Potent 4-arylalkyl-substituted 3-isothiazolol GABAA competitive/noncompetitive antagonists: Synthesis and pharmacology

Article abstract of DOI:10.1021/jm050987l

The GABA_A agonists muscimol (1), 4,5,6,7-tetrahydroisoxazolo[5, 4-c]pyridin-3-ol (THIP, gaboxadol, 3), and the partial GABA_A agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 6a) and their respective 3-isothiazolol analogues thiomuscimol

Full text of DOI:10.1021/jm050987l

1388
J. Med. Chem. 2006, 49, 1388-1396
Potent 4-Arylalkyl-Substituted 3-Isothiazolol GABAA Competitive/Noncompetitive Antagonists:
Synthesis and Pharmacology
Dorte Krehan,† Signe ´ı Storustovu,‡ Tommy Liljefors,† Bjarke Ebert,‡ Birgitte Nielsen,† Povl Krogsgaard-Larsen,† and
Bente Frølund*,†
Department of Medicinal Chemistry, The Danish UniVersity of Pharmaceutical Sciences, DK-2100 Copenhagen, Denmark,
and Department of Electrophysiology, H. Lundbeck A/S, DK-2500 Valby, Denmark
ReceiVed October 4, 2005
The GABAA agonists muscimol (1), 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, gaboxadol, 3),
and the partial GABAA agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 6a) and their respective 3-isothiazolol
analogues thiomuscimol (2), thio-THIP (4), and thio-4-PIOL (7a) are ligands at the GABAA orthosteric
(recognition) site. The structure-activity relationships (SARs) between these structures are key elements of
a 3D-pharmacophore model for GABAA agonists and competitive antagonists [Frølund, B.; Jørgensen, A.
T.; Tagmose, L.; Stensbøl, T. B.; Vestergaard, H. T.; Engblom, C.; Kristiansen, U.; Sanchez, C.; Krogsgaard-
Larsen, P.; Liljefors, T. J. Med. Chem. 2002, 45, 2454-2468]. Prompted by this model, we now report the
synthesis and SAR of a series of analogues of 7a, in which the 4-position of the 3-isothiazolol was substituted
by alkyl or bulky aromatic groups such as naphthylmethyl and diphenylalkyl groups (7b-h). The compounds
have been pharmacologically characterized using receptor binding assays and two-electrode voltage-clamped
Xenopus oocytes expressing R1â3γ2S- and R4â3δ-containing receptors. The compounds show SARs comparable
with those of 6b-h but are generally 5-15 times more potent. The 2-naphthylmethyl, the 1-bromo-2-
naphthylmethyl, and the 3,3-diphenylpropyl analogues, compounds 7e, 7f, and 7h, respectively, show affinity
in the low-nanomolar range (Ki 2-10 nM). Interestingly, 7e and 7h exhibited a mixed antagonist profile
consisting of a noncompetitive component in the picomolar range and a competitive component at
concentrations above 1 nM. This unique profile was shown not to be due to either use dependence or kinetic
effects. This antagonist profile of 7e and 7h was particularly pronounced at R4â3δ-containing GABAA
receptors, which showed three- and 10-fold selectivity for 7h and 6h, respectively.
Introduction
The affinity of the sulfur analogue of THIP (thio-THIP, 4) is
more than 300 times lower than that of THIP itself,6 whereas
the 3-isoxazolol ring in muscimol can be replaced by an
isothiazolol ring to give thiomuscimol (2) without significant
loss of GABAA receptor affinity.7 The affinity of thio-4-PIOL
(7a), on the other hand, is about an order of magnitude higher
than that of 6a.8
These observations have formed part of the basis for the
hypothesis previously proposed for different binding modes in
the receptor binding cavity for muscimol, THIP, and 4-PIOL.9,10
Moreover, according to the resulting 3D-pharmacophore model,
a cavity exists in the vicinity of the 4-position of the 3-isoxazolol
ring of 4-PIOL but not of the corresponding position in
muscimol or THIP.9 Exploration of this cavity has led to a series
of potent competitive GABAA antagonists exemplified by
compounds 6e, f, and h.10,11
The major inhibitory neurotransmitter 4-aminobutyric acid
(GABA) exerts its effect in the central nervous system through
two classes of receptors, the ionotropic GABAA and GABAC
receptors and the metabotropic GABAB receptors. The GABA
neurotransmitter system is involved in a wide range of physi-
ological and pathological processes, and ligands for the het-
eromeric GABAA receptor complex have attracted considerable
interest as potential therapeutics in the treatment of anxiety,
seizures, schizophrenia, depression, and sleep disorders.1
The GABAA receptors belong to a superfamily of ligand-
gated ion channels that also includes the nicotinic acetylcholine,
the glycine, and the serotonin (5-HT3) receptors. The GABAA
receptor is a transmembrane allosteric protein complex com-
posed of five subunits. So far, 16 GABAA receptor subunits
have been identified and divided into seven groups (R1-6, â1-3
,
To validate and further extend this hypothesis/model, we here
describe the synthesis and pharmacological characterization of
a series of thio-4-PIOL analogues (7b-h) in which the
4-position of the 3-isothiazolol is substituted by alkyl and bulky
aromatic groups such as naphthylmethyl and diphenylalkyl
groups.
γ1-3, δ, ꢀ, π, and θ). Although a high number of different
subunit compositions are possible, only a limited number has
been identified, the R1â2γ2 apparently being the most abundant
in the human central nervous system.2,3
Muscimol (1)4 and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-
3-ol (THIP, gaboxadol, 3)4,5 (Figure 1) are both standard
GABAA receptor agonists. Recently, THIP has in in vivo studies
been shown to act as a functionally selective extrasynaptic
GABAA agonist with high potency and relative efficacy at the
extrasynaptically expressed R4â3δ. THIP is currently in phase
III clinical development as a hypnotic.
Results
Chemistry. In the synthesis previously described for thio-
4-PIOL (7a), the 3-isothiazolol moiety was formed by oxidative
cyclization of the corresponding â-thioamide.8 In this study, the
3-isothiazolol moiety was synthesized as shown in Scheme 1,
using a strategy previously described for the synthesis of thio-
THIP (4).12 The (E)-isomer of the R,â-unsaturated amide, 9,
was synthesized using N-benzyl-protected 4-formylpiperidine
* Corresponding author. Phone: (+45) 35306495. Fax: (+45) 35306040.
E-mail: bfr@dfuni.dk.
† The Danish University of Pharmaceutical Sciences.
‡ H. Lundbeck A/S.
10.1021/jm050987l CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/21/2006

Substituted 3-Isothiazolol GABAA Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1389
Scheme 1a
Figure 1. Structures of GABA, the GABAA agonists muscimol (1),
thiomuscimol (2), THIP (3), Thio-THIP (4), the GABAA antagonist
SR 95531 (5), the low-efficacy partial GABAA agonists 4-PIOL (6a)
and thio-4-PIOL (7a), the 4-PIOL analogues 6b-h, and the new
3-isothiazolols (7b-h).
a Reagents: (a) triethylphosphonoacetamide, NaH, DME; (b) ClCO2CH3;
(c) CH3COSH, EtOAc; (d) aq NaOH; (e) 35% aq H2O2, 45 °C; (f) SO2Cl2,
ClCH2CH2Cl; (g) i-PrBr, K2CO3, DMF, 0 °C; (h) ICl, HOAc, H2O, 80 °C;
(i) EtMgBr, THF, -30 °C; (j) RCHO (R, see Figure 1), THF, 0 °C; for
compound 14b, (1) DMF, THF, 0 °C and room temperature, (2) NaBH4,
MeOH, 0 °C and room temperature; (k) TFA, Et3SiH, CH2Cl2, 0 or 50 °C;
(l) 33% HBr, HOAc, 65 °C.
aldehyde, 8, and triethylphosphonoacetamide. Sulfur was in-
troduced by a conjugated addition of thioacetic acid to 10 giving
thioester 11 in good yield. Compound 11 was hydrolyzed to
the corresponding thiol, oxidized to the disulfide, which was
then cyclized using sulfuryl chloride, to give N-protected thio-
4-PIOL, 12.
analogues (7b-h) synthesized in the present study showed
affinity selectively for the GABAA receptor sites (Table 1) and
exhibited similar structure-activity relationships (SARs) as
described for the corresponding 3-isoxazolol analogues 6b-h
(Table 1).10,11 Alkyl groups such as methyl and propyl groups
as well as a benzyl group are tolerated (compounds 7b-d)
showing affinities for the GABAA receptor sites comparable to
or slightly higher than that of 7a. Addition of two phenyl groups
to the terminal carbon of 7c, as in the 3,3-diphenylpropyl
analogue 7h, gave a 46-fold increase in affinity relative to the
propyl analogue 7c. The 2,2-diphenylethyl analogue 7g shows
affinity comparable to that of 7c. In accordance with the results
from the 3-isoxazolol series,11 introduction of a bromo sub-
stituent in the 1-position of the naphthyl ring system of
compound 7e affording compound 7f provided an enhanced
affinity relative to that of the 2-naphthylmethyl analogue, 7e.
Except for the 2,2-diphenylethyl analogue 7g, the affinities of
the 3-isothiazolol series show a 5-15-fold higher affinity for
the GABAA receptor sites compared to that of the 3-isoxazolol
series (Table 1). The 1-bromo-substituted analogue, 7f, showing
a Ki value of 1.8 nM, is the compound of highest affinity in
this study.
With the use of a previously reported metal-iodine exchange
reaction,10 the iodinated analogue 14 underwent iodine-
magnesium exchange using either ethylmagnesium chloride or
bromide followed by reaction with the appropriate electrophile
to give the hydroxy compounds 15b-h (Scheme 1). The methyl
group in compound 7b was introduced as illustrated in Scheme
1 using DMF as the electrophile followed by reduction to give
the hydroxymethyl compound 15b. The benzylic hydroxy group
was subsequently removed by ionic hydrogenation using tri-
fluoroacetic acid and triethylsilane in dichloromethane to give
the compounds 16b-h. Deprotection to give the target com-
pounds 7b-h was accomplished by treatment with hydrogen
bromide in acetic acid.
In Vitro Pharmacology. The compounds were pharmaco-
logically characterized in receptor binding studies based on rat
brain membrane preparations and on human R1â3γ2S and R4â3δ
GABAA receptors expressed in Xenopus oocytes using two-
electrode voltage-clamp electrophysiology. The affinities for
GABAA and GABAB receptor sites, using [3H]muscimol and
[3H]GABA, respectively, were determined using methods
described previously.11 Like 7a, all of the 3-isothiazolol

1390 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Krehan et al.
Table 1. Receptor Binding and in Vitro Electrophysiological Data
Ki (nM) (pKi ( SEM)a
[3H]muscimol bindingb
electrophysiologyc
5
74
240
6ad
6bd
6cd
6dd
6ed
6fe
6gd
6hd
7a
7b
7c
7d
7e
9100
10000
6600
3800
49
10
360
68
1870 (5.73 ( 0.04)
2200 (5.66 ( 0.01)
440 (6.35 ( 0.02)
331 (6.48 ( 0.05)
5.2 (8.28 ( 0.07)
1.8 (8.75 ( 0.14)
388 (6.41 ( 0.09)
9.6 (8.02 ( 0.17)
110000
26000
4600
4000
370
42
810
20
25704 (4.59 ( 0.16)
2880 (5.54 ( 0.08)
230 (6.64 ( 0.06)
465 (6.33 ( 0.09)
22 (7.66 ( 0.04)
3.0 (8.53 ( 0.09)
720 (6.14 ( 0.15)
1.2 (8.92 ( 0.04)
7f
7g
7h
a Ki values were calculated from pKi values found in brackets. b Standard
receptor binding on rat brain synaptic membranes, n g 3. c Two-electrode
voltage-clamp recordings on Xenopus oocytes expressing R1â3γ2S GABAA
receptor subunits n ) 4. d Ref 10. e Ref 11.
Figure 2. (a) GABA concentration-response curves (full lines) in
the absence and presence of a fixed concentration (2, 20, or 200 nM)
of 7h obtained from the same Xenopus oocyte expressing R1â3γ2S
GABAA receptor subunits. Data were normalized with respect to the
maximum response (GABA Imax) and subsequently fitted to eq 2. The
dotted curve represents data from another latter oocyte exposed to 0.2
nM 7h + variable GABA concentrations. From the latter oocyte, a
practically identical GABA concentration-response curve was obtained,
permitting a direct comparison between the data sets. (b) GABA
concentration-response curves in the absence and presence of a fixed
concentration (1, 3, or 10 nM) of 7h obtained from the same Xenopus
oocyte expressing R4â3δ GABAA receptor subunits. Data were normal-
ized with respect to the maximum response (GABA Imax) and
subsequently fitted to eq 2.
The pharmacological profile of the compounds was studied
using a two-electrode voltage-clamp technique on human
R1â3γ2S GABAA receptors expressed in Xenopus oocytes. At
fixed concentrations of compounds, 7b-h, the effect on a
GABA concentration-response curve was investigated. As
shown for the corresponding 3-isoxazolol analogues,10,11 all of
the new compounds were characterized as antagonists and did
induce a rightward shift of the GABA concentration-response
curve, suggesting competitive antagonism. However, compounds
7e and 7h behaved as both competitive and noncompetitive
antagonists. Thus, in addition to a rightward shift of the dose-
response curve, both compounds significantly reduced the
maximum response to GABA to approximately 60% (relative
to the maximum GABA response in the absence of the test
compound). With the use of 7h as a probe, this mixed
antagonism was characterized in further details. As illustrated
in Figure 2a, two distinct and separable mechanisms appeared
to affect the response to GABA at R1â3γ2S-containing receptors.
At very low concentrations, in the picomolar range, a dose-
dependent reduction in the response to GABA was seen. The
reduction was saturable in the sense that increasing the doses
of 7h did not lead to reduction of the maximum GABA
responses below 60% of the control response. At concentrations
above 1 nM, 7h shifted the remaining GABA concentration-
response curve parallel rightward in agreement with classical
competitive interaction theory.
noncompetitive antagonist profile was seen, where the suppres-
sion of the maximal response to GABA is even more pro-
nounced (Emax 40%) (Figure 2b). In line with previous
observations at the R4â3δ receptor subtype, the potency of
compound 7h is 3-fold higher than at the R1â3γ2S receptor
subtype, with approximate Ki values for the competitive
component of 0.35 and 1.0 nM, respectively.
Since a discrepancy of the pharmacological profiles of the
3-isothiazolol analogues of 4-PIOL and the corresponding
3-isoxazolol analogues seems to exist, two 3-isoxazolol ana-
logues, 6e and 6h, were studied in detail at the R1â3γ2S receptor
subtype. As previously observed, the 3-isoxazolol analogue 6e
showed a pure competitive antagonist effect, whereas the
3-isoxazolol analogue 6h in addition to the rightward shift of
the GABA concentration-response curve exhibited a weak
suppression of the maximal effect of GABA (Emax 90%). On
the basis of this observation, the effect of the 3-isoxazolol
analogue 6h at the R4â3δ receptor subtype was examined as
well. As observed on the R1â3γ2S receptor subtype, the 3-isox-
azolol analogue showed a shift of the GABA dose-response
curve, and in agreement with data for 7h, a more pronounced
suppression of the maximal effect of GABA (Emax 70%) was
seen. Furthermore, the 3-isoxazolol analogue 6h exhibits a 10-
fold higher potency at the R4â3δ than at the R1â3γ2S receptor
subtype (Ki ) 2.3 and 26 nM, respectively) consistent with a
more pronounced tendency for subtype selectivity than that
shown for 7h.
This unusual pharmacological profile observed for compound
7e and 7h might be the consequence of a slow on-rate or some
degree of use dependence. To test for a possible use dependence,
the consequence of repeated administration of agonist in the
presence of antagonists was examined. Furthermore, the impact
of an antagonist pretreatment time as a marker for on-rate was
characterized. In both experiments, no differences were detected
between the responses ruling out the two scenarios mentioned
above (Figure 3).
Compound 7h was further characterized at R4â3δ-containing
GABAA receptors. This receptor population, which is exclu-
sively extrasynaptically located, has previously been shown to
be particularly sensitive to GABAA agonists13 and is now
thought to mediate the pharmacological effects of THIP
(gaboxadol), which is under development as a hypnotic. As
observed for the R1â3γ2S receptor subtype, a mixed competitive/
The benzyl analogue, 7d, and the highly potent 1-bromo-2-
naphthylmethyl analogue, 7f, slightly altered the Emax of
GABA, whereas the rest of the series of compounds in the

Substituted 3-Isothiazolol GABAA Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1391
Figure 3. Effect of 7h on the response to 2.4 µM GABA (black bar). The figure displays traces from a voltage-clamped Xenopus oocyte expressing
R1â3γ2S GABAA receptor subunits. The oocyte was clamped at -70 mV, and 7h (grey bar) was added with different pretreatment periods (1, 5, and
10 min).
present study were shown to be purely competitive antagonists.
Ki values for the competitive component of the tested com-
pounds in this study are given in Table 1 and show a fairly
good correlation to the obtained binding affinities.
Discussion
Previous SAR studies have disclosed that the 3-isothiazolol
analogue of muscimol (1), thiomuscimol (2), is slightly weaker
than muscimol, thio-THIP (4) is markedly less active than THIP
(3). Thio-4-PIOL (7a), however, was shown to be a partial
GABAA agonist slightly more potent and efficacious than the
parent compound, 4-PIOL (6a).
Figure 4. Pharmacophore model for GABAA receptor ligands showing
the proposed binding modes of muscimol (2, green carbon atoms),
A summary of these and other SAR studies led to a hypothesis
of the binding mode of heterocyclic GABAA agonists and
4-PIOL (6a, gray carbon atoms), and THIP (3, orange carbon atoms).
provided a 3D-pharmacophore model for this group of com-
The cyan-colored tetrahedrons represent sterically “forbidden” volumes
(receptor essential volumes). Dashed lines indicate hydrogen bonds.
pounds.9,10,14 On the basis of this model, we have previously
described a series of 3-isoxazolol analogues of 4-PIOL (6a) as
competitive GABAA antagonists. Introduction of bulky aromatic
substituents, such as 3,3-diphenylpropyl and 2-naphthylmethyl,
in the 4-position of the 3-isoxazolol ring of 4-PIOL provided
6h and 6e, respectively, showing markedly higher affinity for,
and more potent effects at, the GABAA receptors than the parent
compound. The results of these SAR studies strongly suggest
the existence of a large cavity in the receptor binding site capable
of accommodating these large substituents, with specific sites
for ligand interactions. All of these analogues were shown to
be moderate to potent competitive antagonists at the GABAA
receptor.
Some hydrogens are removed for clarity.
algorithm. The free energies of hydration of the anions of
3-isoxazolol and 3-isothiazolol are calculated to be -77.9 and
-75.7 kcal/mol, respectively, indicating a significantly lower
desolvation energy for the sulfur compound. Although this
rationalizes the higher affinities of the thio-4-PIOL analogues
7a-h as compared to those of the corresponding 4-PIOL
analogues, it cannot explain the virtually identical affinities of
muscimol (1) and thiomuscimol (2) and the significantly (300-
fold) lower affinity of thio-THIP (4) in comparison to that of
THIP (3).
Now, a series of 3-isothiazolol analogues structurally related
to the 3-isoxazolol analogues mentioned above have been
synthesized and pharmacologically characterized. In general, a
similar SAR as seen for the corresponding 3-isoxazolol series
was observed, although the 3-isothiazolol analogues generally
showed higher affinity and potency as compared to that of the
3-isoxazolol analogues. The 2-naphthylmethyl, the 1-bromo-2-
naphthylmethyl, and the 3,3-diphenylpropyl analogues, com-
pounds 7e, 7f, and 7h, showed affinities and potencies in the
low-nanomolar range considerably higher than that of the
standard competitive GABAA antagonist SR 95531 (Table
1).15,16
The higher affinities of the 3-isothiazolol compounds 7a-h
as compared to the corresponding 3-isoxazolol compounds 6a-h
(Table 1) may be rationalized by the higher lipophilicities and
the lower desolvation energies of the sulfur analogues. The log
P values for 3-isoxazolol and 3-isothiazolol are calculated to
be 0.87 and 2.24, respectively, by using Crippen’s fragmentation
method and 0.65 and 2.24, respectively, by using the ClogP
We have previously reported a pharmacophore model for
GABAA receptor ligands based on muscimol (2) and 4-PIOL
(6a).9 The main features of this model are that the 3-isoxazolol
rings of the two compounds do not overlap in their proposed
binding modes and that the two compounds interact with
different conformations of an arginine residue located at the
GABAA recognition site. By docking THIP (3) into this
pharmacophore model it is revealed that the 3-isoxazolol ring
in THIP (3) occupies yet another position in the receptor cavity
as shown in Figure 4. The docking to the pharmacophore model
was done by requiring overlap of the protonated amino groups
of all three compounds, hydrogen bonding by the isoxazolol
group of THIP (3) to one of the arginine conformers, and
avoidance of previously identified sterically “forbidden” receptor
volumes (receptor essential volumes) represented by tetrahedrons
in Figure 4.
In contrast to the ring oxygen in 4-PIOL (6a), the ring
oxygens in muscimol (1) and THIP (3) are both in the vicinity
of a previously identified sterically “forbidden” volume indicated

1392 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Krehan et al.
by an arrow in Figure 4. The ring oxygen in THIP (3) is
significantly closer to the forbidden volume than the corre-
sponding oxygen in muscimol (1). The replacement of the ring
oxygen by the much larger sulfur atom is expected to decrease
the affinity of thio-THIP (4) significantly more than that of
thiomuscimol (2). Thus, the expected affinity increase of
thiomuscimol (2) and thio-THIP (4) in comparison to that of
muscimol (1) and THIP (3) due to favorable lipophilicities and
desolvation energies is counteracted by an affinity decrease due
to steric repulsions between the sulfur atom and a sterically
“forbidden” volume. This affinity decrease is significantly larger
for thio-THIP (4) than for thiomuscimol (2). Thus, the different
affinity changes due to an oxygen-sulfur exchange in muscimol
(1), THIP (3), and 4-PIOL (6a) may be rationalized on the basis
of different positions of their 3-isoxazolol rings in the receptor
cavity as proposed by the pharmacophore model.
Taken into account that thio-4-PIOL (7a) exhibits a higher
efficacy than 4-PIOL (6a) and that the methyl and ethyl
analogues of 4-PIOL show a weak agonist effect,10,17 it was
expected that the corresponding thio-4-PIOL analogues would
be partial agonists of higher affinity and efficacy. Actually, the
methyl and propyl analogues, 7b and 7c, did show higher
affinity but were without detectable efficacy.
thio-4-PIOL (7a) with substituents, such as alkyl and more bulky
aromatic groups, in the 4-position of the 3-isothiazolol ring.
The 3-isothiazolol analogues exhibit similar SARs as shown
for the corresponding 3-isoxazolol analogues but show 5-15-
fold higher affinity and potency at the GABAA receptor. The
2-naphthylmethyl, the 1-bromo-2-naphthylmethyl, and the 3,3-
diphenylpropyl analogues, compounds 7e, 7f, and 7h, respec-
tively, show affinity in the low-nanomolar range (Ki 2-10 nM).
In addition, the 3-isothiazolol analogue 7e exhibit 3-fold
selectivity for the extrasynaptic R4â3δ receptor subtype over
the synaptic R1â3γ2S receptor subtype, whereas a 10-fold
selectivity was shown for the corresponding 3-isoxazolol
compound 6e.
Like the 3-isoxazolol analogues most of the 3-isothiazolol
analogues were shown to be competitive antagonists. However,
the antagonist effects of 7e and 7h were shown to consist of a
competitive and a noncompetitive component operating in
different concentration ranges. In the picomolar range, a
noncompetitive saturable component prevails reducing the
maximal response of GABA, whereas at concentrations in the
nanomolar range, a competitive component becomes significant.
These results indicate an additional high-affinity binding site
in or near the orthosteric site and will be subject of further
studies.
Interestingly, the 2-naphthylmethyl and the 3,3-diphenylpro-
pyl 3-isothiazolol analogues, 7e and 7h, did show a markedly
different pharmacological profile than previously shown for the
corresponding 3-isoxazolol analogues at the synaptic R1â3γ2S
receptor subtype. As expected for a competitive antagonist a
rightward shift of the GABA concentration-response curve was
observed, but in addition the compounds did cause a reduction
of the maximal response of GABA down to 60%. The apparent
dual behavior could be separated into two components, a
noncompetitive saturable component at subnanomolar concen-
trations and a competitive component at low-nanomolar con-
centrations. A similar antagonist profile was shown for the
compound at the extrasynaptic R4â3δ receptor subtype where
the noncompetitive effect was even more pronounced (Emax
40%). At this receptor subtype a minor reduction of the maximal
effect of GABA (Emax 70%) was seen for the 3-isoxazolol
analogue 6e.
It is very unexpected that compound 6e, 7e, and 7h in addition
to a competitive component possess a noncompetitive compo-
nent. Since these compounds are highly lipophilic one might
expect that part of the noncompetitive action could be due to
slow on-rate either to the orthosteric (recognition) site or to a
site located within the chloride channel. However, since
experiments with variation in pretreatment time or repeated
administration showed no indication of use dependence or slow
on-rate, these explanations may be rejected. The insurmountable
inhibition of GABA responses may therefore be the consequence
of interaction with a site, an amino acid residue, located outside
the ion channel and probably very close to the orthosteric
binding site. In agreement with this interpretation are binding
data, which all point toward a purely competitive interaction.
If the secondary binding site is located very close to the
orthosteric binding site, and since the potency at this site is
within the picomolar range, it might be possible to selectively
label it at very low concentrations of either radioactively labeled
compounds or photoaffinity probes based on the structures of
7e and 7h.
Experimental Section
Chemistry. General Procedures. All reactions involving air-
sensitive reagents were performed under a N2 atmosphere using
the syringe-septum technique, and glassware was flame-dried in
vacuo prior to use. Compounds were visualized on TLC (silica gel
60 F254 plates; Merck) using UV light and KMnO4 spraying reagent.
2,4-Dinitrophenylhydrazin (DNP) was used to visualize aldehydes
and ninhydrin to visualize secondary and primary amines. Com-
pounds containing the 3-isothiazolol moiety were visualized using
FeCl3 spraying reagent. Column chromatography (CC) was per-
formed on Millipore A/S silica gel 60A (70-200 µm) and dry
column vacuum chromatography (DCVC) was performed on silica
gel 60A (35-70 µm) from Millipore A/S. All solvents and reagents
were purchased from commercial sources and used without further
purification except for DME, DMF, and THF, which were stored
over 4 Å molecular sieves. Melting points were determined in open
capillary tubes and are uncorrected. NMR spectra were recorded
on a 300 MHz Varian Gemini spectrometer in CDCl3 solutions
using TMS as an internal standard or in CD3OD solutions.
Elemental analyses were carried out at the Analytical Research
Department, H. Lundbeck A/S, Denmark or by J. Theiner, Mi-
croanalytical Laboratory, Institute of Physical Chemistry, University
of Vienna, Austria and are within (0.4% of the calculated values
unless otherwise stated.
(E)-3-(1-Benzyl-4-piperidyl)-2-propenamide (9). NaH (60%,
2.72 g, 67.9 mmol) was added slowly to a suspension of dieth-
ylphosphonoacetamide (13.25 g, 67.9 mmol) in DME (150 mL)
followed by dropwise addition of a solution of compound 818 (5.52
g, 27.2 mmol) in DME (6 mL). The reaction mixture was left for
90 min at room temperature and then quenched with H2O (60 mL).
The pH was adjusted to 2, and the acidic phase was washed with
Et2O (3 × 100 mL). The pH was adjusted to 10 precipitating the
product. The alkaline H2O phase was filtered and extracted with
Et2O (3 × 100 mL). The combined organic phases were dried and
evaporated in vacuo and combined with the filtered product.
Recrystallization (EtOAc) afforded 9 as colorless crystals (4.9 g,
1
70%): mp 153.5-154 °C. H NMR (CDCl3): δ 7.37-7.22 (5H,
m), 6.83 (1H, dd, J ) 15.6 Hz, J ) 6.8 Hz), 5.80 (1H, dd, J )
15.6 Hz, J ) 1.2 Hz), 5.49 (2H, bs), 3.52 (2H, s), 2.97-2.87 (2H,
m), 2.23-1.80 (3H, m), 1.79-1.67 (2H, m), 1.60-1.43 (2H, m).
13C NMR (CDCl3): δ 168.09, 150.11, 138.05, 129.31, 128.29,
127.16, 120.89, 63.25, 53.09, 38.29, 30.82. Anal. (C15H20N2O) C,
H, N.
Conclusion
In conclusion, we have synthesized and pharmacologically
characterized a series of analogues of the partial GABAA agonist

Substituted 3-Isothiazolol GABAA Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1393
(E)-3-(1-Methoxycarbonyl-4-piperidyl)-2-propenamide (10).
Methyl chloroformate (3 mL, 38.7 mmol) was added quickly
through a condenser to compound 9 (5 g, 19.4 mmol). The reaction
was left for 3 h, evaporated in vacuo, and added to H2O (75 mL).
The aqueous phase was extracted with EtOAc (10 × 100 mL). The
combined organic phases were dried and evaporated giving a white
solid. Recrystallization (EtOAc) afforded 10 (2.91 g, 71%) as
s), 3.08 (1H, dt, J ) 12 Hz, J ) 3.6 Hz), 2.90 (2H, bt, J ) 12.6
Hz), 2.03 (2H, bd, J ) 12.6 Hz), 1.66-1.48 (2H, m), 1.40 (6H, d,
J ) 6.3 Hz). 13C NMR (CDCl3): δ 170.20, 166.86, 159.31, 155.88,
72.51, 67.31, 52.63, 43.81, 39.33, 31.54, 21.87. Anal. (C13H19-
IN2O3S) C, H, I, N, S.
4-Hydroxymethyl-3-Isopropoxy-5-(1-methoxycarbonyl-4-pi-
peridyl)isothiazole (15b). To a solution of 14 (1.02 g, 2.49 mmol)
in dry THF cooled to -30 °C was added EtMgBr (1 M, 2.49 mmol)
dropwise at -30 °C. The reaction was left at 0 °C for 2.5 h after
which a solution of dry DMF (0.38 mL, 4.99 mmol) in dry THF
was added dropwise at 0 °C. Stirring was continued at room
temperature overnight. The reaction was quenched with aq saturated
NH4Cl (20 mL) and extracted with EtOAc (3 × 20 mL). The
combined organic phases were dried, evaporated in vacuo, and
purified by DCVC [petroleum ether f EtOAc] affording a yellow
oil (668 mg). The yellow oil was dissolved in MeOH (25 mL),
cooled to 0 °C, and added to NaBH4 (0.12 g, 3.21 mmol) in small
portions. Stirring was continued 20 min at 0 °C and 2.5 h at room
temperature followed by evaporation in vacuo. The residue was
added to H2O (30 mL) and extracted with EtOAc (3 × 30 mL).
The combined EtOAc phases were dried and evaporated in vacuo.
DCVC [petroleum ether f EtOAc] gave 15b as a colorless oil
(551 mg, 70%). 1H NMR (CDCl3): δ 5.15 (1H, hep, J ) 6.3 Hz),
4.48 (1H, s), 4.23 (2H, bs), 3.69 (3H, s), 3.19 (1H, tt, J ) 11.9, J
) 3.6 Hz), 3.20-2.69 (3H, m), 1.97 (2H, bd, J ) 12.3 Hz), 1.58
(2H, dq, J ) 12.5 Hz, J ) 4.1 Hz), 1.36 (6H, d, J ) 6.3 Hz). 13C
NMR (CDCl3): δ 169.45, 166.18, 155.36, 120.57, 71.12, 54.55,
52.43, 43.74, 35.69, 32.87, 21.95. Anal. (C14H22N2O4S) C, H, N,
S.
1
colorless crystals: mp 149-151.5 °C. H NMR (CDCl3): δ 6.82
(1H, dd, J ) 15.6 Hz, J ) 6.6 Hz), 5.83 (1H, dd, J ) 15.6 Hz, J
) 1.5 Hz), 5.69 (1H, bs), 4.16 (2H, bs), 3.70 (3H, s), 2.92-2.77
(2H, m), 2.40-2.13 (2H, m), 1.82-1.70 (2H, m), 1.47-1.25 (2H,
m). 13C NMR (CDCl3): δ 167.87, 156.00, 149.31, 121.13, 52.56,
43.48, 38.23, 30.53. Anal. (C10H16N2O3) C, H, N.
3-Acetylsulfanyl-3-(1-methoxycarbonyl-4-piperidyl)propana-
mide (11). To a suspension of compound 10 (1.1 g, 5.18 mmol)
was added thioacetic acid (410 µL, 5.70 mmol). The reaction was
left for 3 days at room temperature followed by evaporation in
vacuo, and recrystallization (EtOAc) gave 11 (1.15 g, 77%) as
1
colorless crystals: mp 125-126 °C. H NMR (CDCl3): δ 6.08-
5.88 (2H, m), 4.16 (2H, bs), 3.89-3.76 (1H, m), 3.68 (3H, s), 2.80-
2.43 (4H, m), 2.35 (3H, s), 1.96-1.63 (3H, m) 1.40-1.09 (2H,
m). 13C NMR (CDCl3): δ 195.60, 172.76, 155.85, 52.45, 45.43,
43.77, 43.66, 39.29, 38.80, 30.64, 29.39, 28.33. Anal. (C12H20N2O4S)
C, H, N.
5-(1-Methoxycarbonyl-4-piperidyl)isothiazol-3-ol (12). A solu-
tion of NaOH (1.1 g, 27.7 mmol) in H2O (10 mL) was added to
compound 11 (4.0 g, 13.9 mmol), and the reaction was stirred for
4 h at room temperature. The pH was adjusted to 5 with 2 M
H2SO4, and the acidic mixture was extracted with CH2Cl2 (3 × 20
mL). The combined organic phases were dried and evaporated in
vacuo affording a pale yellow solid. The solid was suspended in
H2O (20 mL), heated to 45 °C, and H2O2 (35%, 660 µL, 6.8 mmol)
was added dropwise. The reaction was left for 24 h at 45 °C
followed by evaporation in vacuo giving a white foam, which was
dissolved in ClCH2CH2Cl (18 mL). SO2Cl2 (0.56 mL, 6.89 mmol)
was added slowly to the solution, and the reaction was left for 24
h at room temperature before adding additional SO2Cl2 (0.27 mL,
3.35 mmol). Stirring was continued for 8 h after which additional
SO2Cl2 (0.27 mL, 3.35 mmol) was added. The reaction was left
for 16 h followed by evaporation in vacuo. The residue was added
H2O (50 mL) and extracted with EtOAc (5 × 50 mL). The
combined organic phases were dried and evaporated. Recrystalli-
zation (EtOAc) afforded 128 (1.41 g, 42%) as light brown crystals.
1H NMR (CDCl3): δ 6.33 (1H, s), 4.23 (2H, bs), 3.72 (3H, s),
3.07-2.83 (3H, m), 2.00 (2H, bd, J ) 12.3 Hz), 1.67 (2H, dq, J )
12.3 Hz, J ) 4.5 Hz).
3-Isopropoxy-5-(1-methoxycarbonyl-4-piperidyl)isothiazole (13).
To a solution of 12 (1.12 g, 4.62 mmol) in dry DMF (30 mL) was
added K2CO3 (703 mg, 5.08 mmol), and the mixture was stirred at
60 °C for 30 min. Isopropyl bromide (650 µL, 6.93 mmol) was
added to the mixture, and stirring was continued at 60 °C for 20 h.
The reaction mixture was added to H2O (30 mL) and extracted
with petroleum ether (80-100 °C) (4 × 15 mL). The combined
organic phases were dried and evaporated in vacuo. CC [toluene-
EtOAc (2:1)] gave 13 (731 mg, 56%) as a pale yellow oil. 1H NMR
(CDCl3): δ 6.31 (1H, s), 5.21-5.06 (1H, m), 4.21 (2H, bs), 3.71
(3H, s), 3.03-2.82 (3H, m), 1.98 (2H, bd, J ) 12.6 Hz), 1.71-
1.51 (2H, m), 1.40-1.30 (6H, m). 13C NMR (CDCl3): δ 172.61,
168.43, 155.89, 109.15, 70.94, 52.55, 43.62, 36.26, 32.77, 21.92.
Anal. (C13H20N2O3S) C, H, N, S.
4-(1-Hydroxypropyl)-3-isopropoxy-5-(1-methoxycarbonyl-4-
piperidyl)isothiazole (15c). To a solution of 14 (750 mg, 1.83
mmol) in dry THF (6 mL), cooled to -30 °C, was added EtMgBr
(1 M, 1.83 mmol) dropwise at -30 °C. The reaction was left at 0
°C for 3 h after which a solution of propionaldehyde (0.13 mL,
1.83 mmol) in dry THF (0.75 mL) was added dropwise at 0 °C.
Stirring was continued at room temperature overnight. The reaction
was quenched with saturated NH4Cl (15 mL) and extracted with
EtOAc (3 × 15 mL). The combined organic phases were dried
and evaporated in vacuo. DCVC [petroleum ether f EtOAc] gave
1
15c (464 mg, 74%) as a light yellow oil. H NMR (CDCl3): δ
5.19 (1H, hep, J ) 6.2), 4.57 (1H, bt, J ) 6.9 Hz), 4.24 (2H, bs),
3.70 (3H, s), 3.23 (1H, tt, J ) 12.1 Hz, J ) 3.6 Hz), 3.09 (1H, bs),
2.95-2.74 (2H, m), 2.20-1.83 (3H, m), 1.80-1.66 (1H, m), 1.65-
1.47 (2H, m), 1.37 (6H, d, J ) 6.6 Hz), 0.93 (3H, t, J ) 7.5 Hz).
13C NMR (CDCl3): δ 167.43, 165.32, 155.37, 123.00, 71.29, 69.12,
52.45, 43.88, 35.69, 33.43, 30.13, 22.04, 10.44. Anal. (C16H26N2O4S)
C, H, N, S.
3-Isopropoxy-5-(1-methoxycarbonyl-4-piperidyl)-4-(phenyl-
hydroxymethyl)isothiazole (15d) was synthesized as described for
15c using 14 (400 mg, 0.97 mmol) in dry THF (4 mL), EtMgBr (1
M, 0.97 mmol), and benzaldehyde (99 µL, 0.97 mmol). The crude
product was purified by DCVC [toluene f toluene-EtOAc (1:1)]
affording 15d (214 mg, 56%) as a white foam. 1H NMR (CDCl3):
δ 7.36-7.21 (5H, m), 5.88 (1H, s), 5.16 (1H, hep, J ) 6.2 Hz),
4.15 (2H, bs), 3.67 (3H, s), 3.15 (1H, tt, J ) 12 Hz, J ) 3.6 Hz),
2.86-2.58 (2H, m), 1.88 (1H, bd, J ) 12.9 Hz), 1.67 (1H, bd, J )
12.9 Hz), 1.59-1.36 (2H, m), 1.33 (3H, d, J ) 6.2 Hz), 1.22 (3H,
d, J ) 6.2 Hz). 13C NMR (CDCl3): δ 169.11, 165.81, 155.78,
142.75, 128.19, 127.26, 125.67, 122.88, 71.64, 68.28, 52.48, 43.81,
35.69, 33.31, 32.85, 21.92, 21.71.
4-Iodo-3-isopropoxy-5-(1-methoxycarbonyl-4-piperidyl)isothi-
azole (14). To a solution of compound 13 (840 mg, 2.95 mmol) in
HOAc (5 mL), a solution of ICl (0.22 mL, 4.43 mmol) in HOAc
(7 mL) was added dropwise followed by addition of H2O (20 mL).
The reaction was stirred at 80 °C for 18 h. The reaction mixture
was added to Na2S2O4 (s) and evaporated. The residue was dissolved
in H2O (40 mL) and extracted with Et2O (3 × 40 mL). The
combined organic phases were washed with 2% aq Na2S2O4 (3 ×
50 mL), dried, and evaporated in vacuo. CC [toluene-EtOAc (4:
1)] gave 14 (987 mg, 82%) as a light brown oil. 1H NMR
(CDCl3): δ 5.15 (1H, hep, J ) 6.3 Hz), 4.28 (2H, bs), 3.72 (3H,
3-Isopropoxy-5-(1-methoxycarbonyl-4-piperidyl)-4-(hydroxy-
(2-naphthyl)methyl)isothiazole (15e) was synthesized as described
for 15c using 14 (300 mg, 0.73 mmol) in dry THF (3 mL), EtMgBr
(1 M, 0.73 mmol), and 2-formylnaphthalene (114 mg, 0.73 mmol).
The crude product was purified by CC [toluene-EtOAc (4:1)]
affording 15e (208 mg, 65%) as a white solid: mp 131-135 °C.
1H NMR: δ 7.87-7.75 (4H, m), 7.52-7.40 (3H, m), 6.02 (1H, d,
J ) 7.5 Hz), 5.20 (1H, hep, J ) 6.3 Hz), 4.15 (2H, bs), 3.68 (3H,
s), 3.27 (1H, bd, J ) 7.5 Hz), 3.22-3.08 (1H, m), 2.82-2.57 (2H,
m), 1.95-1.82 (1H, m), 1.80-1.70 (1H, m), 1.63-1.45 (2H, m),
1.34 (3H, d, J ) 6.3 Hz), 1.21 (3H, d, J ) 6.3 Hz). 13C NMR

1394 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Krehan et al.
(CDCl3): δ 169.29, 166.01, 155.84, 139.93, 133.15, 132.79, 128.23,
128.04, 127.71, 126.38, 126.12, 124.43, 124.31, 122.34, 71.91,
68.84, 52.61, 43.89, 35.92, 33.40, 22.08, 21.87. Anal. (C24H28N2O4S)
C, H, N.
was purified by DCVC [petroleum ether f EtOAc] affording 16c
as a yellow oil (370 mg, 87%). H NMR (CDCl3): δ 5.15 (1H,
1
hep, J ) 6.1 Hz), 4.26 (2H, bs), 3.72 (3H, s), 3.00 (1H, tt, J )
12.1 Hz, J ) 3.7 Hz), 2.94-2.74 (2H, m), 2.43-2.33 (2H, m),
2.04-1.82 (2H, m), 1.68-1.44 (4H, m), 1.35 (6H, d, J ) 6.3 Hz),
0.93 (3H, t, J ) 7.4 Hz). 13C NMR (CDCl3): δ 166.73, 165.11,
155.49, 121.76, 70.60, 52.52, 43.95, 35.81, 33.08, 26.85, 22.43,
22.01, 13.87.
4-Benzyl-3-isopropoxy-5-(1-methoxycarbonyl-4-piperidyl)-
isothiazole (16d) was synthesized as described for 16b by using
15d (214 mg, 0.55 mmol), triethylsilane (140 µL, 0.88 mmol), TFA
(1 mL), and 2 h of reaction time at 50 °C. The crude product was
purified by DCVC [toluenef toluene-EtOAc (1:1)] affording 16d
(179 mg, 87%) as a colorless oil. 1H NMR (CDCl3): δ 7.29-7.11
(5H, m), 5.16 (1H, hep, J ) 6.1 Hz), 4.18 (2H, bs), 3.80 (2H, s),
3.69 (3H, s), 3.00 (1H, dt, J ) 12.4 Hz, J ) 3.9 Hz), 2.77 (2H, bt,
J ) 12.4 Hz), 1.76 (2H, bd, J ) 12.4), 1.51 (2H, dq, J ) 12.4 Hz,
J ) 3.9 Hz), 1.31 (6H, d, J ) 6.1 Hz). 13C NMR (CDCl3): δ 67.08,
67.05, 155.82, 139.73, 128.35, 128.19, 126.13, 120.48, 71.00, 60.26,
52.51, 43.88, 35.99, 32.74, 30.42, 21.91.
4-((1-Bromo-2-naphthyl)hydroxymethyl)-3-isopropoxy-5-(1-
methoxycarbonyl-4-piperidyl)isothiazole (15f) was synthesized
as described for 15c by using 14 (300 mg, 0.73 mmol) in dry THF
(3 mL), EtMgBr (1 M, 0.73 mmol), and 1-bromo-2-formylnaph-
thalene (172 mg, 0.73 mmol). The crude product was purified by
DCVC [toluene f toluene-EtOAc (1:1)] affording 15f (248 mg,
65%) as a white foam. 1H NMR (CDCl3): δ 8.30 (1H, d, J ) 8.4
Hz), 7.84-7.76 (3H, m), 7.62-7.47 (2H, m), 6.37 (1H, bd, J )
4.2 Hz), 5.18 (1H, hep, J ) 6.2 Hz), 4.06 (2H, bs), 3.83 (1H, bs),
3.63 (3H, s), 3.06-2.93 (1H, m), 2.72-2.53 (1H, m), 2.52-2.37
(1H, m), 1.78 (1H bd, J ) 12.6 Hz), 1.58 (1H, bd, J ) 12.6 Hz),
1.48-1.34 (2H, m), 1.31 (3H, d, J ) 6.2 Hz), 1.27 (3H, d, J ) 6.2
Hz). 13C NMR (CDCl3): δ 169.75, 165.95, 155.74, 139.33, 133.96,
132.09, 128.04, 127.60, 127.40, 127.28, 126.78, 125.42, 122.52,
121.22, 71.72, 69.21, 52.46, 43.82, 43.70, 35.85, 33.00, 21.91,
21.85.
4-(2,2-Diphenyl-1-hydroxethyl)-3-isopropoxy-5-(1-methoxy-
carbonyl-4-piperidyl)isothiazole (15g) was synthesized as de-
scribed for 15c by using 14 (480 mg, 1.17 mmol) in dry THF (4
mL), EtMgBr (1 M, 1.17 mmol), and 2,2-diphenylethanal (210 µL,
1.17 mmol). The crude product was purified by CC [toluene-
EtOAc (4:1)] affording 15g (254 mg, 45%) as a white foam: mp
3-Isopropoxy-5-(1-methoxycarbonyl-4-piperidyl)-4-(2-naph-
thylmethyl)isothiazole (16e) was synthesized as described for 16b
by using 15e (197 mg, 0.45 mmol), triethylsilane (110 µL, 0.72
mmol), TFA (0.8 mL), and 2 h of reaction time at 50 °C. Crude
16e (173 mg, 94%) was a yellow oil and pure according to NMR.
1H NMR (CDCl3): δ 7.82-7.69 (3H, m), 7.54 (1H, s), 7.48-7.37
(2H, m), 7.30 (1H, dd, J ) 8.6, J ) 1.7 Hz), 5.19 (1H, hep, J )
6.2), 4.16 (2H, bs), 3.96 (2H, s), 3.67 (3H, s), 3.10-2.98 (1H, m),
2.74 (2H, bt, J ) 12.5 Hz), 1.77 (2H, bd, J ) 12 Hz), 1.60-1.38
(2H, m), 1.32 (6H, d, J ) 6.2 Hz). 13C NMR (CDCl3): δ 167.35,
167.12, 155.84, 137.15, 133.44, 132.05, 128.04, 127.61, 127.40,
126.90, 126.23, 126.09, 125.41, 120.24, 71.10, 52.54, 43.85, 36.02,
32.72, 30.57, 21.95.
4-((1-Bromo-2-naphthyl)methyl)-3-isopropoxy-5-(1-methoxy-
carbonyl-4-piperidyl)isothiazole (16f) was synthesized as de-
scribed for 16b by using 15f (248 mg, 0.48 mmol), triethylsilane
(120 µL, 0.76 mmol), TFA (0.9 mL), and 2 h of reaction time at
50 °C. The crude product was purified by DCVC [toluene f
toluene-EtOAc (2:1)] affording 16f (204 mg, 85%) as a colorless
oil. 1H NMR (CDCl3): δ 8.33 (1H, d, J ) 8.4 Hz), 7.78 (1H, d, J
) 8.1 Hz), 7.67 (1H, d, J ) 8.7 Hz), 7.59 (1H, t, J ) 7.7 Hz), 7.49
(1H, d, J ) 7.5 Hz), 7.12 (1H, d, J ) 8.4 Hz), 5.20 (1H, hep, J )
6 Hz), 4.18 (2H, s), 4.12 (2H, bs), 3.66 (3H, s), 3.03 (1H, dt, J )
11.9 Hz, J ) 3.6 Hz), 2.70 (2H, bt, J ) 12.3 Hz), 1.71 (2H, bd, J
) 12.9 Hz), 1.57-1.38 (2H, m), 1.31 (6H, d, J ) 6 Hz). 13C NMR
(CDCl3): δ 168.31, 167.10, 155.78, 137.05, 133.27, 132.32, 128.05,
127.59, 127.53, 127.29, 127.08, 126.17, 123.54, 119.50, 71.14,
52.46, 43.80, 35.97, 32.63, 31.52, 21.92.
4-(2,2-Diphenyl-1-ethyl)-3-isopropoxy-5-(1-methoxycarbonyl-
4-piperidyl)isothiazole (16g) was synthesized as described for 16b
by using 15g (269 mg, 0.61 mmol), triethylsilane (160 µL, 1.0
mmol), TFA (1.1 mL), and 2 h of reaction time at 50 °C. Crude
16g (221 mg, 78%) was a yellow oil and pure according to NMR.
1H NMR (CDCl3): δ 7.37-7.10 (10H, m), 5.23 (1H, hep, J ) 6.2
Hz), 4.24 (1H, t, J ) 8.0 Hz), 4.08 (2H, bs), 3.67 (3H, s), 3.12
(2H, d, J ) 8.0 Hz), 2.64-2.49 (2H, m), 2.37-2.24 (1H, m), 1.39
(6H, d, J ) 6.2 Hz), 1.30-1.15 (4H, m). 13C NMR (CDCl3): δ
167.33, 166.86, 155.88, 144.42, 128.37, 128.22, 126.38, 119.35,
70.95, 52.55, 49.82, 43.98, 35.76, 33.05, 32.11, 22.09.
4-(3,3-Diphenyl-1-propyl)-3-isopropoxy-5-(1-methoxycarbo-
nyl-4-piperidyl)isothiazole (16h) was synthesized as described for
16b by using 15h (210 mg, 0.42 mmol), triethylsilane (110 µL,
mmol), TFA (0.8 mL), and 2 h of reaction time at 50 °C. The crude
product was purified by DCVC [toluene f toluene-EtOAc (1:1)]
affording 16h (184 mg, 91%) as a colorless oil. 1H NMR (CDCl3):
δ 7.32-7.12 (10H, m), 5.17 (1H, hep, J ) 6.2 Hz), 4.13 (2H, bs),
3.88 (1H, bt, J ) 7.5 Hz), 3.69 (3H, s), 2.72-2.20 (7H, m), 1.70
(2H, bd, J ) 12.6), 1.54-1.30 (8H, m). 13C NMR (CDCl3): δ
167.15, 165.82, 155.78, 144.59, 128.47, 127.82, 126.24, 121.33,
70.80, 52.51, 50.48, 43.75, 35.51, 34.36, 32.61, 23.19, 22.03.
1
66-70 °C. H NMR (CDCl3): δ 7.49-7.44 (2H, m), 7.42-7.35
(2H, m), 7.31-7.24 (1H, m), 7.17-7.05 (3H, m), 7.03-6.80 (2H,
m), 5.38-5.21 (1H, m), 4.52 (1H, d, J ) 10.2 Hz), 4.32-3.90
(2H, m), 3.68 (3H, s), 3.04 (1H, d, J ) 9.9 Hz), 2.74 (1H, bt, J )
12.9 Hz), 2.60-2.44 (2H, m), 1.83 (1H, bd, J ) 13.1 Hz), 1.50
(3H, d, J ) 6 Hz), 1.47-1.31 (4H, m), 1.11 (1H, dq, J ) 12.8 Hz,
J ) 4.4 Hz), 0.71 (1H, bd, J ) 13.1 Hz). 13C NMR (CDCl3): δ
168.83, 165.46, 155.84, 141.85, 141.03, 129.07, 128.66, 128.60,
128.41, 126.87, 126.74, 120.97, 71.92, 70.28, 57.34, 52.59, 43.95,
43.90, 35.87, 33.86, 32.72, 22.25, 22.18. Anal. (C27H32N2O4S‚1/
4H2O) C, H, N.
4-(3,3-Diphenyl-1-hydroxypropyl)-3-isopropoxy-5-(1-meth-
oxycarbonyl-4-piperidyl)isothiazole (15h) was synthesized as
described for 15c by using 14 (400 mg, 0.97 mmol) in dry THF (4
mL), EtMgBr (1 M, 0.97 mmol), and 3,3-diphenylpropanal (205
mg, 0.97 mmol). The crude product was purified by DCVC [toluene
f toluene-EtOAc (1:1)] affording 15h (230 mg, 48%) as a white
foam. 1H NMR (CDCl3): δ 7.34-7.14 (10H, m), 5.23 (1H, hep, J
) 6.3 Hz), 4.52-4.42 (1H, m), 4.24-3.96 (3H, m), 3.67 (3H, s),
2.81 (1H, bs), 2.73-2.60 (1H, m), 2.59-2.30 (4H, m), 1.73 (1H,
bd, J ) 13.2 Hz), 1.52 (1H, bd, J ) 13.2), 1.47-1.28 (8H, m). 13
C
NMR (CDCl3): δ 167.70, 165.93, 155.76, 144.45, 144.01, 129.04,
128.62, 128.23, 128.04, 127.83, 126.47, 126.42, 125.29, 122.80,
71.78, 65.43, 52.54, 52.50, 47.40, 43.71, 43.58, 42.88, 35.39, 32.90,
32.50, 22.09, 22.06.
3-Isopropoxy-5-(1-methoxycarbonyl-4-piperidyl)-4-methyl-
isothiazole (16b). To a solution of 15b (447 mg, 1.48 mmol) in
dry CH2Cl2 (5 mL) was added triethylsilane (380 µL, 2.37 mmol),
and the solution was cooled to 0 °C. TFA (2.8 mL) was added
dropwise at 0 °C, and the reaction was allowed to warm to room
temperature followed by heating to 50 °C. Stirring was continued
at 50 °C for 5 h. The reaction was quenched with H2O (7 mL) and
extracted with CH2Cl2 (3 × 7 mL). The combined CH2Cl2 phases
were dried and evaporated in vacuo. The residue was purified by
DCVC [petroleum ether f EtOAc] affording 16b as a colorless
1
oil (258 mg; 58%). H NMR (CDCl3): δ 5.15 (1H, hep, J ) 6.3
Hz), 4.26 (2H, bs), 3.71 (3H, s), 3.00 (1H, tt, J ) 12 Hz, J ) 3.8
Hz), 2.88 (2H, bt, J ) 12 Hz), 1.98 (3H, s), 1.97-1.86 (2H, m),
1.58 (2H, dq, J ) 12.6 Hz, J ) 4.4 Hz), 1.36 (6H, d, J ) 6.3 Hz).
13C NMR (CDCl3): δ 167.43, 165.25, 156.03, 117.69, 71.24, 53.07,
44.51, 36.55, 32.72, 22.70, 10.48.
3-Isopropoxy-5-(1-methoxycarbonyl-4-piperidyl)-4-propyl-
isothiazole (16c) was synthesized as described for 16b by using
15c (446 mg, 1.30 mmol), triethylsilane (330 µL, 2.08 mmol), TFA
(2.5 mL), and 2 h of reaction time at 50 °C. The crude product

Substituted 3-Isothiazolol GABAA Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1395
4-Methyl-5-(4-piperidyl)isothiazol-3-ol Hydrobromide (7b).
Compound 16b (258 mg, 0.86 mmol) was added to 33% HBr in
HOAc (16 mL) and stirred for 72 h at 65 °C. The reaction mixture
was evaporated followed by coevaporation from MeOH-toluene
(1:1) three times. The residue was recrystallized from EtOH
affording 7b (78 mg, 32%) as light gray crystals; mp >200 °C. 1H
NMR (CD3OD): δ 3.55-3.45 (2H, m), 3.44-3.31 (1H, m), 3.25-
3.12 (2H, m), 2.26-2.14 (2H, m), 2.03 (3H, s), 1.91-1.74 (2H,
m). 13C NMR (CD3OD): δ 170.21, 163.16, 118.96, 45.09, 34.88,
30.19, 10.27. Anal. (C9H15BrN2OS) C, H, Br, N, S.
(CD3OD): δ 7.29 (8H, d, J ) 4.2 Hz), 7.22-7.14 (2H, m), 3.91
(1H, t, J ) 7.7 Hz), 3.40 (2H, bd, J ) 12.9 Hz), 2.95-2.72 (3H,
m), 2.52-2.41 (2H, m), 2.39-2.26 (2H, m), 1.97 (2H, bd, J )
13.6 Hz), 1.71 (2H, dq, J ) 13.6 Hz, J ) 3.5 Hz). 13C NMR (CD3-
OD): δ 170.43, 164.35, 146.39, 129.71, 129.19, 127.44, 123.21,
52.09, 45.11, 35.25, 34.72, 30.58, 24.61. Anal. (C23H27BrN2OS‚1/
4H2O) C, H, Br, N, S.
Pharmacology. The receptor binding technique for determining
the affinities for GABAA and GABAB receptors was performed
using rat brain membrane preparations using either [3H]muscimol
or [3H]GABA as the radioligands as described previously.11 Cloning
and sequencing of cDNAs encoding human R1, R4, â3, γ2S, and δ
GABAA receptor subunit proteins have been described else-
where.1,19-21 R1 encoding cDNA was engineered into a pCDM8
vector (Invitrogen, San Diego, CA) and R4, â3, γ2S, and δ into a
pcDNAI/Amp vector (Invitrogen). DNA was a kind gift from Dr.
Paul Whiting, Merck Sharp & Dohme, Terlings Park, Harlow, U.K.
Large scale cDNA preparation and purification was undertaken
using a QIAGEN Plasmid Maxi kit (QIAGEN GmbH, Hilden,
Germany). Plasmids were linearized using HpaI and XbaI restriction
enzymes for R1/R4/δ and â3/γ2S cDNAs, respectively, and tran-
scribed and capped in vitro (mMessage mMachine T7 kit, Ambion,
Inc., Austin, TX). The RNAs were precipitated with LiCl and
redissolved in sterile RNase-free water, diluted to a concentration
of 0.2 µg/µL, and divided into portions that were stored at -80
°C. cRNA was kindly supplied by Jan Egebjerg and Lene Heding,
Department of Molecular Genetics, H. Lundbeck A/S.
An adult female Xenopus laeVis was anaesthetized by immersion
in a 0.4% (w/v) 3-aminobenzoic acid ethyl ester solution (Sigma)
for 15-20 min. Through an incision in the abdominal wall, 2-3
ovarian lobes were removed and stage V and VI oocytes manually
defolliculated with watchmaker’s fine forceps. After mild collage-
nase treatment (type IA (Sigma), 0.5 mg/mL, for 6 min) to remove
remaining follicle cells, each oocyte was injected with 23.0 nL of
cRNA encoding human R1â3γ2S GABAA receptor subunits (0.2 µg/
µL in a subunit ratio of 1:1:1)). Oocytes were incubated for at least
24 h in modified Barth’s saline [88 mM NaCl, 1 mM KCl, 15 mM
HEPES, 2.4 mM NaHCO3, 0.41 mM CaCl2, 0.82 mM MgSO4, 0.3
mM Ca(NO3)2] supplemented with 2 mM sodium pyruvate, 0.1 U/L
penicillin and 0.1 µg/L streptomycin and filtered through nitrocel-
lulose.
Oocytes were placed in a 60 µL bath and perfused with Ringer
[115 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1.8 mM CaCl2, 0.1
mM MgCl2, pH 7.5]. Cells were impaled with agar-plugged 0.5-1
MΩ electrodes containing 3 M KCl and voltage clamped at -70
mV by a GeneClamp 500B amplifier (Axon Instruments). The cells
were continuously perfused with Ringer buffer at 4-6 mL/min,
and the drugs were applied in the perfusate. GABA-containing
solutions were applied until the peak of the response was observed,
usually after 30 s or less. A 4-8 min wash-out period between
GABA applications was allowed in order to minimize desensitiza-
tion. To ensure complete binding, antagonists were preapplied alone
for 1 min before their coapplication with GABA. The competitive
GABAA receptor antagonists were tested with respect to their ability
to shift a GABA concentration-response curve rightward when
present at a fixed concentration. Ki values for these antagonists were
derived from coanalysis of data obtained from each individual
oocyte in the absence and presence of antagonist by use of the
Waud equation:
5-(4-Piperidyl)-4-propylisothiazol-3-ol Hydrobromide (7c)
was synthesized as described for 7b by using 16c (370 mg, 1.13
mmol) and 33% HBr in HOAc (21 mL). The crude product was
purified by recrystallization (EtOH) affording 7c (203 mg, 58%)
as light yellow crystals: mp >200 °C. 1H NMR (CD3OD): δ 3.54-
3.45 (2H, m), 3.44-3.32 (1H, m), 3.19 (2H, dt, J ) 13.1 Hz, J )
3 Hz), 2.53-2.41 (2H, m), 2.25-2.10 (2H, m), 1.94-1.74 (2H,
m), 1.57 (2H, hex, J ) 7.5 Hz), 0.96 (3H, t, J ) 7.4 Hz). 13C
NMR (CD3OD): δ 170.03, 163.82, 123.53, 45.10, 34.80, 31.21,
28.10, 23.56, 14.30. Anal. (C11H19BrN2OS) C, H, Br, N, S.
4-Benzyl-5-(4-piperidyl)isothiazol-3-ol Hydrobromide (7d)
was synthesized as described for 7b by using 16d (179 mg, 0.48
mmol) and 33% HBr in HOAc (10 mL). The crude product was
purified by recrystallization (EtOH) affording 7d (93 mg, 55%) as
1
light gray crystals: mp >200 °C. H NMR (CD3OD): δ 7.29-
7.09 (5H, m), 3.88 (2H, s), 3.45-3.33 (3H, m), 3.08 (2H, dt, J )
13.1 Hz, J ) 3.0 Hz), 1.96 (2H, bd, J ) 13.4 Hz), 1.74 (2H, dq,
J ) 13.4, J ) 3.9). 13C NMR (CD3OD): δ 170.32, 165.76, 141.33,
129.71, 129.42, 127.46, 122.63, 45.01, 35.00, 31.29, 30.77. Anal.
(C15H19BrN2OS) C, H, Br, N, S.
4-(2-Naphthylmethyl)-5-(4-piperidyl)isothiazol-3-ol Hydro-
bromide (7e) was synthesized as described for 7b by using 16e
(173 mg, 0.42 mmol) and 33% HBr in HOAc (10 mL). The crude
product was purified by recrystallization (EtOH) affording 7e (44
1
mg, 26%) as light brown crystals: mp >200 °C. H NMR (CD3-
OD): δ 7.82-7.73 (3H, m), 7.65 (1H, s), 7.47-7.35 (3H, m), 4.05
(2H, s), 3.50-3.32 (3H, m), 3.05 (2H, dt, J ) 13.1 Hz, J ) 3.0
Hz), 1.95 (2H, bd, J ) 14.1 Hz), 1.83-1.65 (2H, m). 13C NMR
(CD3OD): δ 170.39, 166.06, 138.83, 135.16, 133.78, 129.40,
128.77, 128.62, 128.08, 127.33, 126.69, 122.40, 44.94, 35.03, 31.47,
30.74. Anal. (C19H21BrN2OS‚2/5H2O), Calcd: Br, 19.37. Found: Br,
20.20.
4-((1-Bromo-2-naphthyl)methyl)-5-(4-piperidyl)isothiazol-3-
ol Hydrobromide (7f) was synthesized as described for 7b by using
16f (204 mg, 0.41 mmol) and 33% HBr in HOAc (10 mL). The
crude product was purified by recrystallization (EtOH) affording
7f (109 mg, 56%) as light brown crystals: mp > 200 °C. 1H NMR
(CD3OD): δ 8.32 (1H, d, J ) 8.6 Hz), 7.85 (1H, d, J ) 8.1 Hz),
7.79 (1H, d, J ) 8.4 Hz), 7.67-7.59 (1H, m), 7.57-7.49 (1H, m),
7.22 (1H, d, J ) 8.6 Hz), 4.25 (2H, s), 3.42-3.33 (3H, m), 2.97
(2H, dt, J ) 13.1 Hz, J ) 2.9 Hz), 1.96 (2H, bd, J ) 13.8 Hz),
1.84-1.66 (2H, m). 13C NMR (CD3OD): δ 170.24, 166.79, 138.42,
135.04, 133.81, 129.47, 129.22, 128.97, 128.23, 128.18, 127.65,
124.38, 121.39, 45.02, 35.22, 32.80, 30.65. Anal. (C19H20Br2N2-
OS‚1.5H2O) C, H, Br, N, S.
4-(2,2-Diphenyl-1-ethyl)-5-(4-piperidyl)isothiazol-3-ol Hydro-
bromide (7g) was synthesized as described for 7b by using 16g
(221 mg, 0.48 mmol) and 33% HBr in HOAc (11 mL). The crude
product was purified by recrystallization (EtOH) affording 7g (56
1
mg, 26%) as light gray crystals: mp >210 °C. H NMR (CD3-
E ) [Emax[L]n]/[EC50n(1 + [I]/Ki)n + [L]n]
(1)
OD): δ 7.31-7.22 (8H, m), 7.21-7.13 (2H, m), 4.45 (1H, bs),
4.30 (1H, t, J ) 7.8 Hz), 3.29-3.18 (4H, m), 2.89 (2H, bdt, J )
12.9 Hz, J ) 3 Hz), 2.76 (1H, btt, J ) 11.9, J ) 3.8 Hz), 1.56-
1.26 (4H, m). 13C NMR (CD3OD): δ 170.27, 165.37, 146.18,
129.57, 129.52, 127.55, 121.52, 50.98, 45.15, 34.92, 33.07, 31.15.
Anal. (C22H25BrN2OS‚2/5H2O) C, H, Br, N, S.
where Emax is the maximum response, [L] is the concentration of
the ligand, EC50 is the concentration of the ligand eliciting 50% of
the maximal response, n is the slope factor, and [I] is the (fixed)
antagonist concentration. Geometric mean values of the Ki values
were calculated.
For antagonists exhibiting a mixed competitive/noncompetitive
behavior, an approximate Ki value for the competitive component
of the profile was estimated. This was accomplished by fitting only
data points from the initial phase of the GABA concentration-
4-(3,3-Diphenyl-1-propyl)-5-(4-piperidyl)isothiazol-3-ol Hy-
drobromide (7h) was synthesized as described for 7b by using
16h (184 mg, 0.38 mmol) and 33% HBr in HOAc (10 mL). The
crude product was purified by recrystallization (EtOH) affording
1
7h (94 mg, 53%) as colorless crystals: mp >200 °C. H NMR

1396 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Krehan et al.
(10) Frølund, B.; Jørgensen, A. T.; Tagmose, L.; Stensbøl, T. B.;
Vestergaard, H. T.; Engblom, C.; Kristiansen, U.; Sanchez, C.;
Krogsgaard-Larsen, P.; Liljefors, T. Novel class of potent 4-arylalkyl
substituted 3-isoxazolol GABA(A) antagonists: synthesis, pharma-
cology, and molecular modeling. J. Med. Chem. 2002, 45, 2454-
2468.
(11) Frølund, B.; Jensen, L. S.; Guandalini, L.; Canillo, C.; Vestergaard,
H. T.; Kristiansen, U.; Nielsen, B.; Stensbøl, T. B.; Madsen, C.;
Krogsgaard-Larsen, P.; Liljefors, T. Potent 4-aryl- or 4-arylalkyl-
substituted 3-isoxazolol GABAA antagonists: synthesis, pharmacol-
ogy, and molecular modeling. J. Med. Chem. 2005, 48, 427-439.
(12) Krehan, D.; Frølund, B.; Ebert, B.; Nielsen, B.; Krogsgaard-Larsen,
P.; Johnston, G. A.; Chebib, M. Aza-THIP and related analogues of
THIP as GABA C antagonists. Bioorg. Med. Chem. 2003, 11, 4891-
4896.
response curve in the presence of antagonist to the Waud equation,
omitting data points from which a visible reduction of Emax becomes
apparent.
For the most elaborate data sets, a quantitative analysis of the
mixed competitive/noncompetitive interaction was feasible, using
the following equation:
E ) Emax/[1 + Fnc([I]/[1 + Knc]) + [EC50/[L]]n(1 + [I]/Ki)n] (2)
where Knc is a measure of the potency of the noncompetitive
component and Fnc is an arbitrary constant controlling the reduction
(13) Brown, N.; Kerby, J.; Bonnert, T. P.; Whiting, P. J.; Wafford, K. A.
Pharmacological characterization of a novel cell line expressing
human R4â3δ GABA(A) receptors. Br. J. Pharmacol. 2002, 136,
965-974.
(14) Tagmose, L. A Pharmacophore Model for GABAA Receptor Agonists.
Ph.D. Thesis, Royal Danish School of Pharmacy, Copenhagen, 2000.
(15) Wermuth, C. G.; Bizie´re, K. Pyridazinyl-GABA derivatives: A new
class of synthetic GABAA antagonists. Trends Pharmacol. Sci. 1986,
7, 421-424.
(16) Wermuth, C. G.; Bourguignon, J.-J.; Schlewer, G.; Gies, J.-P.;
Schoenfelder, A.; Melikian, A.; Bouchet, M.-J.; Chantreux, D.;
Molimard, J.-C.; Heaulme, M.; Chambon, J.-P.; Biziere, K. Synthesis
and structure-activity relationships of a series of aminopyridazine
derivatives of γ-aminobutyric acid acting as selective GABA-A
antagonists. J. Med. Chem. 1987, 30, 239-249.
(17) Mortensen, M.; Frølund, B.; Jørgensen, A. T.; Liljefors, T.; Krogs-
gaard-Larsen, P.; Ebert, B. Activity of novel 4-PIOL analogues at
human R1â2γ2S GABAA receptorsscorrelation with hydrophobicity.
Eur. J. Pharmacol. 2002, 451, 125-132.
(18) Alfaro-Lopez, J.; Okayama, T.; Hosohata, K.; Davis, P.; Porreca,
F.; Yamamura, H. I.; Hruby, V. J. Exploring the structure-activity
relationships of 1-(4-tert-butyl-3′-hydroxy)benzhydryl-4-benzylpip-
erazine (SL-3111), a high-affinity and selective δ-opioid receptor
nonpeptide agonist ligand. J. Med. Chem. 1999, 42, 5359-5368.
(19) Hadingham, K. L.; Wingrove, P. B.; Wafford, K. A.; Bain, C.; Kemp,
J. A.; Palmer, K. J.; Wilson, A. W.; Wilcox, A. S.; Sikela, J. M.;
Ragan, C. I. Role of the beta subunit in determining the pharmacology
of human gamma-aminobutyric acid type A receptors. Mol. Phar-
macol. 1993, 44, 1211-1218.
(20) Hadingham, K. L.; Wingrove, P.; Le Bourdelles, B.; Palmer, K. J.;
Ragan, C. I.; Whiting, P. J. Cloning of cDNA sequences encoding
human alpha 2 and alpha 3 gamma-aminobutyric acidA receptor
subunits and characterization of the benzodiazepine pharmacology
of recombinant alpha 1-, alpha 2-, alpha 3-, and alpha 5-containing
human gamma-aminobutyric acidA receptors. Mol. Pharmacol. 1993,
43, 970-975.
(21) Wafford, K. A.; Thompson, S. A.; Thomas, D.; Sikela, J.; Wilcox,
A. S.; Whiting, P. J. Functional characterization of human gamma-
aminobutyric acidA receptors containing the R4 subunit. Mol.
Pharmacol. 1996, 50, 670-678.
(22) CPCM as implemented in Gaussian98, with the tesserae area set
explicitly to 0.4 Å2. For the heterocycles, the bonding list used in
the cavity calculation was corrected to the Lewis structure with a
single bond to the negatively charged oxy substituent.
of Emax
.
All experiments were performed on at least four different oocytes
stemming from at least two different oocyte batches. Stock solution
of GABA (1 M) were made in double-distilled water and stored in
aliquots at -20 °C. 4-PIOL analogues were dissolved freshly in
DMSO at a concentration of 10 mM and diluted at the appropriate
concentration in Ringer buffer.
Calculations of log P. The calculations were performed by using
Crippen’s fragmentation method and the ClogP algorithm as
implemented in ChemDrawUltra version 8.0.3 (CambridgeSoft).
Calculations of ∆Gsolv. Solvated structures were obtained by
full optimization at the HF/6-31+G* level employing the CPCM22
solvation method as implemented in Gaussian98.23
Acknowledgment. The Danish Medical Research Council
supported this work.
Supporting Information Available: Data from microanalysis.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Thomsen, C.; Ebert, B. Modulators of the GABA receptor. Novel
therapeutic prospects. Glutamate and GABA Receptors and Trans-
porters. Structure, Function and Pharmacology; Taylor & Francis:
New York, 2002; pp 407-427.
(2) Sieghart, W.; Sperk, G. Subunit composition, distribution and function
of GABA(A) receptor subtypes. Curr. Top. Med. Chem. 2002, 2,
795-816.
(3) McKernan, R. M.; Whiting, P. J. Which GABAA-receptor subtypes
really occur in the brain? Trends Neurosci. 1996, 19, 139-143.
(4) Krogsgaard-Larsen, P.; Johnston, G. A. R.; Curtis, D. R.; Game, C.
J. A.; McCulloch, R. M. Structure and biological activity of a series
of conformationally restricted analogues of GABA. J. Neurochem.
1975, 25, 803-809.
(5) Krogsgaard-Larsen, P.; Johnston, G. A. R.; Lodge, D.; Curtis, D. R.
A new class of GABA agonist. Nature 1977, 268, 53-55.
(6) Krogsgaard-Larsen, P.; Mikkelsen, H.; Jacobsen, P.; Falch, E.; Curtis,
D. R.; Peet, M. J.; Leah, J. D. 4,5,6,7-Tetrahydroisothiazolo[5,4-c]-
pyridin-3-ol and related analogues of THIP. Synthesis and biological
activity. J. Med. Chem. 1983, 26, 895-900.
(7) Krogsgaard-Larsen, P.; Hjeds, H.; Curtis, D. R.; Lodge, D.; Johnston,
G. A. R. Dihydromuscimol, thiomuscimol and related heterocyclic
compounds as GABA analogues. J. Neurochem. 1979, 32, 1717-
1724.
(8) Frølund, B.; Kristiansen, U.; Brehm, L.; Hansen, A. B.; Krogsgaard-
Larsen, P.; Falch, E. Partial GABAA receptor agonists. Synthesis and
in vitro pharmacology of a series of nonannulated analogues of
4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THIP). J. Med. Chem.
1995, 38, 3287-3296.
(9) Frølund, B.; Tagmose, L.; Liljefors, T.; Stensbøl, T. B.; Engblom,
C.; Kristiansen, U.; Krogsgaard-Larsen, P. A novel class of potent
3-isoxazolol GABAA antagonists: design, synthesis, and pharmacol-
ogy. J. Med. Chem. 2000, 43, 4930-4933.
(23) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A.;
Stratmann, R. E., Jr.; Burant, J. C.; Dapprich, S.; Millam, J. M.;
Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.;
Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.;
Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P.
Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M.
A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.;
Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.;
Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A.
Gaussian 98, revision A.7; Gaussian, Inc.: Pittsburgh, PA, 1998.
JM050987L