4-Aryl-5-(4-piperidyl)-3-isoxazolol GABAA antagonists: Synthesis, pharmacology, and structure-activity relationships

Article abstract of DOI:10.1021/jm070038n

A series of 4-aryl-5-(4-piperidyl)-3-isoxazolol GABA_A antagonists have been synthesized and pharmacologically characterized. The meta-phenyl-substituted compounds 9k and 9m and the para-phenoxy-substituted compound 91 all display high affinities (K_i = 10-70 nM) and antagonist potencies in the low nanomolar range (K_i = 9-10 nM). These potencies are significantly higher than those of previously reported 4-PIOL antagonists and considerably higher than that of the standard GABA_A antagonist SR 95531.

Full text of DOI:10.1021/jm070038n

1988
J. Med. Chem. 2007, 50, 1988-1992
4-Aryl-5-(4-Piperidyl)-3-Isoxazolol GABAA Antagonists: Synthesis, Pharmacology, and
Structure-Activity Relationships
Bente Frølund,*,† Lars S. Jensen,† Signe I. Storustovu,‡ Tine B. Stensbøl,‡ Bjarke Ebert,‡ Jan Kehler,‡
Povl Krogsgaard-Larsen,† and Tommy Liljefors†
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, DK-2100 Copenhagen, Denmark, and
Department of Molecular Pharmacology, Electrophysiology, and Medicinal Chemistry, H. Lundbeck A/S, DK-2500 Valby, Denmark
ReceiVed January 11, 2007
A series of 4-aryl-5-(4-piperidyl)-3-isoxazolol GABAA antagonists have been synthesized and pharmacologi-
cally characterized. The meta-phenyl-substituted compounds 9k and 9m and the para-phenoxy-substituted
compound 9l all display high affinities (Ki ) 10-70 nM) and antagonist potencies in the low nanomolar
range (Ki ) 9-10 nM). These potencies are significantly higher than those of previously reported 4-PIOL
antagonists and considerably higher than that of the standard GABAA antagonist SR 95531.
Introduction
4-Aminobutyric acid (GABA), the major inhibitory neu-
rotransmitter in the central nervous system, exerts its effect in
the central nervous system through two different classes of
receptors, the ionotropic GABAA and GABAC receptors and
the metabotropic GABAB receptors. Especially the GABAA
receptors have attracted much attention as therapeutic targets
for the treatment of conditions such as anxiety, epilepsy, and
sleep disorders.1
The GABAA receptor is a member of a superfamily of ligand-
gated ion channels, which also comprises the nicotinic acetyl-
choline, the glycine, and the serotonin (5-HT3) receptors. The
GABAA receptor is a heteropentameric transmembrane allosteric
protein complex, and in addition to the GABA recognition site,
it contains a considerable number of separate but allosterically
interacting binding sites. This is reflected in the structural
diversity of compounds acting at the GABAA receptors, includ-
ing important drugs such as benzodiazepines, neurosteroids, and
barbiturates.
In contrast to the allosteric modulatory sites, the GABA
binding site has very distinct and specific structural requirements
for recognition and activation. Thus, very few different classes
of structures have been reported. Within the series of compounds
showing agonist activity at the GABAA receptor site are the
selective GABAA agonists muscimol (1)2 and 4,5,6,7-tetrahy-
Figure 1. Structures of GABA, the GABAA agonists muscimol (1),
droisoxazolo[5,4-c]pyridin-3-ol (THIP, 2),2,3 which have been
THIP (2), the low-efficacy partial GABAA agonists 4-PIOL (3), the
used for the characterization of the GABAA receptors (Figure
1).4 Recently, 2 has been shown to be functionally selective
for a subpopulation of GABAA receptors and is currently in
clinical trials as a therapeutic for the regulation of sleep.
To date, six R, three â, three γ, and three F, ꢀ, π, θ, and δ
subunits have been identified in humans. Although a high
theoretical number of different pentameric receptor isoforms
are possible, only a limited number have been identified, with
the R1â2γ2 apparently being the most abundant in the human
central nervous system.5,6
GABAA antagonists 4-8, and the new 4-aryl-5-(4-piperidyl)-3-isox-
azolols (9).
model, a series of potent and selective competitive antagonists
have been developed, including compounds 4 and 5, and the
existence of a cavity of considerable dimensions in the vicinity
of the 4-position of the 3-isoxazolol ring of 3 has been
identified.8,9 Most of these potent antagonists have aromatic
rings linked to the 3-isoxazol moiety via a methylene group.
However, in a previous study, we have shown that attaching a
phenyl group directly to the 4-position of the 3-isoxazolol ring
in 3 yields a compound (compound 6) that displays an
unexpectedly high affinity, more than 40-fold higher than that
of the parent compound 3 and 17 times higher than that of the
corresponding 4-benzyl-substituted compound.9 In the present
study, 4-phenyl-5-(4-piperidyl)-3-isoxazolol (6) has been used
as lead compound to investigate the influence of different
substituents in the ortho-, meta-, and para-positions of the
We have previously reported a 3D-pharmacophore model for
GABAA agonists and competitive antagonists based on the
GABAA receptor ligands: 1, 2, and 4-PIOL (3).7,8 Using this
* To whom correspondence should be addressed. Phone: (+45) 35306495.
Fax: (+45) 35306040. E-mail: bfr@farma.ku.dk.
† University of Copenhagen.
‡ H. Lundbeck A/S.
10.1021/jm070038n CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/22/2007

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1989
Scheme 1a
a Reagents and conditions: (a) Pd(PPh3)2Cl2, aq K2CO3, RB(OH)2 (for 9m, compound 13), DMF; (b) 33% HBr, AcOH, room temperature; (c) n-BuLi,
THF, -78 °C, B(O-i-pr)3.
Table 1. Receptor Binding and In Vitro Electrophysiology Data for
4-phenyl group on the binding to the GABAA receptor. The
Selected Compounds
synthesis and pharmacological properties are reported and
[3H]muscimol
bindinga
electro-
structure-activity relationships are discussed in terms of our
previously reported pharmacophore model.
physiologyb
Kic (nM)
Kic (nM)
cmpd
R
(pKi ( SEM)
(pKi ( SEM)
Results and Discussion
4-PIOL
H
9100d
11 000d
Chemistry. The target compounds were synthesized as
outlined in Scheme 1.
4
5
2-naphthylmethyl
1-phenyl-2-
naphthylmethyl
phenyl
2-naphthyl
1-naphthyl
49d
370d
21d
198d
The 4-iodo analogue, 10,8 was used as starting material for
coupling under Suzuki conditions using Pd(PPh3)2Cl2 and the
appropriate aryl boronic acid or ester to give the 4-aryl-
substituted compounds 11 in high yields. The boronic acids were
commercially available, except for the 5-phenyl-3-thienyl
analogue, where the 5,5-dimethyl[1,3,2]dioxaborinan ester (13)
was synthesized from the corresponding 4-bromo-5-phenylth-
iophene (12).10 Deprotection to give the target compounds with
the generic structures 9 was accomplished by treatment with
hydrogen bromide in acetic acid.
6
7
8
220d
159d
36d
141d
820d
334d
9a
2-chlorophenyl
851
(6.07 ( 0.11)
58
(7.24 ( 0.01)
2425
(5.62 ( 0.06)
77
(7.11 ( 0.01)
234
(6.63 ( 0.02)
84
(7.07 ( 0.24)
955
(6.02 ( 0.08)
317
(6.50 ( 0.02)
6977
(5.16 ( 0.07)
195
(6.71 ( 0.11)
10
(8.00 ( 0.09)
70
129
(6.89 ( 0.05)
48
(7.32 ( 0.04)
204
(6.69 ( 0.05)
68
(7.17 ( 0.08)
138
(6.86 ( 0.03)
59
(7.23 ( 0.02)
427
(6.32 ( 0.03)
76
(7.12 ( 0.09)
257
(6.59 ( 0.03)
71
(7.15 ( 0.01)
10
(8.02 ( 0.04)
8
9b
9c
9d
9e
9f
3-chlorophenyl
4-chlorophenyl
3,5-dichlorophenyl
3-nitrophenyl
3-aminophenyl
3-pyridyl
In Vitro Pharmacology. The compounds were characterized
in receptor binding studies using rat membrane preparations and
electrophysiologically using two-electrode voltage clamp on
human R1â3γ2S GABAA receptors expressed in Xenopus oocytes.
The affinities for GABAA and GABAB receptor sites, using [3H]-
muscimol and [3H]GABA, respectively, were determined using
methods described previously.8 Like the lead compound 6, the
entire selected group of compounds showed affinity selectively
for the GABAA receptor sites (Table 1). None of the compounds
showed detectable affinity for the GABAB receptors at test
concentrations of 100 µM.
9g
9h
9i
3-thienyl
4-tert-butylphenyl
4-biphenyl
9j
9k
9l
3-biphenyl
The standard GABAA receptor antagonist SR 95531 was
included in this study for comparison.
4-phenoxyphenyl
3-(5-phenyl)thienyl
(7.15 ( 0.02)
25
(8.10 ( 0.04)
9
9m
Receptor Binding and Structure-Affinity Relationships.
As mentioned in the introduction, the attachment of a phenyl
group directly to the 4-position of the 3-isoxazolol ring in 3
gives compound 6, which displays an unexpectedly high affinity
compared to that of the parent compound 3 (Table 1). The
position of the phenyl ring in terms of our previously reported
pharmacophore model9 is shown as ring A in Figure 2. The
figure also shows the spatial relationships according to the
pharmacophore model between the phenyl ring in 6 and the
naphthyl rings in 5, 7, and 8.
To investigate if other monocyclic aromatic ring systems other
than phenyl are compatible with high affinity, the 3-thienyl
analogue (9h) and the 3-pyridyl analogue (9g) were synthesized
and pharmacologically characterized. The affinity of 9h is
comparable to that of 6, whereas 9g shows a reduction in affinity
(7.61 ( 0.03)
(8.04 ( 0.05)
SR 95531
9n
74d
240d
1-bromo-2-
naphthylmethyl
10d
42d
a Standard receptor binding on rat brain synaptic membranes, n ) 3.
b Two-electrode voltage-clamp recordings on Xenopus oocytes expressing
R1â3γ2S GABAA receptor subunits, n ) 4. c Ki values were calculated from
pKi values found in bracket. d From reference 9.
by a factor of 4. The affinity order follows the relative
hydrophobicities of the ring systems as calculated by ClogP
(benzene 2.14, thiophene 1.78, and pyridine 0.65; Chem-
DrawUltra version 10, CambridgeSoft). On this basis,
further studies were confined to investigate the effects of ortho-,
meta-, and para-substitutions in the 4-phenyl-substituted com-
pound 6.

1990 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Brief Articles
As may be inferred from Figure 2, the distal phenyl group in
the 3-biphenyl-substituted compound 9k perfectly overlaps with
ring C in compound 5. As may be predicted using the
pharmacophore model, the affinity of the 3-biphenyl analogue,
9k, was shown to be the highest one of the group of compounds
tested, revealing a Ki value in the low nanomolar range (10 nM,
Table 1). This corresponds to an increase in affinity compared
to that of 6 by a factor of 22, which nicely validates the
superimpositions in Figure 2.
The 3-nitrophenyl and 3-aminophenyl analogues, compounds
9e and 9f, respectively, show similar affinities as that of the
lead compound 6. This indicates that the receptor region
corresponding to ring C in Figure 2 and/or in the vicinity of
the alternative meta-position (close to ring E in Figure 2) is
water accessible and that the nitro and amino substituents are
both solvated in the binding site. The opposite electronic effects
of these two substituents on ring A and their similar influence
on the affinity also indicate that the interactions between ring
A and the receptor is largely of hydrophobic nature.
Figure 2. Superimposition of the previously deduced bioactive
conformations of 5-8.9
Based on the affinities of compounds 9h and 9k, a hybrid
compound 9m was designed using a 3-(5-phenyl)thienyl group.
As predicted, 9m displays an affinity similar to that of 9k.
Electrophysiology. The pharmacological profile of the
selected group of compounds was studied using a two-electrode
voltage clamp on human GABAA R1â3γ2S receptors expressed
in Xenopus oocytes. As shown for structurally related 4-PIOL
analogues, all of the compounds tested in the present study are
competitive antagonists in that they are able to shift a GABA
concentration-response curve rightward when present in a fixed
concentration.11 The electrophysiological data shows a fairly
good correlation to the obtained binding affinities (Table 1).
However, the 4-tert-butylphenyl and 4-chlorophenyl-substituted
analogues, 9i and 9c, did show higher potency than expected
from the affinity data. The 3-biphenyl-, 4-phenoxyphenyl-, and
3-(5-phenyl)thienyl-substituted analogues, 9k, 9l, and 9m,
showed antagonist potencies in the low nanomolar range (Ki
9-10 nM), considerably higher potencies than that of the
standard GABAA antagonist 2-(3′-(carboxypropyl)-3-amino-6-
(paramethoxyphenyl)pyridazinium bromide (SR 95531;12 Ki 240
nM, Table 1), of the parent compound 6 (Ki 159 nM, Table 1),
and of the earlier reported corresponding 1-bromo-2-naphthyl-
methyl analogue, 9n,9 of compound 3 (Ki 42 nM).
ortho-Substitution. An ortho-chloro substituent (9a) reduces
the affinity of 6 by a factor of 4 (Table 1). This is most probably
due to a steric conflict between the chlorine atom and the axial
hydrogen atoms in the piperidyl ring of the 4-PIOL moiety in
one of the two alternative ortho-positions and with steric and
electrostatic repulsions with the negatively charged isoxazole
oxygen in the other. This is in line with the observation that
the 4-(1-naphthyl) compound 8 displays a 4-fold lower affinity
than 6 (Table 1), most probably due to steric repulsions between
ring B in 8 (Figure 2) and the 4-PIOL moiety. Thus, substitution
in the ortho-position is not compatible with high affinity and
was, therefore, not further investigated.
para-Substitution. A chloro substituent (9c) and a tert-butyl
group (9i) in the para-position of the phenyl ring reduces the
receptor affinity by a factor of 11 and 32, respectively. This
strongly indicates that there are steric restrictions to ligand
binding in the vicinity of the para-position in 6 and close to
ring D in Figure 2. Interestingly, a phenyl substituent in the
para-position (9j) gives retained affinity compared to that of 6
and a phenoxy group (9l) gives a 3-fold affinity increase.
Because the thickness of a phenyl ring (3.4 Å) is somewhat
smaller than twice the van der Waals radius of a chloro
substituent (3.6-4.0 Å), the conformation of the phenyl and
phenoxy substituents with respect to the phenyl ring directly
attached to the 4-PIOL system may be adjusted to avoid the
steric repulsions experienced by a chloro substituent and a tert-
butyl group.
meta-Substitution. According to the superimpositions in
Figure 2, a meta-substituent may in the receptor binding site
be located in the region of ring C in Figure 2. This ring
corresponds to the distal naphthalene ring in 5, which contributes
to the affinity of 5 by a factor of as much as 78, indicating a
potentially favorable interaction site for substituents located in
this area.8 Compound 9b with a chloro substituent in the meta-
position of the phenyl ring shows a 4-fold higher affinity than
that of 6. Introduction of a chloro substituent in both meta-
positions, compound 9d, results in an affinity comparable with
that of compound 9b. The single chloro substituent in 9b is
most likely located in the area of ring C, whereas the second
chloro substituent in the dichloro compound 9d is located in
the area close to ring E. This ring corresponds to the 1-phenyl
ring in compound 5 and contributes by only a factor of 2 to the
affinity of 5. On the basis of these observations, the influence
of different substituents in the meta-position of the phenyl ring
on binding to the GABAA receptor was further investigated.
Conclusions
On the basis of our previous observation that a phenyl group
directly attached to the 4-position of the 3-isoxazolol ring in 3
results in a considerable affinity increase for the GABAA
receptor, a series of 4-aryl-5-(4-piperidyl)-3-isoxazolols and in
particular of ortho-, meta-, and para-substituted 4-phenyl-5-(4-
piperidyl)-3-isoxazolols have been synthesized and pharmaco-
logically characterized by receptor binding and electrophysiol-
ogy. The meta-phenyl-substituted compounds 9k and 9m and
the para-phenoxy-substituted compound 9l all display antagonist
potencies significantly higher than that of previously reported
4-PIOL antagonists and considerably higher than that of the
standard GABAA antagonist SR 95531. The study has further-
more provided additional information on the properties of the
ligand-binding site of the GABAA receptor and also validated
our previously reported pharmacophore model.
Experimental Section
Chemistry. General Procedures. All reactions involving air-
sensitive reagents were performed under N2 using syringe-septum
cap techniques. Column chromatography (CC) was performed on

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1991
Merck silica gel 60 (0.06-0.200 mm). Flash column chromatog-
raphy (FC) was performed using Merck silica gel 60 (0.040-0.063
mm). Dry column vacuum chromatography13 was performed using
Merck silica gel 60 (0.015-0.040 mm). Analytical thin-layer
chromatography (TLC) was carried out using Merck silica gel 60
F254 plates. All compounds were detected as single spots on TLC
plates and visualized using UV light and KMnO4 spraying reagent.
Compounds containing amino groups were also visualized using a
ninhydrin spray reagent. Compounds containing the 3-isoxazolol
ring were visualized using a FeCl3 spraying reagent. Elemental
analyses were performed at Analytical Research Department, H.
Lundbeck A/S, Denmark, or by Mr. J. Theiner, Department of
Physical Chemistry, University of Vienna, Austria, and are within
(0.4% of the calculated values, unless otherwise stated.
4-(3,5-Dichlorophenyl)-5-(4-piperidyl)-3-isoxazolol Hydrobro-
mide (9d). Compound 9d was prepared as described for 9a using
compound 11d (0.11 g, 0.24 mmol) to give the product (60 mg,
64%) as colorless crystals: mp >210 °C. Anal.(C14H14Cl2N2O2‚
HBr) C, H, N.
3-Benzyloxy-4-(3-nitrophenyl)-5-(1-methoxycarbonyl-4-pip-
eridyl)isoxazol (11e). Compound 11e was prepared as described
for 11a using 3-nitrophenyl boronic acid to give the product as
pale yellow crystals (0.05 g, 26%): mp 171-174 °C.
4-(3-Nitrophenyl)-5-(4-piperidyl)-3-isoxazolol Hydrobromide
(9e). Compound 9e was prepared as described for 9a using
compound 11e (0.05 g, 0.11 mmol) to give the product (17 mg,
42%) as colorless crystals: mp >210 °C. Anal. (C14H15N3O4‚HBr‚
0.5H2O) C, H, N.
5-Phenyl-3-(5,5-dimethyl-[1,3,2]dioxaborinan-2-yl)-
thiophene (13). A solution of 4-bromo-5-phenylthiophene (12;10
0.24 g, 1.0 mmol) in dry THF (4 mL) was stirred at -78 °C under
nitrogen atmosphere, and n-BuLi (0.47 mL, 2.30 M, 1.1 mmol)
was added and stirred for 35 min at -78 °C. Triisopropyl borate
(0.38 g, 2.0 mmol) was added, and the stirring was continued for
3 h at -78 °C and then 30 min at room temperature. Acetic acid
(0.15 mL, 2.2 mmol) was added, followed by neopentylglycol (0.23
g, 2.2 mmol), and the mixture was stirred for 25 min at room
temperature. Dichloromethane (15 mL) was added, and the phases
were separated. The organic phase was washed with a solution of
saturated aqueous ammonium chloride and water 1:1 (3 × 15 mL)
and saturated aqueous sodium hydrogen carbonate (2 × 15 mL)
and water (2 × 15 mL), dried, and evaporated. The crude was
recrystallized from petroleum ether 80-100 °C/EtOAc to give the
product as gray crystals (0.11 g, 40%): mp 115-117 °C.
3-Benzyloxy-4-(2-chlorophenyl)-5-(1-methoxycarbonyl-4-pi-
peridyl)isoxazol (11a). A mixture of 10, 2-chlorophenyl boronic
acid (141 mg, 0.9 mmol), Pd(PPh3)2Cl2 (30 mg, 45 mmol), DMF
(4 mL), and aqueous potassium carbonate (0.3 mL, 3 M, 0.9 mmol)
was stirred at 70 °C for 22 h. The reaction mixture was filtrated
through celite. The organic phase was washed with water (15 mL),
aqueous NaOH (2 × 15 mL, 2 M), and water (15 mL), dried, and
evaporated. Dry CC (toluene/EtOAc (4:1)) gave the product as
yellow oil (0.15 g, 78%).
4-(2-Chlorophenyl)-5-(4-piperidyl)-3-isoxazolol Hydrobro-
mide (9a). Compound 11a (0.15 g, 0.35 mmol) was dissolved in a
solution of HBr in AcOH (5 mL, 33%), and the mixture was stirred
at room temperature for 24 h. The reaction mixture was evaporated,
and the residue was recrystallized (MeOH/Et2O) to give 9a (95
mg, 76%) as colorless crystals: mp >210 °C. Anal. (C14H15ClN2O2‚
HBr) C, H, N.
3-Benzyloxy-4-(3-chlorophenyl)-5-(1-methoxycarbonyl-4-pi-
peridyl)isoxazol (11b). Compound 11b was prepared as described
for 11a using 3-chloro-phenyl boronic acid to give the product as
pale yellow crystals (0.14 g, 73%): mp 150-153 °C. Anal. (C23H23-
ClN2O4) C, H, N.
4-(3-Chlorophenyl)-5-(4-piperidyl)-3-isoxazolol Hydrobro-
mide (9b). Compound 9b was prepared as described for 9a using
compound 11b (0.14 g, 0.33 mmol) to give the product (89 mg,
75%) as colorless crystals: mp >210 °C. Anal. (C14H15ClN2O2‚
HBr) C, H, N.
3-Benzyloxy-4-(4-chlorophenyl)-5-(1-methoxycarbonyl-4-pi-
peridyl)isoxazol (11c). Compound 11c was prepared as described
for 11a using 4-chlorophenyl boronic acid to give the product as
brown crystals (0.05 g, 47%): mp 155-157 °C. Anal. (C23H23-
ClN2O4) C, H, N.
4-(4-Chlorophenyl)-5-(4-piperidyl)-3-isoxazolol Hydrobro-
mide (9c). Compound 9c was prepared as described for 9a using
compound 11c (0.09 g, 0.21 mmol) to give the product (45 mg,
60%) as colorless crystals: mp >210 °C. Anal. (C14H15ClN2O2‚
HBr‚H2O) C, H, N.
3-Benzyloxy-4-(3,5-dichlorophenyl)-5-(1-methoxycarbonyl-4-
piperidyl)isoxazol (11d). Compound 11d was prepared as described
for 11a using 3,5-dichlorophenyl boronic acid to give the product
as brown oil (0.11 g, 74%).
3-Benzyloxy-4-(3-aminophenyl)-5-(1-methoxycarbonyl-4-pi-
peridyl)isoxazol (11f). Compound 11f was prepared as described
for 11a using 3-aminophenyl boronic acid to give the product as a
yellow oil (0.17 g, 93%).
4-(3-Aminophenyl)-5-(4-piperidyl)-3-isoxazolol Dihydrobro-
mide (9f). Compound 9f was prepared as described for 9a using
compound 11f (0.17 g, 0.42 mmol) to give the product (0.13 g,
74%) as colorless crystals: mp >210 °C. Anal. (C14H15N3O4‚2HBr‚
H2O) C, H, N.
3-Benzyloxy-4-(3-pyridyl)-5-(1-methoxycarbonyl-4-piperidyl)-
isoxazol (11g). Compound 11g was prepared as described for 11a
using 3-pyridyl boronic acid to give the product as colorless crystals
(0.18 g, 99%): mp 108-111 °C.
4-(3-Pyridyl)-5-(4-piperidyl)-3-isoxazolol Hydrobromide (9g).
Compound 9g was prepared as described for 9a using compound
11g (0.18 g, 0.45 mmol) to give the product (0.12 g, 79%) as
colorless crystals: mp >210 °C. Anal. (C13H15N3O2‚2HBr‚H2O)
C, H, N.
3-Benzyloxy-5-(1-methoxycarbonyl-4-piperidyl)-4-(3-thienyl)-
isoxazol (11h). Compound 11h was prepared as described for 11a
using 3-thienyl boronic acid to give the product as brown crystals
(40 mg, 22%): mp 153-155 °C.
5-(4-Piperidyl)-4-(3-thienyl)-3-isoxazolol Hydrobromide (9h).
Compound 9h was prepared as described for 9a using compound
11h (40 mg, 0.1 mmol) to give the product (28 mg, 85%) as pale
yellow crystals: mp >210 °C. Anal. (C12H14N2O2S‚HBr‚0.5H2O)
C, H, N.
3-Benzyloxy-4-[(1,1-dimethylethyl)phenyl]-5-(1-methoxycar-
bonyl-4-piperidyl)isoxazol (11i). Compound 11i was prepared as
described for 11a using 4-(1,1-dimethylethyl)phenyl boronic acid
to give the product as brown oil (0.17 g, 84%)
4-((1,1-Dimethylethyl)-phenyl)-5-(4-piperidyl)-3-isoxazolol Hy-
drobromide (9i). Compound 9i was prepared as described for 9a
using compound 11f (0.17 g, 0.38 mmol) to give the product (0.11
g, 76%) as colorless crystals: mp >210 °C. Anal. (C18H24N2O2‚
HBr) C, H, N.
3-Benzyloxy-4-(4-biphenyl)-5-(1-methoxycarbonyl-4-piperidyl)-
isoxazol (11j). Compound 11j was prepared as described for 11a
using 4-biphenyl boronic acid to give the product as a brown oil
(0.21 g, 99%).
4-(4-Biphenyl)-5-(4-piperidyl)-3-isoxazolol Hydrobromide (9j).
Compound 9j was prepared as described for 9a using compound
11j (0.21 g, 0.44 mmol) to give the product (0.13 g, 74%) as
colorless crystals: mp >210 °C. Anal. (C20H20N2O2‚HBr‚H2O) C,
H, N.
3-Benzyloxy-4-(3-biphenyl)-5-(1-methoxycarbonyl-4-piperidyl)-
isoxazol (11k). Compound 11k was prepared as described for 11a
using 3-biphenyl boronic acid to give the product as brown oil (0.15
g, 71%).
4-(3-Biphenyl)-5-(4-piperidyl)-3-isoxazolol Hydrobromide (9k).
Compound 9k was prepared as described for 9a using compound
11k (0.15 g, 0.32 mmol) to give the product (0.11 g, 85%) as
colorless crystals: mp >210 °C. Anal. (C20H20N2O2‚HBr‚0.5H2O)
C, H, N.
3-Benzyloxy-4-(4-phenoxyphenyl)-5-(1-methoxycarbonyl-4-pi-
peridyl)isoxazol (11l). Compound 11l was prepared as described

1992 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Brief Articles
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25, 803-809.
(3) Krogsgaard-Larsen, P.; Johnston, G. A. R.; Lodge, D.; Curtis, D. R.
A New Class of GABA Agonist. Nature 1977, 268, 53-55.
(4) Frølund, B.; Ebert, B.; Kristiansen, U.; Liljefors, T.; Krogsgaard-
Larsen, P. GABA(A) receptor ligands and their therapeutic potentials.
Curr. Top. Med. Chem. 2002, 2, 817-832.
(5) McKernan, R. M.; Whiting, P. J. Which GABAA-receptor subtypes
really occur in the brain? Trends Neurosci. 1996, 19, 139-143.
(6) Sieghart, W.; Sperk, G. Subunit composition, distribution and function
of GABA(A) receptor subtypes. Curr. Top. Med. Chem. 2002, 2,
795-816.
(7) 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.
for 11a using 4-phenoxyphenyl boronic acid to give the product as
brown oil (0.15 g, 69%).
4-(4-Phenoxyphenyl)-5-(4-piperidyl)-3-isoxazolol Hydrobro-
mide (9l). Compound 9l was prepared as described for 9a using
compound 11l (0.10 g, 0.21 mmol) to give the product (65 mg,
74%) as colorless crystals: mp >210 °C. Anal. (C20H20N2O3‚HBr)
C, H, N.
3-Benzyloxy-4-[(5-phenyl)-3-thienyl]-5-(1-methoxycarbonyl-
4-piperidyl)isoxazol (11m). Compound 11m was prepared as
described for 11a using 5-phenyl-3-(5,5-dimethyl-[1,3,2]dioxabori-
nan-2-yl)thiophen to give the product as a brown oil (0.08 g, 51%).
4-[(5-Phenyl)-3-thienyl]-5-(4-piperidyl)-3-isoxazolol Hydro-
bromide (9m). Compound 9m was prepared as described for 9a
using compound 11m (0.08 g, 0.17 mmol) to give the product (40
mg, 58%) as colorless crystals: mp >202-205 °C. Anal.
(C18H18N2O2S‚HBr‚1.5H2O) C, H, N.
Experimental Pharmacology. The receptor binding technique
for determining the affinities toward GABAA and GABAB receptors
was determined in rat brain membrane preparations using either
[3H]muscimol or [3H]GABA as the radioligands and performed as
described previously.8
(8) Frølund, B.; Jørgensen, A. T.; Tagmose, L.; Stensbøl, T. B.;
Vestergaard, H. T.; Engblom, C.; Kristiansen, U.; Sanchez, C.;
Krogsgaard-Larsen, P.; Liljefors, T. A novel class of potent 4-ary-
lalkyl substituted 3-isoxazolol GABAA antagonists: Synthesis,
pharmacology, and molecular modeling. J. Med. Chem. 2002, 45,
2454-2468.
Cloning and sequencing of cDNAs encoding human R1, â3, and
γ2S GABAA receptor subunit proteins have been described else-
where.14,15 The R1 encoding cDNA was engineered into a pCDM8
vector (Invitrogen, San Diego, CA), and â3 and γ2S was engineered
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/δ and â3/γ2S cDNAs, respectively, and transcribed
and capped in vitro (mMessage mMachine T7 kit, Ambion, Inc.,
Austin, TX). The RNAs were precipitated with LiCl, 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.
(9) 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 GABA(A) antagonists: Synthesis, pharma-
cology, and molecular modeling. J. Med. Chem. 2005, 48, 427-
439.
(10) Dingemans, T. J.; Murthy, N. S.; Samulski, E. T. Javelin-, hockey
stick-, and boomerang-shaped liquid crystals. Structural variations
on p-quinquephenyl. J. Phys. Chem. B 2001, 105, 8845-8860.
(11) Mortensen, M.; Frølund, B.; Jørgensen, A. T.; Liljefors, T.; Krogs-
gaard-Larsen, P.; Ebert, B. Activity of novel 4-PIOL analogues at
human a1b2g2S GABAA receptorsscorrelation with hydrophobicity.
Eur. J. Pharmacol. 2002, 451, 125-132.
(12) Wermuth, C. G.; Bizie´re, K. Pyridazinyl-GABA derivatives: A new
class of synthetic GABAA antagonists. Trends Pharmacol. Sci. 1986,
7, 421-424.
(13) Pedersen, D. S.; Rosenbohm, C. Dry column vacuum chromatogra-
phy. Synthesis 2001, 2431-2434.
(14) 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.
(15) Hadingham, K. L.; Wingrove, P. B.; Wafford, K. A.; Bain, C. J.;
Kemp, J. A.; Palmer, K. J.; Wilson, A. W.; Wilcox, A. S.; Sikela, J.;
Ragan, C. I.; Whiting, P. J. The role of the beta subunit in determining
the pharmacology of human “gamma”-aminobutyric acid type A
receptors. Mol. Pharmacol. 1993, 44, 1211-1218.
The electrophysiological characterization using a two-electrode
voltage clamp on human R1â3γ2S GABAA receptors expressed in
Xenopus oocytes was performed as described previously.16
Acknowledgment. The Danish Research Council supported
this work.
Supporting Information Available: Spectral data (1H NMR
and 13C NMR) of all synthesized compounds and elemental analyses
for all new target compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(16) Krehan, D.; Storustovu, S. I.; Liljefors, T.; Ebert, B.; Nielsen, B.;
Krogsgaard-Larsen, P.; Frølund, B. Potent 4-arylalkyl-substituted
3-isothiazolol GABA(A) competitive/noncompetitive antagonists:
Synthesis and pharmacology. J. Med. Chem. 2006, 49, 1388-1396.
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