Selective inhibitors of glial GABA uptake: Synthesis, absolute stereochemistry, and pharmacology of the enantiomers of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO) and analogues

Article abstract of DOI:10.1021/jm9904452

3-Methoxy-4,5,6,7-tetrahydro-1,2-benzisoxazol-4-one (20a), or the corresponding 3-ethoxy analogue (20b), and 3-chloro- 4,5,6,7-tetrahydro-1,2-benzisothiazol-4-one (51) were synthesized by regioselective chromic acid oxidation of the respective bicyclic te

Full text of DOI:10.1021/jm9904452

5402
J . Med. Chem. 1999, 42, 5402-5414
Selective In h ibitor s of Glia l GABA Up ta k e: Syn th esis, Absolu te
Ster eoch em istr y, a n d P h a r m a cology of th e En a n tiom er s of
3-Hyd r oxy-4-a m in o-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole (exo-THP O) a n d
An a logu es
Erik Falch,† J ens Perregaard,‡ Bente Frølund,† Birgitte Søkilde,† Anders Buur,‡ Lene M. Hansen,†
Karla Frydenvang,† Lotte Brehm,† Tina Bolvig,§ Orla M. Larsson,§ Connie Sanchez,‡ Harold S. White,3
Arne Schousboe,§ and Povl Krogsgaard-Larsen*,†
Centre for Drug Design and Transport, Departments of Medicinal Chemistry and Pharmacology, The Royal Danish School of
Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark, Departments of Medicinal Chemistry and Chemical and
Pharmaceutical Research, H. Lundbeck A/ S, 9 Ottiliavej, DK-2500 Valby, Denmark, and Department of Pharmacology and
Toxicology, University of Utah, Salt Lake City, Utah 84112
Received August 30, 1999
3-Methoxy-4,5,6,7-tetrahydro-1,2-benzisoxazol-4-one (20a ), or the corresponding 3-ethoxy
analogue (20b), and 3-chloro-4,5,6,7-tetrahydro-1,2-benzisothiazol-4-one (51) were synthesized
by regioselective chromic acid oxidation of the respective bicyclic tetrahydrobenzenes 19a ,b
and 50, and they were used as key intermediates for the syntheses of the target zwitterionic
3-isoxazolols 8-15 and 3-isothiazolols 16 and 17, respectively. These reaction sequences involved
different reductive processes. Whereas (RS)-4-amino-3-hydroxy-4,5,6,7-tetrahydro-1,2-ben-
zisoxazole (8, exo-THPO) was synthesized via aluminum amalgam reduction of oxime 22a or
22b, compounds 9, 11-13, and 15-17 were obtained via reductive aminations. Compound 10
was synthesized via N-ethylation of the N-Boc-protected primary amine 25. The enantiomers
of 8 were obtained in high enantiomeric purities (ee g 99.1%) via the diastereomeric amides
32 and 33, synthesized from the primary amine 23b and (R)-R-methoxyphenylacetyl chloride
and subsequent separation by preparative HPLC. The enantiomers of 9 were prepared
analogously from the secondary amine 27. On the basis of X-ray crystallographic analyses,
the configuration of oxime 22a was shown to be E and the absolute configurations of (-)-8‚
HCl and (+)-9‚HBr were established to be R. The effects of the target compounds on GABA
uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation and
primary cultures of mouse cortical neurons and glia cells (astrocytes). Whereas the classical
GABA uptake inhibitor, (R)-nipecotic acid (2), nonselectively inhibits neuronal (IC50 ) 12 µM)
and glial (IC50 ) 16 µM) GABA uptake and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (1,
THPO) shows some selectivity for glial (IC50 ) 268 µM) versus neuronal (IC50 ) 530 µM) GABA
uptake, exo-THPO (8) was shown to be more potent as an inhibitor of glial (IC50 ) 200 µM)
rather than neuronal (IC50 ) 900 µM) GABA uptake. This selectivity was more pronounced for
9, which showed IC50 values of 40 and 500 µM as an inhibitor of glial and neuronal GABA
uptake, respectively. These effects of 8 and 9 proved to be enantioselective, (R)-(-)-8 and (R)-
(+)-9 being the active inhibitors of both uptake systems. The selectivity of 9 as a glial GABA
uptake inhibitor was largely lost by replacing the N-methyl group of 9 by an ethyl group,
compound 10 being an almost equipotent inhibitor of glial (IC50 ) 280 µM) and neuronal (IC50
) 400 µM) GABA uptake. The remaining target compounds, 11-17, were very weak or inactive
as inhibitors of both uptake systems. Compounds 9-13 and 15 were shown to be essentially
inactive against isoniazide-induced convulsions in mice after subcutaneous administration. The
isomeric pivaloyloxymethyl derivatives of 9, compounds 43 and 44, were synthesized and tested
as potential prodrugs in the isoniazide animal model. Both 43 (ED50 ) 150 µmol/kg) and 44
(ED50 ) 220 µmol/kg) showed anticonvulsant effects, and this effect of 43 was shown to reside
in the (R)-(+)-enantiomer, 45 (ED50 ) 44 µmol/kg). Compound 9 also showed anticonvulsant
activity when administered intracerebroventricularly (ED50 ) 59 nmol).
In tr od u ction
The complicity of central neurotransmitters in epi-
lepsy has been the subject of extensive research.1-3 The
possible role of the inhibitory neurotransmitter 4-ami-
nobutyric acid (GABA) has in particular been brought
into focus,4,5 but so far, impairment of GABA-mediated
neurotransmission in epilepsy has not been proved.
There is, however, indirect evidence of disturbances in
GABA neurotransmission in epileptic phenomena. Thus,
inhibition of GABA synthesis and administration of
GABA antagonists provoke convulsions more or less
similar to epileptic seizures.6 Drug-induced convulsions
* Corresponding author: Prof. Povl Krogsgaard-Larsen. Phone:
(+45) 35306511. Fax: (+45) 35306040. E-mail: nordly@medchem.dfh.dk.
† Department of Medicinal Chemistry, The Royal Danish School of
Pharmacy.
§ Department of Pharmacology, The Royal Danish School of Phar-
macy.
‡ H. Lundbeck A/S.
3 University of Utah.
10.1021/jm9904452 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/04/1999

Selective Inhibitors of Glial GABA Uptake
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5403
Like GABA, (R)-nipecotic acid (2) interacts with the
neuronal and glial GABA transport systems with almost
equal affinity,15 and 2 has been shown to be a substrate
for the neuronal16,17 as well as glial17,18 transporters.
Whereas guvacine (3) has a cellular pharmacological
profile very similar to that of 2,12,15,19 THPO (1) has been
shown not to be a substrate for the glial GABA trans-
porter.17 Furthermore, 1 has been shown to possess a
2-fold higher affinity for the glial over the neuronal
transporter.15 Although 1 is at least an order of mag-
nitude weaker than 2 as an inhibitor of GABA uptake
in vitro,15 compound 1 is capable of increasing extra-
cellular GABA levels more effectively than 2,20 and 1 is
much more effective than 2 as an anticonvulsant
agent.21
We envisage that the very low degree of conforma-
tional flexibility of 1, at least to some extent, can explain
its selectivity for the glial GABA transporter and its lack
of ability to act as a substrate for the glial trans-
porter22,23 and that these characteristics underlie the
pharmacological profile of 1, considered to be thera-
peutically interesting.22 These considerations prompted
us to synthesize and pharmacologically characterize
(RS)-4-amino-3-hydroxy-4,5,6,7-tetrahydro-1,2-benzisoxa-
zole (8, exo-THPO) as an analogue of 1 with a compa-
rably low degree of conformational flexibility and with
an exocyclic amino group at the ring carbon atom
analogous with that linked to the amino group of 1
(Figure 1). Furthermore, (R)-8, (S)-8, and the analogues
9-17 were synthesized and studied pharmacologically.
F igu r e 1. Structures of GABA, the GABA uptake inhibitors
4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO, 1), (R)-
nipecotic acid (2), guvacine (3), and 5,6,7,8-tetrahydro-4H-
isoxazolo[4,5-c]azepin-3-ol (4), a number of structurally related
compounds, and the new 3-isoxazolols (8-15) and 3-isothia-
zolols (16, 17).
can be counteracted by elevation of brain GABA levels,7
although there is no simple correlation between the
onset of convulsions and brain GABA concentrations.8
Inhibitors of GABA transporters, which play a key
role in the termination of the GABA neurotransmission
process, show anticonvulsant effects in animal models,9
and members of this group of GABAergic compounds
have been clinically useful for the treatment of epi-
lepsy.10 These anticonvulsant and antiepileptic effects
may reflect an enhancement of GABA neurotransmis-
sion processes under conditions where GABA is being
released physiologically.10 Thus, the strategies for phar-
macological interventions into these transport mecha-
nisms must be (1) effective blockade of both neuronal
and glial GABA uptake in order to enhance the inhibi-
tory effect of synaptically released GABA or, preferen-
tially, (2) selective blockade of glial GABA uptake in
order to increase the amount of GABA taken up by the
neuronal carrier with subsequent elevation of the GABA
concentration in nerve terminals and, thus, enhanced
release of GABA.
The identification of 4,5,6,7-tetrahydroisoxazolo[4,5-
c]pyridin-3-ol (1, THPO) as an inhibitor of GABA uptake
more than two decades ago11 prompted the discovery of
the structurally related cyclic amino acids, (R)-nipecotic
acid (2)11 and guvacine (3),12 as GABA uptake inhibitors
(Figure 1). Whereas 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-
c]azepin-3-ol (4, THAO) was shown to be approximately
equipotent with THPO (1) as an inhibitor of GABA
uptake,13 the corresponding seven-membered ring amino
acids, 5 and 6, quite surprisingly turned out to be
essentially inactive as inhibitors of GABA uptake, and
likewise, (RS)-4-aminocyclohepteno[1,2-d]isoxazol-3-ol
(7) does not detectably interact with GABA transport-
ers.14
Resu lts
Ch em istr y. Introduction of oxo groups into the
4-position of compounds 19a ,b (Scheme 1) and 50
(Scheme 5) represents key steps in the sequences for
the synthesis of the target compounds. Chromic acid
oxidation of 19a gave the desired ketone 20a in 55%
yield, whereas the undesired isomer 21a was obtained
in 10% yield. In an attempt to increase the yield of the
desired ketone and the regioselectivity of this oxidation
reaction, 19b, which contains a 3-ethoxy group, was
oxidized under the same condition to give ketone 20b
in a markedly higher yield (71%) and 8% of the isomeric
byproduct 21b. Ketones 20a ,b were converted into the
corresponding oximes 22a ,b, both in high yields, but the
aluminum amalgam reduction of 22b gave 23b‚HBr at
significantly higher yield (70%) than that of 23a ‚HBr
(58%) obtained by aluminum reduction of 22a . Com-
pound 23b was dimethylated under Eschweiler-Clarke
conditions to give 24, whereas synthesis of the N-ethyl
analogue of exo-THPO (8) required Boc protection of 23b
prior to the introduction of the ethyl group.
Compounds 9, 11-13, and 15 (Scheme 2) and 16 and
17 (Scheme 5) were synthesized via reductive amina-
tions using the appropriate amines and sodium cy-
anoborohydride. Cleavage of the ethoxy group and
acetylation of the primary alcohol group of 30 to give
12 were accomplished in one step by treatment with
hydrogen bromide in glacial acetic acid. Pure 11 could
only be obtained after Boc protection of crude 11,
purification of intermediate 31, and subsequent depro-
tection (Scheme 2).
The enantiomers of exo-THPO (8) and 9 were synthe-
sized from amines 23b and 27, respectively, via forma-

5404 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Falch et al.
Sch em e 1a
Sch em e 2a
a
Reagents: (i) R1NHR2, NaCNBH3; (ii) HBr in HOAc; (iii) HBr
aq (48%); (iv) (Boc)2O, NaOH; (v) CF3COOH, (COOH)2.
acetic acid in dichloromethane, and the final products
43 and 44 were isolated as oxalate salts. The O-
pivaloyloxymethyl prodrugs of (R)-(+)-9 and (S)-(-)-9,
compounds 45 and 46, were synthesized following an
identical reaction sequence (Scheme 4), and the final
products 45 and 46 were also isolated as oxalate salts.
The 3-isothiazolol analogues of 8 and 9, compounds
16 and 17, respectively, were synthesized as outlined
in Scheme 5. Treatment of the enamine amide 48 with
hydrogen sulfide and subsequent oxidation of the crude
reaction product with bromine gave 49 in relatively low
yield (33%). Attempted synthesis of a 4-oxo intermediate
from 49 for the syntheses of 16 and 17 following a route
analogous with that outlined in Scheme 1 for the
syntheses of 20a ,b led to extensive decomposition.
Chromic acid oxidation of 3-chloro-4,5,6,7-tetrahydro-
1,2-benzisothiazole (50) did, however, provide 51 but in
low yield (27%), but the isomeric byproduct 3-chloro-
4,5,6,7-tetrahydro-1,2-benzisothiazol-7-one was formed
in 23% yield. Compound 51 was isolated chromato-
graphically and converted into 16 and 17 via ketone 52.
X-r ay Cr ystallogr aph ic An alyses. Perspective draw-
ings24 of the molecular structures of oxime 22a , (R)-
(-)-8‚HCl, and (R)-(+)-9‚HBr with atomic labeling are
depicted in Figure 2. The configuration of 22a was
shown to be E, and according to the procedure of
Flack,25,26 the absolute configurations of (-)-8 and (+)-9
were established to be R. Bond lengths and angles are
in agreement with expected values.27 For all three
compounds, the isoxazole rings are planar, and the six-
membered rings are intermediates between a C2[C5,
C6] half-chair and a Cs[C6] envelope conformation28
(Figure 2). The crystal structure of oxime 22a consists
of dimers connected by two hydrogen bonds between the
oxime moieties of two molecules related by a center of
symmetry. The packing of the dimers is governed by van
a
Reagents: (i) CH2N2 (a ) or EtBr, K2CO3 (b); (ii) Na2Cr2O7,
H2SO4; (iii) NH2OH; (iv) Al, HgCl2; (v) HCOOH, HCOONa, H2CO;
(vi) HBr in HOAc; (vii) (Boc)2O, NaOH; (viii) EtBr, NaH.
tion of the diastereomeric R-methoxyphenylacetamides
32 and 33, and 36 and 37, respectively (Scheme 3).
These pairs of diastereomeric amides were separated
by preparative HPLC. Acid-catalyzed deprotection of 32
and 33, chromatographic purification of the Boc-
protected intermediates, and, finally, removal of the Boc
groups gave the respective enantiomerically pure target
compounds (R)-(-)-8 (ee ) 99.7%) and (S)-(+)-8 (ee )
99.8%). Reductive cleavage of 36 and 37 using lithium
triethylborohydride and subsequent deprotection of the
respective secondary amines 38 and 39 by treatment
with hydrogen bromide in glacial acetic acid gave
enantiomerically pure (R)-(+)-9 (ee ) 99.1%) and (S)-
(-)-9 (ee > 99.4%), respectively (Scheme 3). The abso-
lute stereochemistry of the enantiomers of 8 and 9 was
established by X-ray crystallographic analyses (see the
subsequent section).
Boc-protected 9, compound 40, was used as interme-
diate for the synthesis of the isomeric O- and N-
pivaloyloxymethyl prodrugs 43 and 44, respectively
(Scheme 4). The Boc groups of intermediates 41 and 42
were selectively removed by treatment with trifluoro-

Selective Inhibitors of Glial GABA Uptake
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5405
Sch em e 3a
a
Reagents: (i) PhCH(OMe)COCl, TEA; (ii) HBr aq (48%), (Boc)2O, NaOH; (iii) HCl, Et2O; (iv) LiEt3BH; (v) HBr in HOAc.
der Waals interactions. Hydrogen bond geometries are
listed in Table 1. In the crystal structures of the two
salts (R)-(-)-8‚HCl and (R)-(+)-9‚HBr, hydrogen bonds
are observed from the ammonium and the hydroxy
groups to the halide ions. For (R)-(-)-8‚HCl an ad-
ditional hydrogen bond is observed between the am-
monium group and the nitrogen atom of the isoxazole
ring.
of 8 as well as 9 were shown to reside in the (R)-
enantiomers.
The additional glia selectivity of 9 as compared to 1
and 8 was, however, lost by substitution of an ethyl
group for the N-methyl group of 9, the resulting
compound 10 showing an inhibitor profile very similar
to that of 1 (Table 2). Although compounds 11-15,
which contain N-substituents larger than ethyl or
tertiary amino groups, still showed significant affinity
in the synaptosomal GABA uptake assay, none of these
compounds significantly inhibited the uptake of GABA
into cultured neurons or astrocytes. Similarly, the
3-isothiazolol analogues of 8 and 9, compounds 16 and
17, respectively, showed negligible effects on neuronal
as well as glial GABA uptake.
In Vivo P h a r m a cology. Some of the new compounds
tested in Table 2 were tested for anticonvulsant effects
in mice after subcutaneous administration.33 In agree-
ment with the earlier observation that 1 only to a very
limited extent penetrates into the brain after systemic
administration in animals with a fully developed blood-
brain barrier (BBB),34 none of the selected compounds,
(R)-9 and 10-13, showed significant anticonvulsant
effects in mice treated with isoniazide (ED50 > 300 µmol/
kg).
In Vitr o P h a r m a cology. The compounds were ini-
tially tested for inhibitory effect on GABA uptake using
a crude preparation of synaptosomes obtained from rat
brain.30 Compounds showing significant inhibitory effect
in this assay (IC50 < 300 µM) were subsequently studied
for effects on neuronal and glial GABA uptake using
cultured cortical neurons and astrocytes, respectively,
as test systems.17
All of the new compounds tested were weaker than
the classical monocyclic GABA uptake inhibitors (R)-
nipecotic acid (2)11,31 and guvacine (3),12 which nonse-
lectively inhibit neuronal and glial GABA uptake22
(Table 2). THPO (1), which is a 3-isoxazolol bioisostere
of 2 and 3,11 was the first compound showing significant
selectivity for the glial GABA uptake system,32 although
1 is only about 2-fold more potent at the glial than at
the neuronal GABA uptake system.15 exo-THPO (8), an
isomer of 1 containing an exocyclic amino group (Figure
1), was approximately equipotent with 1 but showed a
slightly higher degree of glia selectivity than 1. This
selectivity was further increased by conversion of the
primary amino group of 8 into a methylamino group to
give compound 9 (Table 2). The GABA uptake affinities
The lack of in vivo anticonvulsant effects of the
zwitterionic compounds (R)-9 and 10-13, of which (R)-9
and, to a lesser extent, 10 showed effects in all three in
vitro GABA uptake systems used (Table 2), can most
likely be attributed to the poor biomembrane perme-
ability characteristics of the compounds as indicated by
the low partition coefficient for compound 9 (Table 3).

5406 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Falch et al.
Sch em e 4a
improved permeability across biomembranes. Both com-
pounds 43 and 44 proved stable in aqueous buffer
solution (t1/2 > 4000 min), whereas in the presence of
human plasma they were readily degraded with half-
lives of 7 and 15 min, respectively (Table 3). The
potential utility of compounds 43 and 44 as prodrugs
was further illustrated by systemic administration to
mice. Here the compounds were approximately equally
effective as anticonvulsants, ED50 values being 150 and
220 µmol/kg for 43 and 44, respectively. Subsequently,
the (R)- and (S)-forms of 43, compounds 45 and 46,
respectively, were synthesized (Scheme 4) and tested
in vivo. These studies disclosed that the anticonvulsant
effects of 43 reside in the (R)-enantiomer 45 (ED50
)
44 µmol/kg), whereas the corresponding (S)-enantiomer
46 did not show significant anticonvulsant effects in this
in vitro system.
To verify that the anticonvulsant activity of com-
pounds 43 and 44 was the result of hydrolytic produc-
tion of 9, it was shown that intracerebroventricular
administration35 of 9 effectively protected mice against
audiogenic seizures (ED50 ) 59 nmol).
Discu ssion
The heterocyclic amino acids (R)-nipecotic acid (2)11,36
and guvacine (3)12 (Figure 1) are inhibitors of GABA
uptake showing approximately equal effects on the glial
and neuronal GABA transporters (Table 2). Both of
these inhibitors have been shown also to be substrates
for GABA transporters,12,16 making analyses of the
pharmacology of these compounds difficult. The 3-isox-
azolol bioisostere of these amino acids, THPO (1),11 on
the other hand, has been shown not to be a substrate
for the glial GABA transporter,37 and although 1 is only
a weak inhibitor of GABA uptake, it shows potent
anticonvulsant effects after intracerebroventricular ad-
ministration in animals.21 These effects of 1 have been
associated with its weak but significant selectivity for
the glial GABA uptake systems and its lack of ability
to act as a substrate for this GABA transporter.20-23
Introduction of bulky and lipophilic groups, such as
4,4-diphenyl-3-butenyl and structurally related groups,
on the amino groups of 1 and 2 as well as 3 has led to
compounds showing markedly more potent effects on
GABA uptake than the parent amino acids, and all of
these analogues show anticonvulsant effects after sys-
temic administration.9,10,22,37 The N-4,4-diphenyl-3-bute-
nyl analogue of nipecotic acid has been shown not to be
a substrate for GABA transporters,38 and it is unlikely
that any of the GABA uptake inhibitors containing this
or any of the structurally related N-substituents are
substrates for GABA transporters.22 The results of
structure-activity studies strongly suggest that it is the
amino acid units of these compounds that are recognized
by the GABA uptake systems,22 and it may be proposed
that the in vitro pharmacological profiles of these amino
acids also determine the pharmacology of the corre-
sponding analogues containing the bulky lipophilic
N-substituents.
a
Reagents: (i) (Boc)2O, NaOH; (ii) (CH3)3COOCH2I, (CH3)3COK;
(iii) CF3COOH.
Sch em e 5a
a
Reagents: (i) PhCH2NH2; (ii) H2S, Br2; (iii) POCl3, H3PO4; (iv)
Na2Cr2O7, H2SO4; (v) MeONa; (vi) NH4OAc, NaCNBH3, HBr in
HOAc; (vii) MeNH2, NaCNBH3, HBr in HOAc.
These considerations and the proposal that selective
inhibitors of glial GABA uptake may have particular
pharmacological and therapeutic interest22 have
prompted us to try to develop cyclic amino acids or
bioisosteres thereof showing a degree of glia selectivity
This prompted us to synthesize (Scheme 4) and test the
O- and N-pivaloyloxymethyl derivatives of racemic 9,
compounds 43 and 44, as potential prodrugs possessing
higher lipophilicity at physiological conditions and hence

Selective Inhibitors of Glial GABA Uptake
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5407
Ta ble 1. Hydrogen Bond Geometries (Å, deg) for the
Compounds (R)-(-)-8‚HCl, (R)-(+)-9‚HBr, and Oxime 22a a
X-H‚‚‚Y
X-H
H‚‚‚Y
X‚‚‚Y
<XHY
Compound 8‚HCl
N1-H1a‚‚‚Cli
N1-H1b‚‚‚Clii
N1-H1c‚‚‚N2i
O2-H2‚‚‚Cl
0.92(4)
2.43(4)
2.34(4)
2.16(3)
2.20(3)
3.208(2)
3.237(2)
2.982(2)
2.943(1)
143(3)
166(3)
162(3)
166(3)
0.92(4)
0.86(3)
0.76(3)
Compound 9.HBr
N1-H1b‚‚‚Br
N1-H1a‚‚‚Briii
O2-H2‚‚‚Briv
0.93(3)
0.87(3)
0.84(3)
2.40(3)
2.42(3)
2.41(3)
3.282(2)
3.283(2)
3.245(2)
158(3)
173(3)
169(3)
Compound 22a
0.94(2) 1.930(2)
O4-H4‚‚‚N1v
2.808(1)
154(2)
a
Estimated standard deviations are given in parentheses.
Symmetry code: (i) -x + 2, y + 0.5, -z + 1; (ii) -x + 2, y - 0.5,
-z + 1; (iii) y - 1, x, 1 - z; (iv) 1 + x - y, 3 - y, 0.667 - z;
(v) 1 - x, 1 - y, 2 - z.
Ta ble 2. Inhibition of GABA Uptake into Synaptosomes and
Cultured Cortex Neurons and Astrocytes (Values ( SEM,
n ) 3-6)
uptake inhibition IC50 (µM)
compd
synaptosomes
neurons
astrocytes
2
3
1
8
5 ( 1
12 ( 3
162 ( 15
181 ( 21
118 ( 10
>300
63 ( 7
39 ( 3
>300
105 ( 9
98 ( 12
125 ( 17
173 ( 20
291 ( 36
201 ( 19
290 ( 41
242 ( 20
12a
16a
29a
32a
530a
883 ( 100
681 ( 87
nd
423 ( 57
510 ( 60
nd
268a
208 ( 38
281 ( 66
nd
28 ( 7
60 ( 12
nd
278 ( 52
>500
>500
>500
>500
>500
>500
>500
(R)-8
(S)-8
9
(R)-9
(S)-9
10
11
12
13
14
15
16
17
391 ( 25
>1000
>1000
>1000
>1000
>1000
>1000
>1000
a
Compounds 1-3 are competitive inhibitors of neuronal and
glial GABA uptake,15 and consequently the IC50 values, corre-
sponding to a GABA concentration of 1 µM, were calculated from
the respective Ki values15 using the equation of Cheng and
Prusoff.29 The Ki values were calculated from apparent Km values
with SD values of less than 15%; nd, not dertermined.
Ta ble 3. Physicochemical Characteristics of Compound 9 and
Its Pivaloyloxymethyl Prodrugs 43 and 44
t1/2 (min)
log D
compd aqueous buffer human plasma n-octanol cyclohexane
9
43
44
stable
4500
16000
stable
15
7
-2.3
0.65
0.05
nda
-0.17
-1.4
a
nd, not determined.
higher than that of 1. This line of research involved the
synthesis of exo-THPO (8), having an amino group at
the same ring carbon atom as in 1, but with an exocyclic
orientation (Figure 1). Compound 8 turned out to be
approximately equipotent with 1 as an inhibitor of
GABA uptake, but with a marginally higher degree of
selectivity for the glial transporter (Table 2). The
conversion of the primary amino group of 8 into a
secondary amino group by N-methylation gave 9, show-
ing a slight increase in potency but a significantly higher
degree of glia selectivity, 9 being about 12 times more
potent as an inhibitor of glial than of neuronal GABA
uptake (Table 2). Substitution of an ethyl group for the
F igu r e 2. Hydrogen-bonding patterns in the crystals of the
oxime 22a (A), (R)-(-)-8‚HCl (B), and (R)-(+)-9‚HBr (C). The
molecules are shown in perspective drawings.24 Displacement
ellipsoids enclose 50% probability. Hydrogen atoms are rep-
resented by spheres of arbitrary size. Hydrogen bonds are
indicated by dashed lines.

5408 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Falch et al.
Deter m in a tion of Ster eoch em ica l P u r ity. The enantio-
meric excess (ee) of (S)-(+)-8 was determined at ambient
temperature on a 150 × 4 mm Crownpak CR(-) column
(Daicel). The column was eluted with 0.4 mL/min of aqueous
perchloric acid (pH 1.0). Determination of the ee of (R)-(-)-8
was performed using the same column and eluent, but at 1.5
°C. The instrumentation used consisted of a J asco 880-PU
pump, a Rheodyne 7125 injector, and a Waters M480 UV
detector set at 200 nm connected to a Hitachi-Merck D-2000
chromato-integrator.
Determination of ee values for (R)-(+)-9 and (S)-(-)-9 was
performed at ambient temperature on a 100 × 4.0 mm
CHIRAL-AGP column (ChromTech, Sweden). The column was
eluted with 0.5 mL/min of 10 mM potassium phosphate buffer,
pH 7.0, using a Waters M510 pump connected to a Waters
U6K injector and a Waters 991 photodiode array detector.
N-methyl group of 9 to give 10 markedly reduced the
effect on glial GABA uptake and thus the glia selectivity
of 10. Further increase in the size of the N-alkyl group
led to analogues showing little if any effect on neuronal
or glial GABA uptake (Table 2).
The effects of 8 as well as 9 on GABA uptake were
shown to reside in the (R)-enantiomers of the com-
pounds (Table 2, Figure 2). Interestingly, it also is the
(R)-form of nipecotic acid that is the active GABA
uptake inhibitor.36
Compound 1 does not easily penetrate the BBB,
although the BBB of young animals is more penetrable
for 1 than the BBB of adult animals.34 Like 1, the
bicyclic 3-isoxazolols listed in Table 2 have predomi-
nantly zwitterionic structures, and neither (R)-9 nor
10-13 showed detectable anticonvulsant effects after
subcutaneous administration. However, as expected,
compound 9 potently protected Frings mice35 against
audiogenic seizures when administered intracerebroven-
tricularly following a published procedure.35 These
observations together with the low lipophilicity of 9 as
described in terms of the distribution coefficient between
n-octanol and aqueous buffer of pH 7.4 (Table 3)
prompted us to synthesize the two isomeric pivaloyl-
oxymethyl derivatives 43 and 44 as potential prodrugs
of 9 (Scheme 4). Both these compounds are significantly
more lipophilic than the parent compound, and both are
quite stable in aqueous buffer but relatively unstable
in the presence of human plasma (Table 3). They show
approximately equipotent anticonvulsant effects in mice
after subcutaneous administration. This effect of 43 was
shown to reside in the (R)-enantiomer, 45, whereas the
corresponding (S)-form, 46, was inactive. Since neither
45 nor 46 per se showed effects on GABA uptake (data
not shown); these observations support the view that
the pharmacological effects of 45 are caused by (R)-9,
assumed to be formed in the mouse central nervous
system after decomposition of 45.
Similarly, using the same column and instrumentation as
for (R)-(+)-9 and (S)-(-)-9, the enantiomeric purities of 45 and
46 were determined at ambient temperature. The column was
eluted with 0.5 mL/min of 100 mM phosphate buffer (pH 7.0)/
MeOH (80:20). The enantiomeric purity was based on peak
areas.
Ch a r a cter iza tion of P r od r u gs. The hydrolysis of the O-
and N-pivaloyloxymethyl prodrugs 43 and 44, respectively,
was studied in 0.02 M aqueous phosphate buffer (pH 7.4) at
37 °C (Table 3). The rate of hydrolysis was determined by using
a reversed-phase HPLC procedure. The HPLC system con-
sisted of a Hitachi-Merck pump model L6200, a variable
wavelength UV detector (type Hitachi-Merck L-4000), and a
Hitachi-Merck AS-4000 autosampler. Data acquisition and
processing were performed using the Hitachi-Merck HPLC
manager (model 6000). The analytical column used was a
Lichrospher 100-RP8 (250 × 4 mm) which was kept in a
column oven at 40 °C. The mobile phase consisted of MeOH
mixed with 0.05 M phosphate (pH 2.1) to which was added
200 µL of TEA/L, in the proportion 40-60% v/v. The flow rate
was kept at 1.0 mL/min and the column effluent was monitored
at 220 nm. The retention times for the prodrugs were 7.8 and
6.0 min for 43 and 44, respectively. Quantification of the
prodrugs was done by measuring peak area in relation to those
of standards chromatographed under the same conditions. The
reactions were initiated by adding about 50 µL of a stock
solution of a prodrug in MeCN-H2O (90:10) to 10.0 µL of
phosphate buffer, preheated at 37 °C, in screw-capped test
tubes, the final prodrug concentration being (2-3) × 10-5 M.
The solutions were kept in a water bath at 37 ( 0.2 °C. At
appropriate times samples were taken and immediately chro-
matographed. Pseudo-first-order rate constants were deter-
mined from the slopes of linear plots of the logarithm of
residual prodrug against time.
Thus, although the selectivity of (R)-9 for the glial
GABA uptake system, as compared to the neuronal
system, is only 8-fold, compound (R)-9 still is the most
glia-selective inhibitor so far described. The conversion
of (R)-9 into analogues containing various bulky and
lipophilic N-substituents, including the 4,4-diphenyl-3-
butenyl group, is in progress, and the synthesis and
pharmacology of these compounds will be reported
separately.
Hydrolysis of the prodrugs was also studied in 0.02 M
phosphate buffer (pH 7.4) containing 80% human plasma at
37 °C. Initial concentrations of the compounds were (2-3) ×
10-5 M. At appropriate times, samples of 250 µL were
withdrawn and deproteinized by mixing with 1000 µL of EtOH.
After centrifugation for 3-5 min the clear supernatant fraction
was analyzed by HPLC as described above.
Exp er im en ta l Section
The apparent partition coefficients (D) of 9 and its prodrugs
were determined in two systems, i.e., n-octanol-0.05 M
phosphate buffer (pH 7.40) and cyclohexane-0.05 M phosphate
buffer (pH 7.40) at 21 °C (Table 3). The aqueous and organic
phases were mutually saturated at 21 °C before use. The
compounds were dissolved in the aqueous buffer phase ((2-3)
× 10-5 M) and the organic phase-buffer mixtures were shaken
Ch em istr y. Melting points were determined in capillary
tubes and are uncorrected. Elemental analyses were performed
by Mr. G. Cornali, Microanalytical Laboratory, LEO Pharma-
ceutical Products, Denmark, or by the Analytical Department,
H. Lundbeck A/S, Denmark, and are within (0.4% of the
calculated values, unless otherwise stated. Flash chromatog-
raphy (FC) was performed on Merck silica gel 60 H (5-40 µM)
and column chromatography (CC) on Merck silica gel 60 (70-
230 mesh, ASTM). Analytical thin-layer chromatography
(TLC) was carried out using Merck silica gel F254 plates.
Compounds containing the 3-isoxazolol moiety were visualized
using an FeCl3 spray reagent and compounds containing amino
groups were visualized using a ninhydrin spray reagent. 1H
NMR spectra were recorded on a Bruker AC 200F (200 MHz),
a Bruker AC 250F (250 MHz), or a Varian 360L (60 MHz)
spectrometer. Optical rotations were measured in thermo-
stated cuvettes on a Perkin-Elmer 241 polarimeter.
for 10-15 min to reach
a distribution equilibrium. The
volumes of each phase were chosen so that the solute concen-
tration in the aqueous phase, before and after distribution,
could be measured readily using the HPLC method described
above. At distribution equilibrium the two phases were
separated by centrifugation for 3-5 min. During the entire
procedure less than 2% of the prodrugs were degraded as
determined by HPLC.
3-Meth oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole (19a ).
To a solution of 1839 (5.1 g, 37 mmol) in ether (100 mL) was

Selective Inhibitors of Glial GABA Uptake
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5409
added a solution of diazomethane (0.3 mol) in ether (100 mL).
After stirring for 2 h, HOAc was added and the mixture was
evaporated. CC [tol-EtOAc (3:1)] gave 2.94 g (52%) of 19a as
an oil: 1H NMR (200 MHz, CDCl3) δ 3.98 (3H, s), 2.57 (2H, t,
J ) 6 Hz), 2.29 (2H, t, J ) 6 Hz), 1.85-1.65 (4H, m).
3-Eth oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole (19b). To
a solution of 1839 (100 g, 0.72 mol) in acetone (3 L) was added
K2CO3 (200 g, 1.45 mol) and the mixture was stirred at 50 °C
for 1 h. Ethyl bromide (170 mL, 2.2 mol) in acetone was added
during 1.5 h and stirring at 50 °C was continued overnight.
Filtration and evaporation followed by CC [EtOAc-heptane
(2:3)] afforded 65 g (54%) of 19b as an oil: 1H NMR (200 MHz,
CDCl3) δ 4.26 (2H, q, J ) 7 Hz), 2.56 (2H, t, J ) 6 Hz), 2.28
(2H, t, J ) 6 Hz), 1.85-1.65 (4H, m), 1.38 (3H, t, J ) 7 Hz).
3-Met h oxy-4,5,6,7-t et r a h yd r o-1,2-b en zisoxa zol-4-on e
(20a ) a n d 3-Meth oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zol-
7-on e (21a ). To a mixture of 19a (2.70 g, 17.6 mmol), HOAc
(55 mL), and concentrated H2SO4 (2.1 mL) was added a
solution of Na2Cr2O7‚2H2O (3.7 g, 12 mmol) in HOAc (30 mL)
during 45 min. After stirring for 2 h, the mixture was
neutralized by addition of 2 M NaOH. Extraction with Et2O
(4 × 100 mL), drying, evaporation, and CC [tol-EtOAc (10-
20%)] gave 21a (0.28 g, 10%): mp 100-101 °C (tol-light
petroleum); 1H NMR (200 MHz, CDCl3) δ 4.06 (3H, s), 2.65
(4H, m), 2.2 (2H, m). Anal. (C8H9NO3) C, H, N.
2.65 (2H, m), 2.20-1.75 (4H, m), 1.40 (3H, t, J ) 7 Hz). Anal.
(C9H14N2O2‚HBr) C, H, Br, N.
(RS)-4-Am in o-3-h ydr oxy-4,5,6,7-tetr ah ydr o-1,2-ben zisox-
a zole Hyd r obr om id e (8). A solution of 23a (2.49 g, 10 mmol)
in HBr in HOAc (33%, 20 mL) was stirred at 80 °C for 20 min.
Evaporation and treatment of the residue with HBr in HOAc
at 80 °C for 20 min gave after evaporation and recrystallization
8 (1.83 g, 78%): mp 187-190 °C (EtOH-Et2O); 1H NMR (200
MHz, D2O) δ 4.42 (1H, m), 2.68 (2H, m), 2.2-1.85 (4H, m).
Anal. (C7H10N2O2‚HBr) C, H, Br, N.
(RS)-3-E t h oxy-4-(d im et h yla m in o)-4,5,6,7-t et r a h yd r o-
1,2-ben zisoxa zole Hyd r obr om id e (24). Extraction with
CH2Cl2 of a mixture of 23b (526 mg, 2.0 mmol) and excess 1
M NaOH gave, after drying and evaporation, the free base.
Sodium formate (3.0 g, 44 mmol), formic acid (3.0 mL, 78
mmol), and a 30% solution of formaldehyde (3.0 mL) were
added and the mixture was heated at 100 °C for 20 h and
evaporated. Water (20 mL) and NaOH were added to the
residue. Extraction with CH2Cl2 (3 × 40 mL), drying, and
evaporation gave an oil. To a solution of the oil in ether (20
mL) was added HBr in HOAc and 24 was precipitated.
Recrystallization (MeCN-Et2O) gave pure 24 (422 mg, 72%):
mp 158-160 °C; 1H NMR (60 MHz, D2O) δ 4.53 (1H, m), 4.45
(2H, q, J ) 7 Hz), 3.00 (6H, s), 2.75 (2H, m), 2.05 (4H, m),
1.46 (3H, t, J ) 7 Hz). Anal. (C11H18N2O2‚HBr) C, H, Br, N.
(RS)-4-(Dim eth yla m in o)-3-h yd r oxy-4,5,6,7-tetr a h yd r o-
1,2-ben zisoxa zole Hyd r obr om id e (14). Compound 24 was
deprotected as described for 8: yield 86%; mp 184-186 °C
Later fractions contained 20a (1.55 g, 55%): mp 121-123
1
°C (tol-light petroleum); H NMR (200 MHz, CDCl3) δ 4.05
(3H, s), 2.96 (2H, t, J ) 7 Hz), 2.54 (2H, t, J ) 7 Hz), 2.25 (2H,
1
(EtOH-Et2O); H NMR (60 MHz, D2O) δ 4.50 (1H, m), 3.00
m). Anal. (C8H9NO3) C, H, N.
(6H, s), 2.83 (2H, m), 2.05 (4H, m). Anal. (C9H14N2O2‚HBr‚
0.33H2O) C, H, Br, N.
3-Eth oxy-4,5,6,7-tetr ah ydr o-1,2-ben zisoxazol-4-on e (20b)
a n d 3-Eth oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zol-7-on e
(21b). By using a similar procedure as above compounds 20b
and 21b were synthesized from 19b (35 g, 0.21 mol).
(RS)-4-[(ter t-Bu tyloxycar bon yl)am in o]-3-eth oxy-4,5,6,7-
tetr a h yd r o-1,2-ben zisoxa zole (25). A solution of 23b (2.9
g, 11 mmol) in water (30 mL) and dioxane (30 mL) was cooled
to 10 °C, and a solution of NaOH (0.88 g, 22 mmol) in water
(10 mL) was added. After addition of a solution of di-tert-butyl
dicarbonate (2.60 g, 12 mmol) in dioxane (12 mL), the mixture
was stirred at room temperature for 1.5 h. Water (100 mL)
and a small amount of NaOH (to pH > 10) were added and
the mixture was stirred for 30 min. The mixture was washed
with ether (150 mL) and the ether was discarded. The aqueous
phase was adjusted to pH ) 3 by addition of KHSO4 and was
extracted with ether (2 × 75 mL). Drying, evaporation, and
recrystallization (Et2O-light petroleum) afforded 25 (2.6 g,
84%): mp 115-117 °C; 1H NMR (60 MHz, CDCl3) δ 4.85 (1H,
m), 4.45 (2H, q, J ) 7 Hz), 2.75 (2H, m), 2.00 (4H, m), 1.65
(9H, s), 1.55 (3H, t, J ) 7 Hz). Anal. (C14H22N2O4) C, H, N.
(R S )-4-[(t er t -B u t y lo x y c a r b o n y l)-N -e t h y la m in o ]-3-
eth oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole (26). A solu-
tion of 25 (0.57 g, 2.0 mmol) and ethyl iodide (1.6 mL, 20 mmol)
in THF (25 mL) was cooled in an ice bath and sodium hydride
(60% in mineral oil, 240 mg, 6.0 mmol) was added. After
stirring at room temperature overnight, EtOH was added and
the mixture was evaporated. Addition of water (5 mL),
extraction with EtOAc (2 × 10 mL), and evaporation afforded
26 (0.53 g, 86%) as an oil: 1H NMR (60 MHz, CDCl3) δ 5.1
(1H, br s), 4.15 (2H, q, J ) 7 Hz), 3.0 (2H, m), 2.60 (2H, m),
2.1-1.7 (4H, m), 1.45 (9H, s), 1.20 (6H, m).
20b: yield 71%; mp 99-101 °C (EtOAc-Et2O-light petro-
1
leum); H NMR (200 MHz, CDCl3) δ 4.38 (2H, q, J ) 7 Hz),
2.90 (2H, t, J ) 6 Hz), 2.51 (2H, t, J ) 6 Hz), 2.19 (2H, m),
1.46 (3H, t, J ) 7 Hz). Anal. (C9H11NO3) C, H, N.
21b: yield 8%; mp 69-71 °C (EtOAc-Et2O-light petro-
1
leum); H NMR (200 MHz, CDCl3) δ 4.37 (2H, q, J ) 7 Hz),
2.63 (4H, m), 2.18 (2H, m), 1.44 (3H, t, J ) 7 Hz). Anal. (C9H11
NO3) C, H, N.
-
4-(H yd r oxyim in o)-3-m et h oxy-4,5,6,7-t et r a h yd r o-1,2-
ben zisoxa zole (22a ). A mixture of 20a (1.84 g, 11 mmol),
hydroxylamine hydrochloride (4.6 g, 66 mmol), Na2CO3 (3.5
g, 33 mmol), EtOH (80 mL), and water (120 mL) was refluxed
for 2.5 h. Evaporation, addition of water (50 mL), and extrac-
tion with CH2Cl2 (3 × 100 mL) afforded 22a (1.75 g, 87%):
mp 208-210 °C (EtOAc-light petroleum); 1H NMR (200 MHz,
CDCl3) δ 4.04 (3H, s), 2.76 (4H, m), 2.01 (2H, m). Anal.
(C8H10N2O3) C, H, N.
3-Eth oxy-4-(h yd r oxyim in o)-4,5,6,7-tetr a h yd r o-1,2-ben -
zisoxa zole (22b). Prepared from 20b (1.56 g, 8.60 mmol) by
the same procedure as above: yield 1.38 g (82%); mp 214-
1
217 °C (EtOAc-light petroleum); H NMR (60 MHz, CDCl3-
DMSO-d6 [9:1]) δ 4.30 (2H, q, J ) 7 Hz), 2.70 (4H, m), 1.95
(2H, m), 1.35 (3H, t, J ) 7 Hz). Anal. (C9H12N2O3) C, H, N.
(RS)-4-Am in o-3-m et h oxy-4,5,6,7-t et r a h yd r o-1,2-b en -
zisoxa zole Hyd r obr om id e (23a ). Aluminum strips (4.32 g,
160 mmol) were treated with an aqueous solution of HgCl2
(5%) for 1 min, filtered, and washed with EtOH. To these
pieces was added a solution of 22a (1.46 g, 8.0 mmol) in
aqueous MeOH (75%, 75 mL), and the mixture was stirred at
room temperature for 5 days, filtered, and evaporated. The
resulting oil (1.20 g, 89%) was dissolved in ether (50 mL) and
HBr in HOAc (33%) was added to precipitate 23a . Recrystal-
lization (EtOH-MeCN-Et2O) afforded 23a (1.16 g, 58%): mp
203-205 °C; 1H NMR (200 MHz, D2O) δ 4.36 (1H, m), 3.95
(3H, s), 2.66 (2H, m), 2.20-1.75 (4H, m). Anal. (C8H12N2O2‚
HBr) C, H, Br, N.
(RS)-4-(Eth yla m in o)-3-h yd r oxy-4,5,6,7-tetr a h yd r o-1,2-
ben zisoxa zole (10). Treatment of 26 with HBr in HOAc as
described for 8 gave 10 (66%): mp 189-191 °C (EtOH-
1
MeCN-Et2O); H NMR (60 MHz, D2O) δ 4.35 (1H, m), 3.30
(2H, q, J ) 7 Hz), 2.70 (2H, m), 2.05 (4H, m), 1.30 (3H, t, J )
7 Hz). Anal. (C9H14N2O2‚HBr) C, H, Br, N.
(RS)-3-Eth oxy-4-(m eth yla m in o)-4,5,6,7-tetr a h yd r o-1,2-
ben zisoxa zole (27). To a mixture of methylamine in EtOH
(33%, 5 mL, 40 mmol), methylamine hydrochloride (15 g, 220
mmol), and molecular sieves (3 Å, 25 g) was added a solution
of 20b (4.50 g, 24.8 mmol) in MeOH (100 mL). The mixture
was stirred for 30 min at room temperature and sodium
cyanoborohydride (7.0 g, 85 mmol) was added portionwise
during 20 min. After stirring for 20 h, the mixture was filtered
and the filtrate evaporated. Water (50 mL) was added and the
pH was adjusted to 10 by addition of NaOH. Extraction with
(RS)-4-Am in o-3-eth oxy-4,5,6,7-tetr ah ydr o-1,2-ben zisox-
a zole Hyd r obr om id e (23b). From 22b (1.30 g, 6.5 mmol):
yield 1.19 g (70%); mp 209-211 °C (EtOH-MeCN-Et2O); 1H
NMR (60 MHz, D2O) δ 4.40 (1H, m), 4.30 (2H, q, J ) 7 Hz),

5410 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Falch et al.
1
EtOAc (7 × 50 mL), drying, and evaporation gave crude 27
(4.80 g, 98%) as an oil: 1H NMR (60 MHz, CDCl3) δ 4.35 (2H,
q, J ) 7 Hz), 3.70 (1H, m), 2.70 (2H, m), 2.50 (3H, s), 1.95
(4H, m), 1.40 (3H, t, J ) 7 Hz).
fractions contained 32 (11.5 g): mp 96-97 °C; H NMR (250
MHz, CDCl3) δ 7.45-7.30 (5H, m), 6.85 (1H, br d), 4.95 (1H,
dt), 4.60 (1H, s), 4.25 (2H, q), 3.30 (3H, s), 2.75-2.45 (2H, m),
2.05-1.80 (4H, m), 1.30 (3H, t).
By using the same procedure the following compounds were
synthesized.
(S)-4-[(ter t-Bu tyloxycar bon yl)am in o]-3-h ydr oxy-4,5,6,7-
tetr a h yd r o-1,2-ben zisoxa zole (35). A solution of 33 (3.7 g,
11 mmol) in aqueous HBr (48%, 175 mL) and water (175 mL)
was refluxed for 1.5 h and evaporated. The residue was
dissolved in water, washed with CH2Cl2, and evaporated
leaving impure (S)-(+)-8 hydrobromide (4.0 g) as an oil. This
compound was Boc-protected by the method described for 25.
CC [heptane-EtOAc-EtOH (7:3:1)] gave 35 (0.9 g, 32%): mp
135 °C; 1H NMR (250 MHz, CDCl3) δ 5.05 (1H, br d), 4.50 (1H,
dt), 2.70-2.45 (2H, m), 2.10-1.75 (4H, m), 1.45 (9H, s).
(R)-4-[(ter t-Bu tyloxycar bon yl)am in o]-3-h ydr oxy-4,5,6,7-
tetr a h yd r o-1,2-ben zisoxa zole (34). The compound was pre-
pared as described for 35: mp 134 °C; 1H NMR spectrum was
identical with that of 35.
(S)-(+)-3-Hyd r oxy-4-a m in o-4,5,6,7-tetr a h yd r o-1,2-ben -
zisoxa zole Hyd r och lor id e [(S)-(+)-8‚HCl]. A solution of 35
(0.9 g, 3.5 mmol) in Et2O (200 mL) saturated with HCl was
stirred at room temperature for 1.5 h. Evaporation and
crystallization (EtOH-Et2O) gave (S)-(+)-8‚HCl (0.5 g, 93%):
mp 209-210 °C; 1H NMR (250 MHz, DMSO-d6) δ 8.40 (4H, br
s), 4.25 (1H, br t), 2.65-2.55 (2H, m), 2.05-1.75 (4H, m); ee )
99.8%; [R]D ) +19.4 (c ) 1.0, MeOH). Anal. (C7H10N2O2‚HCl)
C, H, Cl, N.
(RS)-3-Eth oxy-4-(2-p r op en -1-yla m in o)-4,5,6,7-tetr a h y-
d r o-1,2-ben zisoxa zole (28): oil; 1H NMR (250 MHz, CDCl3)
δ 6.05-5.90 (1H, m), 5.30-5.10 (2H, m), 4.35 (2H, q), 3.75 (1H,
t), 3.45-3.30 (2H, m), 2.70-2.45 (2H, m), 2.45 (1H, br s), 2.05-
1.95 (1H, m), 1.85-1.70 (3H, m), 1.40 (3H, t).
(RS)-3-Eth oxy-4-(1-p yr r olid in yl)-4,5,6,7-tetr a h yd r o-1,2-
ben zisoxa zole (29): oil; 1H NMR (250 MHz, CDCl3) δ 4.30
(2H, dq), 3.25 (1H, t), 2.80-2.50 (4H, m), 2.25-2.00 (2H, m),
1.90-1.70 (6H, m), 1.55-1.40 (2H, m), 1.40 (3H, t).
(RS)-3-Eth oxy-4-(2-h ydr oxyeth ylam in o)-4,5,6,7-tetr ah y-
d r o-1,2-ben zisoxa zole (30): mp 72-74 °C; 1H NMR (250
MHz, CDCl3) δ 4.30 (2H, q), 3.75 (1H, t), 3.70-3.60 (2H, m),
2.85 (2H, t), 2.70-2.45 (2H, m), 2.45 (2H, br s), 2.0-1.65 (4H,
m), 1.40 (3H, t).
Compounds 27-30 were deprotected with HBr in HOAc as
described for the synthesis of 8.
(RS)-3-H yd r oxy-4-(m et h yla m in o)-4,5,6,7-t et r a h yd r o-
1,2-ben zisoxa zole h yd r obr om id e (9): yield 3.6 g (74%); mp
1
184-186 °C (EtOH-Et2O); H NMR (250 MHz, DMSO-d6) δ
8.60 (1H, br s), 4.20 (1H, m), 2.70-2.60 (2H, m), 2.65 (3H, s),
2.15-1.75 (4H, m). Anal. (C8H12N2O2‚HBr) C, H, Br, N.
(RS)-3-Eth oxy-4-(1-p yr r olid in yl)-4,5,6,7-tetr a h yd r o-1,2-
ben zisoxa zole h yd r obr om id e (15): mp 209-210 °C; 1H
NMR (250 MHz, DMSO-d6) δ 4.35 (1H, br s), 3.70-3.10 (4H,
m), 2.75-2.60 (2H, m), 2.30-1.70 (8H, m). Anal. (C11H16N2O2‚
HBr) C, H, Br, N.
(R)-(-)-4-Am in o-3-h yd r oxy-4,5,6,7-tetr a h yd r o-1,2-ben -
zisoxa zole Hyd r och lor id e [(R)-(-)-8‚HCl]. Prepared from
34 as described above: mp 209-210 °C; ee ) 99.7%; [R]D
)
-20.0 (c ) 1.0, MeOH); 1H NMR spectrum was identical with
that of compound (S)-(+)-8‚HCl. Anal. (C7H10N2O2‚HCl) C, H,
Cl, N.
Compound 11 obtained by this method was impure and was
purified by treatment with di-tert-butyl dicarbonate by the
method described for the synthesis of 25. CC afforded (RS)-
4-[N-(ter t-bu tyloxyca r bon yl)-N-(2-p r op en -1-yl)a m in o]-3-
h yd r oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole (31) as an
oil: 1H NMR (250 MHz, CDCl3) δ 5.90-5.75 (1H, m), 5.20-
5.00 (3H, m), 4.00-3.85 (1H, m), 3.65-3.50 (1H, m), 2.55 (2H,
br s), 2.10-1.95 (2H, m), 1.85-1.70 (2H, m), 1.40 (9H, s).
(RS)-3-Hydr oxy-4-(2-pr open -1-ylam in o)-4,5,6,7-tetr ah y-
d r o-1,2-ben zisoxa zole Oxa la te (11). Deprotection of 31 with
trifluoroacetic acid and addition of oxalic acid gave 11: mp
182-183 °C (acetone); 1H NMR (250 MHz, DMSO-d6) δ 6.00-
5.85 (1H, m), 5.45 (1H, d), 5.40 (1H, d), 4.15 (1H, br s), 3.65
(2H, d), 2.75-2.60 (2H, m), 2.20-1.75 (4H, m). Anal. (C10H14N2O2‚
C2H2O4) C, H, N.
(R)-N-(r-Meth oxyp h en yla cetyl) Der iva tives of (R)-3-
Eth oxy-4-(m eth yla m in o)-4,5,6,7-tetr a h yd r o-1,2-ben zisox-
a zole (36) a n d (S)-3-E t h oxy-4-(m et h yla m in o)-4,5,6,7-
tetr a h yd r o-1,2-ben zisoxa zole (37). The compounds were
prepared by the method described for 32 and 33. The first
eluted compound was 37 followed by 36.
(R)-(+)-3-Hyd r oxy-4-(m eth yla m in o)-4,5,6,7-tetr a h yd r o-
1,2-ben zisoxa zole Hyd r obr om id e [(R)-(+)-9‚HBr ]. To a
solution of 36 (9.0 g, 26 mmol) in THF was added a solution
of lithium triethylborohydride in THF (1 M, 80 mL) dropwise
during 20 min at 0-5 °C. After stirring at room temperature
overnight, the mixture was poured on ice (500 g) and pH was
adjusted to 2 by addition of HCl. THF was removed by
evaporation and the aqueous mixture was washed with EtOAc
(2 × 50 mL). Concentrated NaOH was added to pH ) 11 and
extraction with EtOAc (100 mL), drying, and evaporation gave
4.6 g of crude 38.
(RS)-4-(2-Acetyloxyeth yla m in o)-3-h yd r oxy-4,5,6,7-tet-
r a h yd r o-1,2-ben zisoxa zole Hyd r obr om id e (12). Treatment
1
of 30 with HBr in HOAc resulted in 12: mp 164-165 °C; H
A solution of 38 in HBr in HOAc (33%, 150 mL) was heated
to 90 °C for 1 h and evaporated. Crystallization of the residue
(EtOH-Et2O) gave 4.2 g (65%) of (R)-(+)-9‚HBr: mp 207-
209 °C; ee ) 99.1%; [R]D ) +5.6 (c ) 1.0, MeOH); 1H NMR
spectrum was identical with that of 9. Anal. (C8H12N2O2‚HBr)
C, H, Br, N.
(S)-(-)-3-Hyd r oxy-4-(m eth yla m in o)-4,5,6,7-tetr a h yd r o-
1,2-ben zisoxa zole Hyd r obr om id e [(S)-(-)-9‚HBr ]. The
compound was prepared analogously from 37: mp 208-209
°C; ee > 99.4%; [R]D ) -5.9 (c ) 1.0, MeOH); 1H NMR
spectrum was identical with that of (R)-(+)-9. Anal. (C8H12N2O2‚
HBr) C, H, Br, N.
(RS)-4-[N-(ter t-Bu tyloxyca r bon yl)m eth yla m in o]-3-h y-
d r oxy-4,5,6,7-t et r a h yd r o-1,2-b en zisoxa zole (40). Com-
pound 9 (7.0 g, 28 mmol) was Boc-protected by the method
described for 25: yield 5.3 g (74%); mp 151-152 °C; 1H NMR
(250 MHz, CDCl3) δ 5.15 (1H, br s), 2.75 (3H, s), 2.65-2.55
(2H, m), 2.10-1.65 (4H, m), 1.50 (9H, s).
(RS)-4-[(ter t-Bu tyloxycar bon yl)-N-m eth ylam in o]-3-[(piv-
a loyloxy)m e t h yloxy]-4,5,6,7-t e t r a h yd r o-1,2-b e n zisox-
a zole (41) a n d (RS)-4-[(ter t-Bu tyloxyca r bon yl)-N-m eth -
yla m in o]-2-[(p iva loyloxy)m eth yl]-4,5,6,7-tetr a h yd r o-1,2-
ben zisoxa zol-3-on e (42). Potassium tert-butoxide (2.5 g, 22
mmol) was cautiously added to a suspension of 40 (5.0 g, 19
NMR (250 MHz, DMSO-d6) δ 4.40-4.20 (3H, m), 3.30 (2H, t),
2.70-2.55 (2H, m), 2.10 (3H, s), 2.20-2.00 (2H, m), 1.95-1.75
(2H, m). Anal. (C11H16N2O4‚HBr) C, H, Br, N.
(RS)-3-H yd r oxy-4-(2-h yd r oxyet h yla m in o)-4,5,6,7-t et -
r a h yd r o-1,2-ben zisoxa zole Hyd r obr om id e (13). A solution
of 12 (1.3 g, 4.0 mmol) in water (50 mL) and aqueous HBr
(48%, 7 mL) was heated at 100 °C for 1 h. Evaporation and
crystallization from EtOH afforded 13 (0.9 g, 80%): mp 172-
1
173 °C; H NMR (250 MHz, DMSO-d6) δ 5.20 (1H, br s), 4.25
(2H, br s), 3.70 (2H, q), 3.10 (2H, t), 2.75-2.55 (2H, m), 2.30-
1.95 (2H, m). Anal. (C9H14N2O3‚HBr) C, H, Br, N.
(R)-N-(r-Met h oxyp h en yla cet yl) Der iva t ives of (S)-4-
Am in o-3-eth oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole (33)
an d (R)-4-Am in o-3-eth oxy-4,5,6,7-tetr ah ydr o-1,2-ben zisox-
a zole (32). To an ice-cold solution of 23b (18 g, 100 mmol) in
CH2Cl2 (500 mL) was added triethylamine (30 mL, 215 mmol)
followed by dropwise addition of a solution of (R)-(-)-R-
methoxyphenylacetyl chloride (from the acid (17 g, 100 mmol)
by reaction with SOCl2) in CH2Cl2 (80 mL). The mixture was
stirred for 2 h at room temperature and water (2 L) was added.
The organic phase was separated, washed with dilute HCl,
dried, and evaporated. The two diastereomers were separated
by preparative HPLC on silical gel [heptane-EtOAc (3:2)]. The
first eluted fractions contained 33 (13 g) as an oil. The later

Selective Inhibitors of Glial GABA Uptake
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5411
mmol) in acetone (50 mL). A solution of iodomethyl pivalate
(7.5 g, 31 mmol) in acetone (10 mL) was added and the mixture
was stirred overnight. After filtration, the filtrate was evapo-
rated to give a mixture of 41 and 42. The compounds were
separated by CC [heptane-EtOAc (3:2)]. The first fractions
contained 41 (oil, 3.9 g, 54%): 1H NMR (250 MHz, CDCl3) δ
5.85 (2H, dd), 5.30-5.00 (1H, m), 2.60-2.50 (3H, m), 2.10-
1.50 (6H, m), 1.50 (9H, s), 1.30 (9H, s).
The latter fractions contained 42 (2.1 g, 29%) as an oil: 1H
NMR (250 MHz, CDCl3) δ 5.75 (2H, s), 5.10-5.00 (1H, m), 2.65
(3H, s), 2.50-2.40 (2H, m), 2.10-1.60 (4H, m), 1.50 (9H, s),
1.20 (9H, s).
(R S )-4-(Me t h yla m in o)-3-[(p iva loyloxy)m e t h yloxy]-
4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole Oxa la te (43). To a
solution of 41 (3.7 g, 10 mmol) in CH2Cl2 was added trifluo-
roacetic acid (19 mL). The mixture was stirred for 1 h and
evaporated. Water (100 mL) and Et2O (100 mL) were added
followed by K2CO3 to pH > 9. The organic phase was
separated, dried, and evaporated. To a solution of the free base
in EtOH was added excess oxalic acid to precipitate 43 (1.6 g,
51%): mp 126-128 °C; 1H NMR (250 MHz, DMSO-d6) δ 5.90
(2H, dd), 3.85 (1H, t), 2.80-2.55 (2H, m), 2.40 (3H, s), 2.05-
1.65 (4H, m), 1.15 (9H, s). Anal. (C14H22N2O4‚C2H2O4) C, H,
N.
(RS)-4-(Meth yla m in o)-2-[(p iva loyloxy)m eth yl]-4,5,6,7-
tetr a h yd r o-1,2-ben zisoxa zol-3-on e Hem ioxa la te (44). Pre-
pared as described for 43: mp 177-178 °C (acetone); 1H NMR
(250 MHz, DMSO-d6) δ 5.80 (2H, dd), 3.80 (1H, t), 2.60-2.45
(2H, m), 2.50 (3H, s), 2.05-1.60 (4H, m), 1.15 (9H, s). Anal.
(C14H22N2O4‚0.5C2H2O4) C, H, N.
(R)-(+)-4-(Met h yla m in o)-3-[(p iva loyloxy)m et h yloxy]-
4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole Hem ioxa la te (45).
The compound was prepared from (R)-(+)-9 by the same
procedure as described above for the synthesis of 43. The
hemioxalate salt was crystallized from acetone: mp 210-213
°C; ee ) 98.9%; [R]D ) +5.6 (c ) 1.0, MeOH). Anal. (C14H22N2O4‚
0.5C2H2O4) C, H, N.
(S)-(-)-4-(Met h yla m in o)-3-[(p iva loyloxy)m et h yloxy]-
4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole Hem ioxa la te (46).
Prepared from (S)-(-)-9 by the same procedure as described
above for the synthesis of 43 and crystallized from acetone:
mp 211-213 °C; ee ) 99.3%; [R]D ) -5.4 (c ) 1.0, MeOH).
Anal. (C14H22N2O4‚0.5C2H2O4) C, H, N.
2-(Ben zyla m in o)-3,4,5,6-tetr a h yd r oben za m id e (48). A
mixture of 4740 (10.0 g, 71 mmol), benzylamine (8.4 g, 78
mmol), toluene (35 mL), and molecular sieves (3 Å, 2 g) was
refluxed for 2 h, water being removed using a Dean-Stark
trap. The reaction mixture was filtered, and the filtrate was
evaporated. The residue was crystallized (light petroleum) to
give 48 (16 g, 98%): mp 73-74 °C; 1H NMR (200 MHz, DMSO-
d6) δ 10.16 (1H, t, J ) 6 Hz), 7.38-7.10 (5H, m), 6.25 (2H, s),
4.31 (2H, d, J ) 6 Hz), 2.22-1.90 (4H, m), 1.49 (4H, s). Anal.
(C14H18N2O) H, N; C: calcd, 72.98; found, 72.45.
3-Hyd r oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoth ia zole (49).
To a solution of 48 (15 g, 65 mmol) in HOAc (100 mL) was
added excess hydrogen sulfide at 80 °C for 4 h. The reaction
mixture was evaporated and Et2O was added to the residue
which initiated crystallization. The crystals were dissolved in
EtOAc (30 mL) and a solution of Br2 (8.3 mL, 150 mmol) in
EtOAc (30 mL) was dropwise added at room temperature. The
mixture was stirred for 20 h at room temperature and
evaporated. CC [EtOAc-EtOH (1:1) containing 1% HOAc]
gave the title compound (3.3 g, 33%): mp 150-151 °C; 1H NMR
(200 MHz, CDCl3-DMSO-d6 [3:1]) δ 8.5 (1H, br s), 2.83-2.70
(2H, m), 2.48-2.31 (2H, m), 1.96-1.69 (4H, m). Anal. (C7H9-
NOS) C, H, N, S.
EtOAc (2 × 150 mL) and the combined organic phases were
dried and evaporated to give an oil. CC [tol-EtOAc (1:1)] gave
50 (2.8 g, 53%) as a yellow oil: 1H NMR (200 MHz, CDCl3) δ
2.93-2.77 (2H, m), 2.51-2.49 (2H, m), 2.00-1.72 (4H, m).
Anal. (C7H8ClNS) C, H, N.
3-Ch lor o-4,5,6,7-t et r a h yd r o-1,2-b en zisot h ia zol-4-on e
(51). A solution of sodium dichromate (4.4 g, 16 mmol) in HOAc
(30 mL) was added dropwise over 1 h to a solution of 50 (2.7
g, 16 mmol) and concentrated H2SO4 (1.8 mL) in HOAc (80
mL). The reaction mixture was stirred at room temperature
for an additional 2 h and neutralized with a saturated solution
of NaHCO3. Extraction with EtO2 (3 × 150 mL), drying, and
evaporation gave an oil. CC [tol-EtOAc (1:1)] eluted first
3-chloro-4,5,6,7-tetrahydro-1,2-benzisothiazol-7-one (680 mg,
23%) as an oil.
The later fractions contained 51 (780 mg, 27%): mp 84-85
°C; 1H NMR (200 MHz, CDCl3) δ 3.17 (2H, t, J ) 6.2 Hz), 2.63
(2H, t, J ) 6.2 Hz), 2.30-2.15 (2H, m).
3-Me t h oxy-4,5,6,7-t e t r a h yd r o-1,2-b e n zisot h ia zol-4-
on e (52). A mixture of 51 (600 mg, 3.2 mmol) and a solution
of Na (506 mg, 22 mmol) in MeOH (22 mL) was stirred at 90
°C for 1 h. The reaction mixture was evaporated and water
(20 mL) was added to the residue. Extraction with CH2Cl2 (3
× 30 mL), drying, and evaporation gave an oil. CC [tol-EtOAc
(4:1)] gave 52 (251 mg, 43%): mp 45-46 °C; 1H NMR (200
MHz, CDCl3) δ 4.11 (3H, s), 3.08 (2H, t, J ) 6.1 Hz), 2.58 (2H,
t, J ) 6.1 Hz), 2.20-2.15 (2H, m). Anal. (C8H9NO2S) C, H, N.
(RS)-4-Am in o-3-h yd r oxy-4,5,6,7-t et r a h yd r o-1,2-b en z-
isoth ia zole Hyd r obr om id e (16). To a solution of 52 (185 mg,
1.0 mmol) and NH4OAc (780 mg, 10.1 mmol) in MeOH (7 mL)
was portionwise added sodium cyanoborohydride (44 mg, 0.71
mmol). The mixture was stirred at room temperature for 48 h
and acidified with concentrated HCl. The mixture was evapo-
rated and water (3 mL) was added to the residue. The aqueous
solution was washed with Et2O (3 × 15 mL) and solid KOH
was added until pH > 10. Extraction with Et2O (3 × 15 mL),
drying, and evaporation gave an oil. The oil was dissolved in
EtOH and excess of HCl in EtOAc was added to precipitate
(RS)-4-amino-3-methoxy-4,5,6,7-tetrahydro-1,2-benzisothia-
zole hydrochloride (68 mg, 31%): mp 195 °C dec; 1H NMR (200
MHz, D2O) δ 4.45-4.32 (1H, m), 3.98 (3H, s), 2.90-2.72 (2H,
m), 2.28-1.73 (4H, m).
A solution of HBr in HOAc (33%, 3 mL) was added to this
compound (60 mg, 0.3 mmol) and the mixture was stirred at
room temperature for 48 h. Evaporation and recrystallization
of the residue (methanol-ether) gave 16 (28 mg, 41%): mp
160-165 °C; 1H NMR (200 MHz, D2O) δ 4.39-4.23 (1H), 2.87-
2.72 (2H, m), 2.28-2.05 (1H, m), 2.04-1.78 (3H, m). Anal.
(C7H10N2OS‚HBr‚2.5H2O) H, Br, N; C: calcd, 28.39; found,
28.87.
(RS)-3-H yd r oxy-4-(m et h yla m in o)-4,5,6,7-t et r a h yd r o-
1,2-ben zisoth ia zole Hyd r obr om id e (17). (RS)-3-methoxy-
4-(methylamino)-4,5,6,7-tetrahydro-1,2-benzisothiazole hydro-
chloride was synthesized as described above, using 52 (200
mg, 22 mmol) in MeOH (5 mL), a 33% solution of methylamine
(220 µL) in EtOH, methylamine hydrochloride (662 mg, 9.8
mmol), molecular sieves (3 Å), and sodium cyanoborohydride
(234 mg, 3.8 mmol). The resulting oil was dissolved in Et2O
and excess of HCl in EtOAc was added to precipitate the
compound (172 mg, 68%): mp 146-148 °C; 1H NMR (200 MHz,
D2O) δ 4.42-4.28 (1H, m), 3.98 (3H, s), 3.00-2.78 (2H, m),
2.69 (3H, s), 2.25-1.81 (4H, m).
Deprotection with HBr in HOAc and recrystallization gave
1
17 (53 mg, 47%): mp 192 °C dec; H NMR (200 MHz, D2O) δ
4.30-4.16 (1H, m), 2.92-2.68 (2H, m), 2.72 (3H, s), 2.24-1.82
(4H, m). Anal. (C8H12N2OS‚HBr) C, H, Br, N.
3-Ch lor o-4,5,6,7-tetr a h yd r o-1,2-ben zisoth ia zole (50). A
mixture of 49 (4.74 g, 30 mmol), pyridinium hydrochloride
(12.7 g, 109 mmol), phosphoric acid (2.1 g, 21 mmol), and
phosphorus oxychloride (25 mL, 270 mmol) was stirred at 90
°C for 5 h. The reaction mixture was evaporated and EtOAc
(130 mL) was added to the residue. A saturated solution of
NaHCO3 (130 mL) was added and after 10 min of stirring the
phases were separated. The aqueous phase was extracted with
X-r a y Cr ysta llogr a p h ic An a lysis of (R)-(-)-4-Am in o-
3-h yd r oxy-4,5,6,7-tetr a h yd r o-1,2-ben zisoxa zole Hyd r o-
ch lor id e [(R)-(-)-8‚HCl]. Crystal data: [C7H11N2O2]+, Cl-,
Mr ) 190.63, mp 209-210 °C (EtOH-EtOAc), colorless prism,
monoclinic, space group P21 (No. 4), a ) 7.168(2) Å, b )
6.079(1) Å, c ) 9.882(2) Å, â ) 91.44(2)°, V ) 430.5(2) Å3, Z )
2, Dc ) 1.471 Mg m-3, F(000) ) 200, µ(Cu KR) ) 3.64 mm-1
,
T ) 122.0(5) K, crystal dimensions ) 0.10 × 0.14 × 0.38 mm.

5412 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Falch et al.
Diffraction data were collected on an Enraf-Nonius CAD-4
diffractometer using graphite monochromated Cu KR radiation
(λ ) 1.54184 Å).41 Intensities were collected using the ω/ 2θ
scan mode. Unit cell dimensions were determined by least-
squares refinement of 22 reflections (θ range 38.58-43.62°).41
The reflections were measured in the range -8 e h e 7, -7 e
k e 7, -12 e l e 12 (4.48° < θ < 74.76°). Data were reduced
using the programs of Blessing (DREADD).42,43 The intensities
of five standard reflections were monitored every 104 s (decay
0.8%, not corrected). Absorption correction was applied using
the program ABSORB (Tmin ) 0.429; Tmax ) 0.713).44 A total
of 3510 reflections were averaged according to the point group
symmetry 2 resulting in 1773 unique reflections (Rint ) 0.037
on Fo2).
mp 208-210 °C (H2O), triclinic, space group P1 (No. 2), a )
5.0678(3) Å, b ) 9.600(1) Å, c ) 9.6965(9) Å, R ) 116.171(8)°,
â ) 93.305(7)°, γ ) 103.453(8)°, V ) 404.76(6) Å3, Z ) 2, Dc )
1.495 Mg m-3, F(000) ) 192, µ(Cu KR) ) 0.980 mm-1, T )
122.0(5) K, crystal dimensions ) 0.05 × 0.10 × 0.35 mm.
Diffraction data were collected on an Enraf-Nonius CAD-4
diffractometer using graphite monochromated Cu KR radiation
(λ ) 1.54184 Å).41 Intensities were collected using the ω/2θ
scan mode. Unit cell dimensions were determined by least-
squares refinement of 20 reflections (θ range 39.5-48.5°).41
A
total of 1661 unique reflections were measured in the
range -6 e h e 6, -11 e k e 10, 0 e l e 12 (5.17° < θ <
74.91°). Data were reduced using the programs of Blessing
(DREADD).42,43 Three standard reflections were monitored
every 104 s (decay 2.2%, not corrected). No absorption correc-
tion was applied.
The structure was solved by the Patterson method using
the program SHELXS9745,46 and refined using the program
SHELXL97.26 Full matrix least-squares refinement on F2 was
The structure was solved using the program MULTAN11/
82 in the Enraf-Nonius Structure Determination Package48,49
and refined using the program SHELXL93.50 Full matrix least-
2
performed, minimizing ∑w(Fo - Fc2)2, with anisotropic dis-
placement parameters for the non-hydrogen atoms. The posi-
tions of the hydrogen atoms were located on intermediate
difference electron density maps and refined with isotropic
displacement parameters fixed at 0.05 Å2. Extinction correction
was applied, extinction coefficient: 0.118(4).26 The refinement
(143 parameters, 1773 reflections) with the molecule having
2
squares refinement on F2 was performed, minimizing ∑w(Fo
- Fc2)2, with anisotropic displacement parameters for the non-
hydrogen atoms. The positions of the hydrogen atoms were
located on intermediate difference electron density maps and
refined with isotropic displacement parameters. Refinement
was carried out on all reflections, except for a small number
of reflections (010 and 1-12 with Fc . Fo and three weak
reflections with ∆F2/esd > 7.5), which were excluded from the
final cycles of refinement. The refinement (158 parameters,
2
the (R)-configuration converged at RF ) 0.026, wRF ) 0.069
for 1765 reflections with Fo > 4σ(Fo); w ) 1/[σ(Fo2) + (0.0481P)2
2
+ 0.1138P], where P ) (Fo + 2Fc2)/3; S ) 1.069. The Flack
absolute structure factor: 0.00(1).25,26 In the final difference
Fourier map maximum and minimum electron densities were
0.23 and -0.26 e Å-3, respectively. Complex atomic scattering
factors for neutral atoms were as incorporated in SHELXL97.26,47
2
1656 reflections) converged at RF ) 0.037, wRF ) 0.096 for
1604 reflections with Fo > 4σ(Fo); w ) 1/[σ(Fo2) + (0.0544P)2
2
+ 0.1495P], where P ) (Fo + 2Fc2)/3; S ) 1.097. In the final
X-r a y Cr ysta llogr a p h ic An a lysis of (R)-(+)-3-Hyd r oxy-
4-(m eth ylam in o)-4,5,6,7-tetr ah ydr o-1,2-ben zisoxazole Hy-
d r obr om id e [(R)-(+)-9.HBr ]. Crystal data: [C8H13N2O2]+,
Br-, Mr ) 249.11, mp 207-209 °C (CH2Cl2-Et2O), colorless
triangles, trigonal, space group P3121 (No. 152), a ) 7.807(2)
Å, b ) 7.807(2) Å, c ) 29.078(7) Å, V ) 1534.8(7) Å3, Z ) 6, Dc
) 1.617 Mg m-3, F(000) ) 756, µ(Cu KR) ) 5.27 mm-1, T )
122.0(5) K, crystal dimensions ) 0.07 × 0.10 × 0.50 mm.
difference Fourier map maximum and minimum electron
densities were 0.30 and -0.23 e Å-3, respectively. Complex
atomic scattering factors for neutral atoms were as incorpo-
rated in SHELXL93.50
In Vitr o GABA Up ta k e Assa ys. In the initial determina-
tion of the affinity of compounds for the GABA uptake systems,
the effects on GABA uptake in crude synaptosomes, prepared
from adult rat brain, were tested.51 These uptake experiments
were performed at a GABA concentration of 0.05 µM with
preincubation of the tissue preparation for 10 min at 20 °C in
the presence of inhibitor.22 The effects of compounds on
neuronal GABA uptake were measured using neurons cultured
from cerebral cortices of 15-day-old mouse embryos, and the
uptake experiments were carried out as previously described.17
Effects of compounds on glial GABA uptake were measured
using astrocytes cultured from cerebral cortices of newborn
mice, and the uptake experiments were performed as previ-
ously described.17
Deter m in a tion of An ticon vu lsa n t Effects. Mice weigh-
ing 20-25 g were used. Test compounds were given subcuta-
neously, and isoniazide (300 µmol/kg) was administered
subcutaneously at the same time as two separate injections.33
Five mice were used per dose, and dose-response relationship
was determined in at least two separate trials with overlapping
doses. A control group, receiving isoniazide only, was included
at each testing. This dose of isoniazide induces intermittent
tonic-clonic siezures. The animals were placed individually
in Macrolon type II cages, and the time from injection to the
first convulsions was recorded. The experiment was terminated
at the occurrence of the first convulsions, or after 60 min. The
number of animals showing no convulsions within 60 min was
recorded, and the ED50 values were calculated by log probit
analyses.33
Diffraction data were collected on an Enraf-Nonius CAD-4
diffractometer using graphite monochromated Cu KR radiation
(λ ) 1.54184 Å).41 Intensities were collected using the ω/2θ
scan mode. Unit cell dimensions were determined by least-
squares refinement of 24 reflections (θ range 39.41-39.84°).41
The reflections were measured in the range -9 e h e 9, -9 e
k e 9, -36 e l e 36 (4.56° < θ < 74.75°). Data were reduced
using the programs of Blessing (DREADD).42,43 The intensities
of five standard reflections were monitored every 104 s (decay
14.5%). Appropriate scaling was performed using the program
SCALE3.42,43 Absorption correction was applied using the
program ABSORB (Tmin ) 0.358; Tmax ) 0.731).44 A total of
12669 reflections were averaged according to the point group
symmetry 321 resulting in 2148 unique reflections (Rint ) 0.060
on Fo2).
The structure was solved by the Patterson method using
the program SHELXS9745,46 and refined using the program
SHELXL97.26 Full matrix least-squares refinement on F2 was
2
performed, minimizing ∑w(Fo - Fc2)2, with anisotropic dis-
placement parameters for the non-hydrogen atoms. The posi-
tions of the hydrogen atoms were located on intermediate
difference electron density maps and refined with isotropic
displacement parameters fixed at 0.05 Å2. Extinction correction
was applied, extinction coefficient: 0.0017(1).26 The refinement
(158 parameters, 2110 reflections) with the molecule having
In another set of experiments, the anticonvulsant activity
of compound 9 was tested in Frings mice after intracere-
broventricular administration as described previously.35 The
number of animals protected against sound-induced convul-
sions was recorded and the ED50 value for protection calcu-
lated.35
2
the (R)-configuration converged at RF ) 0.016, wRF ) 0.041
for 2106 reflections with Fo > 4σ(Fo); w ) 1/[σ(Fo2) + 0.8132P],
2
where P ) (Fo + 2Fc2)/3; S ) 1.115. The Flack absolute
structure factor: 0.00(2).25,26 In the final difference Fourier
map maximum and minimum electron densities were 0.28 and
-0.23 e Å-3, respectively. Complex atomic scattering factors
for neutral atoms were as incorporated in SHELXL97.26,47
Ack n ow led gm en t. This work was supported by
grants from the Lundbeck Foundation, H. Lundbeck
A/S, and the Danish State Biotechnology Programme
X-r a y Cr ysta llogr a p h ic An a lysis of (E)-4-(Hyd r oxy-
im in o)-3-m et h oxy-4,5,6,7-t et r a h yd r o-1,2-b en zisoxa zole
(22a ). Crystal data: C8H10N2O3, Mr ) 182.18, colorless prism,

Selective Inhibitors of Glial GABA Uptake
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5413
lular γ-aminobutyrate levels in rat thalamus. Eur. J . Pharmacol.
1997, 331, 139-144.
(21) Gonsalves, S. F.; Twitchell, B.; Harbaugh, R. E.; Krogsgaard-
Larsen, P.; Schousboe, A. Anticonvulsant activity of intracere-
broventricularly administered glial GABA uptake inhibitors and
other GABA mimetics in chemical seizure models. Epilepsy Res.
1989, 4, 34-41.
(22) Krogsgaard-Larsen, P.; Falch, E.; Larsson, O. M.; Schousboe,
A. GABA uptake inhibitors: relevance to antiepileptic drug
research. Epilepsy Res. 1987, 1, 77-93.
(23) Falch, E.; Larsson, O. M.; Schousboe, A.; Krogsgaard-Larsen,
P. GABA-A agonists and GABA uptake inhibitors: structure-
activity relationships. Drug Dev. Res. 1990, 21, 169-188.
(24) J ohnson, C. K. ORTEP II. A Fortran Thermal-Ellipsoid Plot
Programme for Crystal Structure Illustrations; Report ORNL-
5138; Oak Ridge National Laboratory: Oak Ridge, TN, 1976.
(25) Flack, H. D. On enantiomorph-polarity estimation. Acta Crys-
tallogr. 1983, A39, 876-881.
(1991-1995). The technical assistance of Flemming
Hansen, Department of Chemistry, University of Copen-
hagen, with the collection of X-ray data is gratefully
acknowledged. We also express our gratitude to Tina
Lindgreen and Anne Nordly for technical and secretarial
assistance, respectively.
Su p p or tin g In for m a tion Ava ila ble: Tables for the com-
pounds (R)-(-)-8, (R)-(+)-9, and 22a listing final atomic
coordinates, equivalent isotropic displacement parameters,
anisotropic displacement parameters for non-hydrogen atoms,
full lists of bond lengths and bond angles, and lists of structure
factors. This material is available free of charge via the
Internet at http://pubs.acs.org.
(26) Sheldrick, G. M. SHELXL97. Program for Crystal Structure
Refinement; University of Go¨ttingen: Go¨ttingen, Germany, 1997.
(27) Allen, F. H.; Kennard, O.; Watson, D. G.; Brammer, L.; Orpen,
A. G.; Taylor, R. Typical interatomic distances: organic com-
pounds. In International Tables for Crystallography; Wilson, A.
J . C., Ed.; Kluwer Academic Publishers: Dordrecht, The Neth-
erlands, 1995; Vol. C, pp 685-706.
(28) Spek, A. L. PLATON, an integrated tool for the analysis of the
results of a single-crystal structure determination. Acta Crys-
tallogr. 1990, A46, C34.
(29) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor, which causes
50% inhibition (I50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
(30) Krogsgaard-Larsen, P.; Nielsen, L.; Falch, E.; Curtis, D. R. GABA
agonists. Resolution, absolute stereochemistry, and enantiose-
lectivity of (S)-(+)- and (R)-(-)-dihydromuscimol. J . Med. Chem.
1986, 28, 1612-1617.
(31) J ohnston, G. A. R.; Krogsgaard-Larsen, P.; Stephanson, A. L.;
Twitchin, B. Inhibition of the uptake of GABA and related amino
acids in rat brain slices by the optical isomers of nipecotic acid.
J . Neurochem. 1976, 26, 1029-1032.
(32) Schousboe, A.; Larsson, O. M.; Hertz, L.; Krogsgaard-Larsen,
P. Heterocyclic GABA analogues as new selective inhibitors of
astroglial GABA transport. Drug Dev. Res. 1981, 1, 115-127.
(33) Christensen, A. V.; Larsen, J .-J . Antinociceptive and anticon-
vulsive effect of THIP, a pure GABA agonist. Pol. J . Pharmacol.
Pharm. 1982, 34, 127-134.
(34) Wood, J . D.; J ohnson, D. D.; Krogsgaard-Larsen, P.; Schousboe,
A. Anticonvulsant activity of the glial-selective GABA uptake
inhibitor, THPO. Neuropharmacology 1983, 22, 139-142.
(35) White, H. S.; Wolf, H. H.; Swinyard, E. A.; Skeen, G. A.; Sofia,
R. D. A neuropharmacological evaluation of felbamate as a novel
anticonvulsant. Epilepsia 1992, 33, 564-572.
(36) J ohnston, G. A. R.; Krogsgaard-Larsen, P.; Stephanson, A. L.;
Twitchin, B. Inhibition of the uptake of GABA and related amino
acids in rat brain slices by the optical isomers of nipecotic acid.
J . Neurochem. 1976, 26, 1029-1032.
(37) Knutsen, L. J . S.; Andersen, K. E.; Lau, J .; Lundt, B. F.; Henry,
R. F.; Morton, H. E.; Nærum, L.; Petersen, H.; Stephensen, H.;
Suzdak, P. D.; Swedberg, M. D. B.; Thomsen, C.; Sørensen, P.
O. Synthesis of novel GABA uptake inhibitors. 3. Diaryloxime
and diarylvinyl ether derivatives of nipecotic acid and guvacine
as anticonvulsant agents. J . Med. Chem. 1999, 42, 3447-3462.
(38) Larsson, O. M.; Falch, E.; Krogsgaard-Larsen, P.; Schousboe,
A. Kinetic characterization of inhibition of GABA uptake into
cultured neurons and astrocytes by 4,4-diphenyl-3-butenyl
derivatives of nipecotic acid and guvacine. J . Neurochem. 1988,
50, 818-823.
(39) J acquier, R.; Petrus, C.; Petrus, F.; Verducci, J . EÄ tude dans la
serie des azoles. LXII.- Synthe`se univoque d′isoxazolones-3.
EÄ tude de leur tautomeric. Bull. Soc. Chim. Fr. 1970, 5, 1978-
1985.
(40) Drewes, H. R. Process for the preparation of cyclohexanone-2-
carboxamide. U.S. 4169952 to du Pont de Nemours; Chem. Abstr.
1979, 92, P 22150a.
(41) Enraf-Nonius. CAD-4 Software, version 5.0; Enraf-Nonius: Delft,
The Netherlands, 1989.
(42) Blessing, R. H. Data reduction and error analysis for accurate
single-crystal diffraction intensities. Crystallogr. Rev. 1987, 1,
3-58.
(43) Blessing, R. H. DREADD - data reduction and error analysis
for single-crystal diffractometer data. J . Appl. Crystallogr. 1989,
22, 396-397.
Refer en ces
(1) Morselli, P. L., Lo¨scher, W., Lloyd, K. G., Meldrum, B., Reynolds,
E. H., Eds. Neurotransmitters, Seizures, and Epilepsy; Raven
Press: New York, 1981.
(2) Fariello, R. G., Morselli, P. L., Lloyd, K. G., Quesney, L. F.,
Engel, J ., Eds. Neurotransmitters, Seizures, and Epilepsy II;
Raven Press: New York, 1984.
(3) Nistico, G., Morselli, P. L., Lloyd, K. G., Fariello, R. G., Engel,
J ., Eds. Neurotransmitters, Seizures, and Epilepsy III; Raven
Press: New York, 1986.
(4) Meldrum, B. S. Epilepsy and γ-aminobutyric acid-mediated
inhibition. Int. Rev. Neurobiol. 1975, 17, 1-36.
(5) Meldrum, B. S. Pharmacology of GABA. Clin. Neuropharmacol.
1982, 5, 293-316.
(6) Bowery, N. G., Nistico, G., Eds. GABA: Basic Research and
Clinical Applications; Pythagora Press: Rome, 1989.
(7) Nanavati, S. M.; Silverman, R. B. Design of potential anticon-
vulsant agents: mechanistic classification of GABA aminotrans-
ferase inactivators. J . Med. Chem. 1989, 32, 2413-2421.
(8) Schechter, P. J . Clinical pharmacology of vigabatrin. Br. J . Clin.
Pharmacol. 1989, 27, 19S-22S.
(9) Yunger, L. M.; Fowler, P. J .; Zarevics, P.; Setler, P. E. Novel
inhibitors of γ-aminobutyric acid (GABA) uptake: anticonvul-
sant actions in rats and mice. J . Pharmacol. Exp. Ther. 1984,
228, 109-115.
(10) Andersen, K. E.; Braestrup, C.; Grønwald, F. C.; J ørgensen, A.
S.; Nielsen, E. B.; Sonnewald, U.; Sørensen, P. O.; Suzdak, P.
D.; Knutsen, L. J . S. The synthesis of novel GABA uptake
inhibitors. 1. Elucidation of the structure-activity studies
leading to the choice of (R)-1-[4,4-bis(3-methyl-2-thienyl)-3-
butenyl]-3-piperidinecarboxylic acid (tiagabine) as an anticon-
vulsant drug candidate. J . Med. Chem. 1993, 36, 1716-1725.
(11) Krogsgaard-Larsen, P.; J ohnston, G. A. R. Inhibition of GABA
uptake in rat brain slices by nipecotic acid, various isoxazoles
and related compounds. J . Neurochem. 1975, 25, 797-802.
(12) J ohnston, G. A. R.; Krogsgaard-Larsen, P.; Stephanson, A. Betel
nut constituents as inhibitors of γ-aminobutyric acid uptake.
Nature (London) 1975, 258, 627-628.
(13) Krogsgaard-Larsen, P.; J ohnston, G. A. R. Structure-activity
studies on the inhibition of GABA binding to rat brain mem-
branes by muscimol and related compounds. J . Neurochem.
1978, 30, 1377-1382.
(14) Krogsgaard-Larsen, P.; Thyssen, K.; Schaumburg, K. Inhibitors
of GABA uptake. Syntheses and proton NMR spectroscopic
investigations of guvacine, (3RS,4SR)-4-hydroxypiperidine-3-
carboxylic acid, and related compounds. Acta Chem. Scand., Ser.
B 1978, B32, 327-334.
(15) Larsson, O. M.; Falch, E.; Schousboe, A.; Krogsgaard-Larsen,
P. GABA uptake inhibitors: kinetics and molecular pharmacol-
ogy. Adv. Biosci. (Oxford) 1991, 82, 197-200.
(16) J ohnston, G. A. R.; Stephanson, A. L.; Twitchin, B. Uptake and
release of nipecotic acid by rat brain slices. J . Neurochem. 1976,
26, 83-87.
(17) Larsson, O. M.; Krogsgaard-Larsen, P.; Schousboe, A. Charac-
terization of the uptake of GABA, nipecotic acid and cis-4-
hydroxynipecotic acid in cultured neurons and astrocytes.
Neurochem. Int. 1985, 7, 853-860.
(18) Larsson, O. M.; Krogsgaard-Larsen, P.; Schousboe, A. High-
affinity uptake of (RS)-nipecotic acid in astrocytes cultured from
mouse brain. Comparison with GABA transport. J . Neurochem.
1980, 34, 970-977.
(19) Schousboe, A.; Thorbek, P.; Hertz, L.; Krogsgaard-Larsen, P.
Effects of GABA analogues of restricted conformation on GABA
transport in astrocytes and brain cortex slices and on GABA
receptor binding. J . Neurochem. 1979, 33, 181-189.
(20) J uha´sz, G.; Ke´kesi, K. A.; Nyitrai, G.; Dobolyi, A.; Krogsgaard-
Larsen, P.; Schousboe, A. Differential effects of nipecotic acid
and 4,5,6,7-tetrahydro-isoxazolo[4,5-c]pyridin-3-ol on extracel-
(44) DeTitta, G. T. ABSORB: An absorption correction programme
for crystals enclosed in capillaries with trapped mother liquor.
J . Appl. Crystallogr. 1985, 18, 75-79.
(45) Sheldrick, G. M. SHELXS97. Program for the Solution of Crystal
Structures; University of Go¨ttingen: Go¨ttingen, Germany, 1997.

5414 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Falch et al.
(46) Sheldrick, G. M. Phase annealing in SHELX-90: Direct methods
for larger structures. Acta Crystallogr. 1990, A46, 467-473.
(47) International Tables for Crystallography; Wilson, A. J . C., Ed.;
Kluwer Academic Publishers: Dordrecht, The Netherlands,
1995; Vol. C, Tables 4.2.6.8 and 6.1.1.4.
(48) Main, P.; Fiske, S. J .; Hull, S. E.; Lessinger, L.; Germain, G.;
Declercq, J .-P.; Woolfson, M. M. MULTAN11/82. A System of
Computer Programs for the Automatic Solution of Crystal
Structures from X-ray Diffraction Data; Universities of York:
England, and Lovain: Belgium, 1982.
(49) Frenz, B. A.; Associates Inc. SDP Structure Determination
Package; College Station: Texas, and Enraf-Nonius: Delft, The
Netherlands, 1985.
(50) Sheldrick, G. M. SHELXL93. Program for Crystal Structure
Refinement; University of Go¨ttingen: Go¨ttingen, Germany, 1993.
(51) Fjalland, B. Inhibition by neuroleptics of uptake of [3H]GABA
into rat brain synaptosomes. Acta Pharmacol. Toxicol. 1987, 42,
73-76.
J M9904452