50=398 nM) was
more potent than was the (R)-(+)-isomer of TBIPPS (IC50=1220 nM) in rat receptors, while the potencies of (S)-TBIPPS
(IC50=104 nM) and (R)-TBIPPS (IC50=94.4 nM) in house¯y receptors were almost the same. The di€erent enantiospeci®cities of
rat and house¯y receptors indicate that the three-dimensional structure of the binding site might be di€erent between these receptors.
In a region of the rat binding site there might be a steric bulk that interacts less favorably with (R)-TBIPPS than with (S)-TBIPPS,
while in the corresponding region of the house¯y binding site there might not be such a steric bulk that leads to speci®city for these
compounds. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
for house¯y receptors, as determined by binding assays
using the GABA antagonist [3H]1-(4-ethynylphenyl)-4-
rat
Electrophysiological and biochemical studies have
demonstrated that bicyclophosphorothionates (2,6,7-
trioxa-1-phosphabicyclo[2.2.2]octane 1-sul®des) act as
noncompetitive antagonists of ionotropic g-aminobutyric
acid (GABA) receptors in both invertebrate and verte-
brate nervous systems.1 7 Noncompetitive antagonists
inhibit the inhibitory action of GABA during synaptic
neurotransmission by binding to an allosteric site within
GABA receptor-operated chloride channels to exert toxi-
city in animals.8 One of the antagonists selective for insect
GABA receptors, ®pronil, is used as an insecticide.9
n-propyl-2,6,7-trioxabicyclo[2.2.2]octane (EBOB) (IC /
50
¯y
50
IC =0.0384). The addition of an isopropyl group into
the 3-position of TBPS led to tert-butyl(isopropyl)bicyclo-
phosphorothionate (TBIPPS), or 4-tert-butyl-3-isopropyl-
2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane 1-sul®de, with
rat
50
¯y
50
an IC /IC ratio of 13.8. The selectivity value for house-
¯y receptors was thus increased 359-fold by the introduc-
tion of an isopropyl group, compared with the selectivity
value of TBPS. These ®ndings indicate that the structure
of the binding site might be di€erent between rats and
house¯ies; that is, there might be an ample space that
accommodates the 3-isopropyl group of TBIPPS in the
house¯y binding site but not in the rat binding site.
We recently reported that the introduction of an isopropyl
group into the 3-position of bicyclophosphorothionates
leads to noncompetitive antagonists with increased an-
ity and selectivity for house¯y (Musca domestica L.)
GABA receptors versus rat GABA receptors.10 We noted
that tert-butylbicyclophosphorothionate (TBPS), which
has a 4-tert-butyl group but no substituent at the 3-
position, was much more selective for rat receptors than
As TBIPPS is a chiral compound, the separation of the
enantiomers may provide more information regarding
species di€erences in the structure of the 3-substituent-
interacting area that allows the receptor selectivity. To
gain a better understanding of the molecular interaction
of TBIPPS with the rat and house¯y binding sites, we
prepared the enantiomers of TBIPPS, investigated their
abilities to compete with [3H]EBOB at the binding site,
and report herein the results.
*Corresponding author. Tel.: +81-852-32-6573; fax: +81-852-32-6092;
e-mail: ozoe-y@life.shimane-u.ac.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00162-0

2338
X.-L. Ju, Y. Ozoe / Bioorg. Med. Chem. 8 (2000) 2337±2341
Results and Discussion
observed at a higher ®eld position than that of the (S,S)-
diastereomer, because the former is more shielded by
the p cloud of the phenyl group of the MTPA moiety
(Fig. 1). In contrast, the proton signals of the isopropyl
group of the (R,S)-diastereomer were centered at a
lower ®eld position than those of the (S,S)-diastereo-
mer, because the latter is more shielded by the p cloud
of the phenyl group of the MTPA moiety. (R,S)- and
(S,S)-3 were then converted into triols (R)-(+)- and (S)-
( )-4, respectively, using diisobutylaluminum hydride
(DIBAL) as a reducing agent. The target compounds
((R)-(+)- and (S)-( )-TBIPPS) were ®nally obtained by
the reaction of (R)-(+)- and (S)-( )-4 with thiophos-
phoryl chloride, respectively. The overall yields of (R)-
and (S)-TBIPPS in nine steps from diethyl tert-butyl-
malonate were 1.4 and 1.3%, respectively.
Chemistry
The enantiomers of TBIPPS were prepared as outlined in
Scheme 1. Racemic alcohol 1 was synthesized as pre-
viously described.10 (S)-( )-2-Methoxy-2-tri¯uoromethyl-
phenylacetic acid (MTPA) reacted via its acid chloride
with 1 to give a pair of diastereomeric MTPA esters 2.
Isopropylidene ketal 2 underwent hydrolysis in acetic
acid to a€ord a diastereomeric mixture of diols (R,S)-
and (S,S)-3, which were separated using a chiral HPLC
column (Sumichiral OA-2000) with hexane:2-propanol
(49:1). The absolute stereochemistry of the diaster-
eomers was determined based on their NMR chemical
shifts using Mosher's method.11,12 The proton signal of
the tert-butyl group of the (R,S)-diastereomer was
Scheme 1. Preparation of enantiomers of TBIPPS.
Figure 1. NMR spectra of (A) diastereomeric 3, (B) (R,S)-3, and (C) (S,S)-3.

X.-L. Ju, Y. Ozoe / Bioorg. Med. Chem. 8 (2000) 2337±2341
2339
Receptor binding
receptors, although the binding site of noncompetitive
antagonists is thought to be located in a highly
homologous region within GABA receptor-operated
chloride channels.13 17 In a region of the rat binding site
there might be a steric bulk that interacts less favorably
with (R)-TBIPPS than with (S)-TBIPPS, while in the
corresponding region of the house¯y binding site there
might not be such a steric bulk exhibiting speci®city for
these compounds. In particular, the 3-subsitituent-
interacting area in house¯y receptors appears to be
wider than that in rat receptors, because the former can
accommodate both enantiomers. These ®ndings support
the ®ndings in our previous work that there are structural
di€erences in the noncompetitive antagonist-binding site
between insect and mammalian receptors.10,18,19 Further
synthesis of the enantiomers of other bicyclophos-
phorothionate GABA antagonists and the application
of three-dimensional quantitative structure±activity
relationship analysis would help us to establish model-
ing of the binding site and to understand the molecular
topography of the antagonist-binding sites of rat and
house¯y ionotropic GABA receptors.
We examined (R)- and (S)-TBIPPS for their potencies to
inhibit speci®c binding of [3H]EBOB, a noncompetitive
GABA receptor antagonist, to membranes prepared
from rat brains and house¯y heads. (S)-TBIPPS (IC50=
398 nM) was more potent than (R)-TBIPPS (IC50=
1220 nM) in rat brain membranes (Table 1); their con-
centration±inhibition curves are shown in Figure 2. In
contrast, the potencies of (S)-TBIPPS (IC50=104 nM)
and (R)-TBIPPS (IC50=94.4 nM) were almost the same
in house¯y head membranes (Table 1); these concentra-
tion±inhibition curves are shown in Figure 3. The pre-
viously reported IC50 value of racemic TBIPPS was
intermediate between those of (R)- and (S)-TBIPPS in
the case of rat receptors, while the IC50 value of each
enantiomer was not signi®cantly di€erent from that of
racemic TBIPPS in the case of house¯y receptors (Table 1).
The selectivity for house¯y GABA receptors estimated
in terms of ICr5a0t/IC was >3-fold higher in (R)-
TBIPPS than in (S)-TBIPPS.
¯y
50
The di€erent enantiospeci®cities of rat and house¯y
receptors indicate that the three-dimensional structure
of the binding site might be di€erent between these
Experimental
Chemistry
Table 1. Potencies of (R)- and (S)-TBIPPS in inhibiting [3H]EBOB
binding to rat brain and house¯y head membranes
General. Optical rotations were determined with a
1
JASCO DIP-140 digital polarimeter. H NMR spectra
rat
50
¯y
50
Compound
IC50 (nM)
IC /IC
were obtained in CDCl3 at 400 MHz with a JEOL JNM-
A-400 NMR spectrometer, and chemical shifts are
reported in parts per million relative to tetramethylsilane
as an internal standard. Mass spectra were obtained on
a Hitachi M-80B spectrometer. Melting points were
determined with a Yanako PM-500D apparatus and are
uncorrected. Elemental analyses were performed by
Elemental Analysis Center, Faculty of Science, Kyushu
Rat
House¯y
(R)-TBIPPS
(S)-TBIPPS
Racemic TBIPPSb
1220 (990±1500a)
398 (319±497)
706 (565±883)
94.4 (52.8±169)
104 (64.5±166)
51.2 (33.5±78.2)
12.9
3.83
13.8
a95% con®dence interval.
bData from ref 10.
Figure 2. Concentration±inhibition curves of (R)- and (S)-TBIPPS in
inhibiting speci®c [3H]EBOB binding to rat brain membranes.
Figure 3. Concentration±inhibition curves of (R)- and (S)-TBIPPS in
inhibiting speci®c [3H]EBOB binding to house¯y head membranes.

2340
X.-L. Ju, Y. Ozoe / Bioorg. Med. Chem. 8 (2000) 2337±2341
University. Reagents were purchased from Wako Pure
Chemical Industries, Ltd. [3H]EBOB (1406 GBq/mmol)
was purchased from NEN Life Science Products, Inc.
nylacetoxy)-2-methylpropyl]-1,3-propanediol ((R,S)-3)
was obtained as a colorless solid: retention time
22.7 min; mp 75±77 ꢀC; CIMS (isobutane) m/z 421
1
(M+1); H NMR d 0.96 (9H, s, (CH3)3C), 1.03 (6H, t,
(S)-2-Methoxy-2-tri¯uoromethylphenylacetyl chloride
(MTPA-Cl). A mixture of (S)-( )-2-methoxy-2-tri¯uoro-
methylphenylacetic acid (MTPA) (3.0 g, 12.8 mmol),
thionyl chloride (6 mL), and sodium chloride (36 mg) was
re¯uxed for 50h. After excess thionyl chloride was
removed by vacuum evaporation, the residue was distilled
under reduced pressure to give 2.7 g (84%) of MTPA-Cl:
bp 72±74 ꢀC (3 mm Hg) [lit.11 bp 54±56 ꢀC (1 mm Hg)].
J=7.1 Hz, (CH3)2CH), 2.34 (1H, m, (CH3)2CH), 2.47
(2H, br, OH), 3.55 (1H, d, CH2O), 3.57 (3H, s, CH3O),
3.65 (1H, d, CH2O), 3.77 (1H, d, CH2O), 3.91 (1H, d,
CH2O), 5.29 (1H, d, J=1.5 Hz, (CH3)2CHCH), 7.44
(3H, m, Ar), 7.58 (2H, m, Ar). 2-tert-Butyl-2-[(1S)-1-
((2S) - 2 - methoxy - 2 - tri¯uoromethylphenylacetoxy) - 2 -
methylpropyl]-1,3-propanediol ((S,S)-3) was obtained
as a colorless liquid: retention time 25.6 min; CIMS
1
(isobutane) m/z 421 (M+1); H NMR d 0.94 (3H, d,
5-tert-Butyl-5-[1-((2S)-2-methoxy-2-tri¯uoromethylphenyl-
acetoxy)-2-methylpropyl]-2,2-dimethyl-1,3-dioxane (2). 5-
tert-Butyl-5-(1-hydroxy-2-methylpropyl)-2,2-dimethyl-
1,3-dioxane (1) was synthesized as previously reported.10
A mixture of (S)-MTPA-Cl (0.21 g, 0.83 mmol), 5-tert-
butyl-5-(1-hydroxy-2-methylpropyl)-2,2-dimethyl-1,3-
dioxane (0.14 g, 0.57 mmol), 4-dimethylaminopyridine
(140 mg), dry pyridine (1 mL), and dry carbon tetra-
chloride (1 mL) was stirred for 48 h at room temperature.
After the addition of water (10 mL), the solution was
extracted with ethyl acetate (10 mLÂ3). The extract was
washed with saturated brine, dried (Na2SO4), and con-
centrated under reduced pressure. The residue was puri®ed
by chromatography on silica gel with hexane:ethyl acetate
(9:1) to give 0.15 g (58%) of a diastereomeric mixture of
2 as a colorless liquid: CIMS (isobutane) m/z 461
(M+1); 1H NMR d 0.84, 0.88 (2s, (CH3)3C), 0.96, 0.97,
1.08, 1.09 (4d, J=6.8 Hz, (CH3)2CH), 1.31 (s, (CH3)2C),
2.52 (m, (CH3)2CH), 3.41±3.76 ((CH2O)2), 3.49, 3.54
(2CH3O), 5.22, 5.24 (2d, J=2.9, 3.2 Hz, (CH3)2CHCH),
7.41 (m, Ar), 7.58 (m, Ar).
J=6.8Hz, (CH3)2CH), 0.98 (9H, s, (CH3)3C), 1.01 (3H, d,
J=6.8 Hz, (CH3)2CH), 2.33 (1H, m, (CH3)2CH), 2.59
(2H, br, OH), 3.47 (3H, s, CH3O), 3.69 (1H, d,
J=12.2 Hz, CH2O), 3.74 (1H, d, J=12.2 Hz, CH2O), 3.80
(1H, d, J=12.0 Hz, CH2O), 3.92 (1H, d, J=12.0 Hz,
CH2O), 5.38 (1H, d, J=1.7 Hz, (CH3)2CHCH), 7.46 (3H,
m, Ar), 7.60 (2H, m, Ar).
(R)- and (S)-2-tert-Butyl-2-hydroxymethyl-4-methyl-1,3-
pentanediol (4). A 1.5-M solution of DIBAL in toluene
(0.5 mL, 0.75 mmol) was added to a solution of (R,S)-3
(45 mg, 0.11 mmol) in dry ether (2 mL) with stirring at
0 ꢀC under a nitrogen atmosphere. After stirring at
ambient temperature for 2 days, the reaction solution
was added to a 10% sulfuric acid solution (5 mL), and
the reaction solution was extracted with ether (5mLÂ4).
The combined ether extract was dried (Na2SO4) and con-
centrated. The residue was puri®ed by chromatography
on silica gel with hexane:ethyl acetate (1:1) to give 13.3 mg
(59%) of (R)-4 as a colorless solid: mp 49±50 ꢀC; CIMS
1
(isobutane) m/z 205 (M+1); H NMR d 0.98 (9H, s,
(CH3)3C), 1.07 (6H, 2d, J=6.8 Hz, (CH3)2CH), 2.23
(1H, m, (CH3)2CH), 3.02 (3H, br, OH), 3.79 (1H, s),
3.85 (1H, d, J=11.7 Hz), 4.03 (2H, s), 4.13 (1H, d,
J=11.7 Hz); [a]2d0 (c 1.33, ethanol)=+18.1ꢀ. A similar
reaction of (S,S)-3 (41 mg) a€orded 13.2 mg (66%) of
(S)-4: [a]2d0 (c 1.32, ethanol)= 17.2ꢀ. (S)-4 gave very
similar MS and NMR spectra to those of (R)-4.
2-tert-Butyl-2-[1-((2S)-2-methoxy-2-tri¯uoromethylphenyl-
acetoxy)-2-methylpropyl]-1,3-propanediol (3). A mixture
of 2 (0.20 g, 0.43 mmol), acetic acid (2 mL), and water
(1 mL) was heated to 60 ꢀC for 1 h with stirring. After
the mixture was cooled, a saturated sodium hydrogen
carbonate solution (5 mL) was added to it. The solution
was extracted with ether (5 mLÂ4), and the extract was
dried (Na2SO4) and concentrated. The residue was
puri®ed by chromatography on silica gel with hexane:
ethyl acetate (5:1) to give 117 mg (65%) of a diaster-
eomeric mixture of 3 as a colorless liquid: CIMS (iso-
butane) m/z 421 (M+1); 1H NMR d 0.94 (d, J=6.8 Hz,
(CH3)2CH)), 0.96 (s, (CH3)3C), 0.98 (s, (CH3)3C), 1.01
(d, J=6.8 Hz, (CH3)2CH), 1.03 (t, J=7.1 Hz,
(CH3)2CH), 2.34 (m, (CH3)2CH), 2.47 (m, OH), 2.56 (t,
OH), 2.62 (t, OH), 3.47 (m, CH3O), 3.55 (dd, CH2O),
3.57 (m, CH3O), 3.64 (dd, CH2O), 3.71 (d, CH2O), 3.72 (d,
CH2O), 3.78 (dd, CH2O), 3.80 (dd, CH2O), 3.91 (dd,
CH2O), 3.92 (dd, CH2O), 5.29 (dd, J=1.7Hz,
(CH3)2CHCH), 5.38 (dd, J=2.0 Hz, (CH3)2CHCH),
7.45 (m, Ar), 7.59 (m, Ar).
(R)- and (S)-4-tert-Butyl-3-isopropyl-2,6,7-trioxa-1-phos-
phabicyclo[2.2.2]octane 1-sul®de (TBIPPS). (R)-TBIPPS
(4.5 mg, 26%) was prepared from (R)-4 (13.3 mg) and
thiophosphoryl chloride (11 mg) using a previously
reported method:10 [a]d20 (c 1.20, chloroform)=+33.3ꢀ;
HREIMS m/z calcd for C11H21O3PS 264.0949 (M+),
found 264.0952. Anal. calcd for C11H21O3PS: C, 49.98;
H, 8.01. Found: C, 50.10, H, 7.83. (S)-TBIPPS (3.7 mg,
22%) was prepared from (S)-4 (13.2 mg) and thiophos-
phoryl chloride (11 mg) in the same manner: [a]2d0 (c
1.01, chloroform)= 33.6ꢀ; HREIMS m/z calcd for
C11H21O3PS 264.0949 (M+), found 264.0928. Anal.
calcd for C11H21O3PS: C, 49.98; H, 8.01. Found: C,
49.34; H, 7.69. The 1H NMR spectra of these two
enantiomers were the same as that of racemic TBIPPS.10
Separation of the diastereomers of 3. The diastereomers
of 3 were separated using a Sumichiral OA-2000 HPLC
column (Sumika Chemical Analysis Service, Ltd., 5 mm,
8 mm idÂ25 cm); mobile phase, hexane:2-propanol
(49:1); ¯ow rate, 3 mL/min; detector, 254 nm. 2-tert-
Butyl-2-[(1R)-1-((2S)-2-methoxy-2-tri¯uoromethylphe-
Binding assays
Rat brain and house¯y head P2 membranes were prepared
by a previously reported method.10 Experiments of
[3H]EBOB binding to rat brain and house¯y head

X.-L. Ju, Y. Ozoe / Bioorg. Med. Chem. 8 (2000) 2337±2341
2341
membranes were performed in a manner similar to the
methods of Cole and Casida20 and Deng et al.,21 respec-
tively. In brief, a mixture of DMSO (4 mL), [3H]EBOB
(5 nM) in 10 mM phosphate bu€er (pH 7.5) containing
300 mM sodium chloride (0.1 mL), and rat brain mem-
branes (125 mg protein/0.9 mL of the same bu€er) was
used for determination of total binding. The DMSO
was replaced with unlabeled 1.25 mM EBOB in DMSO
(4 mL) for determination of nonspeci®c binding, and
di€erent concentrations of (R)- and (S)-TBIPPS in
DMSO (4 mL) were substituted for DMSO to determine
the inhibition caused by the compounds. These mixtures
were incubated for 90min at 37 ꢀC, followed by Brandel
M-24 harvester ®ltration on Whatman GF/B ®lters and
two rinses with 5 mL of ice-cold binding bu€er. The ®lters
were subjected to counting in toluene±Methylcellosolve-
based scintillation ¯uid with a Beckman LS 6000 SE
scintillation spectrometer. Experiments with house¯y
head membranes (200 mg protein) were performed using
the same procedure except that the incubation time and
temperature were 70 min and 22 ꢀC, respectively. The
percent inhibition values were calculated. Six to eight
concentrations of each test compound were used for
determination of the IC50 values, which were obtained
by the Probit method.
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Acknowledgements
This work was supported in part by a Grant-in-Aid for
Scienti®c Research from the Ministry of Education,
Science, Sports and Culture of Japan.
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References and Notes
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