Novel 4-(piperidin-4-yl)-1-hydroxypyrazoles as γ-aminobutyric acidA receptor ligands: Synthesis, pharmacology, and structure-activity relationships

Article abstract of DOI:10.1021/jm100106r

A series of substituted 1-hydroxypyrazole analogues of the GABA_A receptor partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL) have been synthesized and pharmacologically characterized. Several of the analogues displayed K_i in the low nanomolar range at the native GABA _A receptors and potent antagonism of the α₁β ₂γ₂ receptor. It appears that several regions situated in proximity to the core of the orthosteric binding site of the GABA_A receptor are able to accommodate large hydrophobic substituents.

Full text of DOI:10.1021/jm100106r

J. Med. Chem. 2010, 53, 3417–3421 3417
DOI: 10.1021/jm100106r
Novel 4-(Piperidin-4-yl)-1-hydroxypyrazoles as γ-Aminobutyric AcidA Receptor Ligands: Synthesis,
Pharmacology, and Structure-Activity Relationships
Henriette A. Møller, Tommy Sander, Jesper L. Kristensen, Birgitte Nielsen, Jacob Krall, Marianne L. Bergmann,
Bolette Christiansen, Thomas Balle, Anders A. Jensen, and Bente Frølund*
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, 2 Universitetsparken, DK 2100 Copenhagen
Received August 25, 2009
A series of substituted 1-hydroxypyrazole analogues of the GABAA receptor partial agonist
5-(4-piperidyl)-3-isoxazolol (4-PIOL) have been synthesized and pharmacologically characterized.
Several of the analogues displayed Ki in the low nanomolar range at the native GABAA receptors
and potent antagonism of the R1β2γ2 receptor. It appears that several regions situated in proximity to the
core of the orthosteric binding site of the GABAA receptor are able to accommodate large hydrophobic
substituents.
Introduction
γ-Aminobutyric acid (GABA,a Figure 1) is the major inhi-
bitory neurotransmitter in the central nervous system, exert-
ing its physiological effects through the ionotropic GABAA
and the metabotropic GABAB receptors. The plethora of
GABAA receptor subtypes is assembled from 19 different
subunits (R1-6, β1-3, γ1-3, δ, ε, π, θ, and F1-3) to form
pentameric channels with a central chloride-selective pore.1,2
So far, the existence of at least 26 native GABAA receptor
subtypes has been proposed,3 of which the three most
dominant synaptic subtypes are believed to be the R1β2γ2,
R3β3γ2, and R2β3γ2 combinations,4 where GABA binds at
Figure 1. Structures of GABA, muscimol, THIP, 4-PIOL (1a) and
the interface of the R and the β subunits in the receptor
complex.5-7 The GABAA receptors are widely distributed in
the central nervous system where they play essential roles in
numerous physiological processes. Therapeutic interven-
tion in GABAA receptor signaling has proven beneficial in
several disorders such as anxiety, epilepsy, schizophrenia, and
depression.8
The GABAA receptors belong to the Cys-loop receptor
superfamily of ligand-gated ion channels, which also compri-
ses the nicotinic acetylcholine receptors, the serotonin 5-HT3
receptors, and the glycine receptors.3,9 For nicotinic ligands,
the elucidation of binding modes has been aided significantly
by structural insight from the homologous acetylcholine
binding protein, resulting in reliable templates for construc-
tion of homology models. For the other Cys-loop receptors,
including GABAA receptors, homology models of the extra-
cellular domains are still based on templates characterized by
low sequence identities.10 Consequently, structure-activity
relationship (SAR) studies and pharmacophore models of
orthosteric GABAA ligands are still essential to probe the struc-
a general structure of the new 1-hydroxypyrazole compounds 2a-p.
tural basis for receptor-ligand interactions and the molecular
determinants of ligand affinity, potency, efficacy, and subtype
selectivity.
In previous studies, we constructed a pharmacophore model
of orthostericligandbinding tothe GABAA receptor based on
SAR studies of analogues of the standard GABAA ligands mus-
cimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP),
and 5-(4-piperidyl)-3-isoxazolol (4-PIOL) (Figure 1).13,14 By
use of this model, a series of potent GABAA antagonists were
developed by introducing bulky substituents in the 4-position
of the 3-isoxazolol ring of 4-PIOL.11,12,15 This suggested that
there is a spacious cavity situated in the vicinity of the core of
the orthosteric site of the GABAA receptor, which is able to
accommodate the binding of large substituents (Figure 2).
In the present study we have investigated the so far un-
explored area of the receptor binding pocket corresponding to
the area near the isoxazole oxygen in 1a (Figure 1). For this
purpose, we have applied compounds containing a new iso-
stere to the isoxazolol moiety of 1a, the 1-hydroxypyrazole
moiety (2a-p, Figure 1), as lead structure. To verify the bioiso-
steric potential of the 1-hydroxypyrazole ring compared to the
3-isoxazolol ring of 4-PIOL, a series of 5-substituted 1-hydro-
xypyrazoles 2b-e, corresponding to a selected subgroup of
previously reported 4-substituted 4-PIOL analogues,11,12
*To whom correspondence should be addressed. Phone: (þ45)
35336495. Fax: (þ45) 35336040. E-mail: bfr@farma.ku.dk.
a Abbreviations: DCVC, dry column vacuum chromatography;
FLIPR, fluorescent imaging plate reader; FMP, FLIPR membrane
potential; GABA, γ-aminobutyric acid; 5-HT, 5-hydroxytryptamine;
4-PIOL, 5-(4-piperidyl)-3-isoxazolol; SAR, structure-activity relation-
ship; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol.
r
2010 American Chemical Society
Published on Web 03/31/2010
pubs.acs.org/jmc

3418 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Møller et al.
Scheme 2a
Figure 2. Structures and 3D superimposition of 1d (gray carbons)
and 2p (cyan carbons), illustrating the new substitution position
introduced by the 1-hydroxypyrazole ring compared to the isoxa-
zole scaffold of the 4-PIOL series.
Scheme 1a
a Reagents: (i) ICl, AcOH/H2O; (ii) iPrMgCl, DMF, THF, 0 °C; (iii)
NaBH4, MeOH; (iv) Et3SiH, TFA, CH2Cl2, 50 °C; (v) R-B(OH)2,
PdCl2(PPh)2, toluene/EtOH (10:1), K2CO3 (3 M); (vi) iPrMgCl, R-CHO,
THF, 0 °C; (vii) 35% aq HCl, 130 °C or 47% aq HBr, 110 °C; (viii) LDA,
2-naphthaldehyde, THF, -78 °C.
Results and Discussion
Chemistry. Compounds 2a-p were all synthesized from
the same lead molecules 5a,b (Schemes 1 and 2). Preparation
of 5a took place through a Grignard reaction using
1-(benzyloxy)-4-iodopyrazole16 and commercially available
1-ethoxycarbonyl-4-piperidone 3 (Scheme 1). The resulting
hydroxyl group was removed using trifluoroacetic acid and
triethylsilane in dichloromethane to give 5a.17
The 5-lithiated version of 5a was prepared using lithium
diisopropylamide (LDA). Addition of the corresponding
aldehydes and subsequent reduction of the hydroxyl group
gave rise to 6b,c (Scheme 1). Compound 7 was prepared by
lithiation of 5a with LDA and subsequent addition of iodine.
Cross-coupling of 7 using phenylboronic acid and 3-biphenyl-
boronic acid resulted in 6d and 6e, respectively. Acidic depro-
tection of 6b-e yielded the target compounds 2b-e.
For the synthesis of the 3-substituted analogues, iodine
was selectively introduced by refluxing 5a with iodine
monochloride in acetic acid and water to give 8a (Scheme 2).
Compound 6f could not be prepared by Suzuki cross-coupling
and was instead synthesized through a Grignard reaction using
a Reagents: (i) iPrMgCl, THF, 0 °C; (ii) Et3SiH, TFA, CH2Cl2, 50 °C;
(iii) 35% aq HCl, 130 °C; (iv) LDA, R-CHO, THF, -78 °C; (v) LDA, I2,
THF, -78 °C; (vi) R-B(OH)2, PdCl2(PPh)2, DMF, K2CO3 (3 M).
was first synthesized. Then, to investigate unexplored areas
of the GABAA receptor ligand binding site, a series of
3-substituted and 3,5-disubstituted 1-hydroxypyrazole ana-
logues (2f-p) was prepared. The pharmacological properties
of the synthesized compounds have been characterized at
native and recombinant GABAA receptors, and the findings
are discussed in terms of our previously reported pharmaco-
phore model.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3419
Table 1. Pharmacological Data for 1a-e and 2a-p: GABAA Receptor Binding Affinities at Rat Synaptic Membranes and Functional Characterization
at the Human R1β2γ2 GABAA Receptor Transiently Expressed in tsA201Cells in the FMP Red Assay
[3H]muscimol binding
Ki (μM)a [pKi ( SEM ]
R1β2γ2 tsA201 cell line
compd
R1
R2
IC50 (μM)b [pIC50 ( SEM]
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
H
2-naphthylmethyl
9.1c
>500 [<3.30]d
1.8 [5.74 ( 0.05]
nd
0.049c
1-bromo-2-naphthylmethyl
phenyl
3-biphenyl
0.010c
0.22c
nd
0.010e
0.078 [7.11 ( 0.04]
>500 [<3.30]d
0.79 [6.10 ( 0.05]
0.48 [6.32 ( 0.05]
0.15 [6.81 ( 0.04]
0.024 [7.62 ( 0.03]
66 [4.18 ( 0.04]
1.9 [5.73 ( 0.06]
2.6 [5.58 ( 0.02]
1.9 [5.72 ( 0.06]
1.4 [5.85 ( 0.03]
0.21 [6.67 ( 0.04]
1.4 [5.85 ( 0.04]
8.3 [5.08 ( 0.04]
0.17 [6.78 ( 0.04]
3.2 [5.50 ( 0.06]
nd
H
2-naphthylmethyl
H
10 [4.99 ( 0.02]
0.033 [7.48 ( 0.03]
0.0095 [8.07 ( 0.09] f
0.022 [7.65 ( 0.02]
0.0028 [8.55 ( 0.03]
5.0 [5.31 ( 0.05]
0.27 [6.57 ( 0.01]
0.67 [6.17 ( 0.03]
0.24 [6.62 ( 0.03]
0.32 [6.50 ( 0.06]
0.030 [7.55 ( 0.09]
0.42 [6.39 ( 0.05]
0.36 [6.45 ( 0.03]
0.0030 [8.55 ( 0.10]
1.5 [5.84 ( 0.05] f
0.022 [7.66 ( 0.02] f
H
1-bromo-2-naphthylmethyl
phenyl
H
H
3-biphenyl
H
H
methyl
phenyl
o-tolyl
m-tolyl
p-tolyl
2g
2h
2i
H
H
H
2j
H
2k
2l
H
3-biphenyl
4-biphenyl
benzyl
H
2m
2n
2o
2p
H
H
2-naphthylmethyl
phenyl
2-naphthylmethyl
2-naphthylmethyl
phenyl
a IC50 values were calculated from inhibition curves and converted to Ki values. Data are given as the mean [mean pKi ( SEM] of three to four
independent experiments. b For the characterization of the antagonists, assay concentrations of GABA of 10-15 μM (5- to 8-fold higher than the EC50
concentration) were used. nd: not determined. c Reference 11. d The compound was also tested in the agonist assay but showed no activity (>500 [<3.30]).
e Reference 12. f Tested in 10% DMSO.
N,N-dimethylformamide followed by reduction. Suzuki cross-
coupling of 8a with the corresponding boronic acids led to
6g-l. In order to introduce the alkylaryl substituents in 6m,n,
compound 8a was converted into the Grignard reagent and
quenched with benzaldehyde and 2-naphthaldehyde, res-
pectively, followed by reduction. Deprotection of 6f-n resulted
in 2f-n.
Finally, 6o and 6p, with substituents in the 3- and the 5-
position of the 1-hydroxypyrazole ring, were prepared from
6g and 8b, respectively, by introduction of the 2-naphthyl-
methyl substituent in the 5- or the 3-position as described for
6b and 6n. Deprotection under acidic conditions resulted in
2o and 2p (Scheme 2).
Pharmacology. The synthesized compounds were pharma-
cologically characterized in binding studies using rat brain
membrane preparations, where the binding affinities of the
compounds at native GABAA receptors were measured by
displacement of [3H]muscimol. The functional characteriza-
tion of the compounds at the human R1β2γ2S GABAA
receptors transiently expressed in tsA201 cells was per-
formed using the FLIPR membrane potential (FMP) red
assay as described in Supporting Information. Data for
2a-p are listed in Table 1.
The effect of the compounds on the uptake of GABA
through the GABA transporters was also examined. None of
the compounds displayed inhibitory activity at the four
human GABA transporters (hGAT-1, hBGT-1, hGAT-2,
and hGAT-3) transiently expressed in tsA201 cells (data not
shown).
Receptor Binding and Structure-Affinity Relationships.
The 1-hydroxypyrazole analogue of 4-PIOL (2a) displayed
GABAA receptor affinity in the low micromolar range com-
parable to that of 4-PIOL (1a). The 5-substituted 1-hydroxy-
pyrazole analogues (2b-e) all showed high affinity for the
GABAA receptor sites and exhibited similar SAR as pre-
viously described for the corresponding 3-isoxazolol ana-
logues 1b-e.11,12
These results support the presence of the previously
suggested cavity in the orthosteric site and, considering the
structural similarity of the compounds, imply a shared
binding mode for the 1-hydroxypyrazole series of com-
pounds. According to this hypothesis, the binding of the
3-substituted 1-hydroxypyrazoles, 2f-n, would include an
area in the binding pocket in the vicinity of the respective ring
position, which has not previously been explored. Introduc-
tion of a methyl group (2f) in this position increased the
affinity slightly compared to that of 2a. This effect was more
pronounced when introducing aromatic groups as in 2g,
where a phenyl group led to approximately 40-fold increased
affinity compared to 2a. Similar effects were observed for the
tolyl analogues (2h-j) and the benzyl analogue (2m), all
showing affinities in the same range as 2g. This was also the
case for 2l, which is generated by enlargement of the
aromatic system by a phenyl group in the 4-position of the
phenyl substituent in 2g. Interestingly, introduction of a
phenyl group in the 3-position of the phenyl ring of 2g,
compound 2k, resulted in a 10-fold increase in affinity
compared to 2g. Introduction of a naphthylmethyl group

3420 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Møller et al.
(2n) led to the highest affinity of all the 3-substituted
1-hydroxypyrazole analogues. Thus, there appears to be
ample space in this particular area of the orthosteric site.
In fact, the area surrounding the relevant positions is clearly
large enough to encompass a benzyl group and larger
aromatic substituents favoring hydrophobic interactions.
This is particularly evident for 2n and 2k, containing a
2-naphthylmethyl and 3-biphenyl substituent, respectively,
exhibiting binding affinities in the low nanomolar range (Ki
of 3.0 and 30 nM).
The 1-hydroxypyrazole analogue 2a and the correspond-
ing 3-isoxazolol 1a were essentially devoid of activity in the
FMP red assay in concentrations up to 500 μM. All of the
3- and 5-substituted 1-hydroxypyrazole analogues, 2b-o,
and the 3-isoxazolol analogues, 1b,e, were characterized as
antagonists with potencies in the range of moderate to highly
potent (IC50=66-0.024 μM). In general, the functional data
are in good agreement with the binding affinity data.
Conclusions
Finally, large aromatic substituents in the 3- and 5-posi-
tion of the 1-hydroxypyrazole ring led, for 2o, to significant
reduction in receptor affinity compared to the monosubsti-
tuted 1-hydroxypyrazole analogues 2b,g. The disubstituted
analogue 2p showed markedly higher affinity than 2o, similar
or 10-fold lower compared to the affinity shown for the corres-
ponding monosubstituted analogues 2d and 2n, respectively
(Table 1).
We have identified the 1-hydroxypyrazole ring system as a
bioisostere for the 3-hydroxyisoxazole ring in 4-PIOL within
the GABAA receptor area. A series of 1-hydroxypyrazoles
(2a-p) has been synthesized and characterized pharmaco-
logically at GABAA receptors. All of the 3- and 5-substituted
1-hydroxypyrazoles (2b-p) were shown to be moderately to
highly potent antagonists(Ki of 5 μM to3 nM). On the basisof
the similar SARs obtained for the 5-substituted 1-hydroxy-
pyrazoles (2f-n) and 4-substituted 3-hydroxyisoxazoles, a
hypothesis of a common binding mode in the orthosteric site
of the GABAA receptor for the two series of compounds was
proposed, indicating space above and below the orthosteric
binding site able to accommodate large hydrophobic substit-
uents. These results offer new knowledge to the architecture of
the binding site and pharmacological tools for focused studies
regarding sequence alignment and receptor flexibility. In
particular, the SAR data are being employed in ongoing
efforts to create a validated structural model of the orthosteric
GABAA receptor binding site and to propose detailed binding
modes for this series of ligands, with the ultimate goal of
being able to perform structure-based ligand design and better
understand the mechanisms underlying agonism and anta-
gonism at the GABAA receptors.
Since a certain resemblance of the overall SAR pattern for
5- and 3-substituted 1-hydroxypyrazoles exists, one could
speculate that the 3-substituted 1-hydroxypyrazoles would
rotate 180° around the piperidine-pyrazole bond to place
the large substituents in the cavity already described in the
pharmacophore model. However, several observations seem
to be in conflict with this hypothesis: First, the relative bind-
ing affinities and antagonistic activities of the 2-naphthyl-
methyl and 3-biphenyl 1-hydroxypyrazole analogues 2b and
2e are similar to those of the 3-isoxazolol counterparts 1b
and 1e. This is in contrast to the pattern observed for the
3-substituted 1-hydroxypyrazoles 2n and 2k with regard to
binding affinity and antagonistic activity. Second, the fact
that the 3,5-disubstituted analogues 2o and 2p bind to the
receptor unequivocally suggests the presence of a relatively
large pocket on both sides of the heterocyclic core. The
affinity of 2o (Ki=1.5 μM) is not as high as for the analogous
compounds 2g and 2b, but since this compound has
two groups fitting into different cavities, it becomes more
restrained and less flexible, so the decrease is not surprising.
However, the high affinity displayed by 2p (Ki = 22 nM),
which correlates well with the corresponding monosubsti-
tuted analogues 2d and 2n, indicates that the position
of these substituents is more favorable compared to that
of 2o.
These data strongly imply that the binding site can accom-
modate large/bulky substituents in the region corresponding
to the 3- and 5-position of the 1-hydroxypyrazole ring.
Functional Characterization. The functional characteriza-
tion of the compounds was performed using the FMP red
assay (Table 1). In previous studies we have applied the FMP
blue assay in the F1 GABAA receptor and other Cys-loop
receptors,18-21 and in recent studies the FMP assay has been
validated for the functional characterization of the R1β2γ2
GABAA receptor.22,23 The functional data displayed by 1b
and 1e at the R1β2γ2 receptor transiently expressed in tsA201
cells in the FMP assay were found to be in good agreement
with findings of previous electrophysiological studies.12 The
measurements are given as IC50 instead of Ki because of the
mixed antagonist profile we observed in a previous study.15
The GABA concentrations used for the characterization of
the antagonist in the assay were 5- to 8-fold higher than the
EC50 for the agonist. Thus, if the compounds are indeed
completely competitive antagonists, as we expect them to
be, the respective functional Ki for the compounds would be
∼10 times lower than their IC50.
Experimental Section
Chemistry. The syntheses of selected compounds are descri-
bed below as representative. Procedures for the chemistry in gene-
ral, detailed experimentals, and syntheses of all other com-
pounds are in the Supporting Information. Elemental analyses
were performed at Analytical Research Department, H. Lund-
beck A/S Denmark or by J. Theiner, Department of Physical
Chemistry, University of Vienna, Austria, and the results were
within (0.4% of the calculated values unless otherwise stated.
General Procedures for Aryl-Heteroaryl Coupling. Proce-
dure A (6b,c,o). Diisopropylamine (1.2 equiv) was dissolved in
THF and cooled to -30 °C where n-BuLi (1.2 equiv) was added.
The mixture was cooled to -78 °C where 5a (for 6b,c) or 6g (for
6o) (1 equiv) in THF was added. After 10 min the appropriate
aldehyde (1.2 equiv) in THF was added and the mixture was
allowed to slowly reach room temperature overnight. The reac-
tion was quenched with saturated NH4Cl/H2O (1:1) and extrac-
ted with 3 ꢀ Et2O. The combined organic phase was dried over
MgSO4 and concentrated in vacuo.
Procedure B (6g-l). A mixture of 8a (1 equiv), RB(OH)2 (2.4
equiv), and PdCl2(PPh)2 (0.08 equiv) in toluene/EtOH (10:1)
and K2CO3 (3 M) was stirred at 100 °C for 20 h and then cooled
to room temperature. Et2O was added, and extraction was
performed using H2O, NaOH (1 M), and then H2O. The com-
bined organic phase was dried over MgSO4 and concentrated in
vacuo. The crude product was purified by DCVC.
Procedure C (6f,m,n,p). Compound 8a (for 6f,m,n) or 8b (for
6p) (1 equiv) was dissolved in THF and cooled to 0 °C where
iPrMgCl (1.5 equiv) was added. The reaction was monitored by
TLC, and after 1 h the appropriate aldehyde (1.5 equiv) was
added upon which the mixture was removed from the ice bath.
After 2 h at room temperature, saturated NH4Cl and water (1:1)

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3421
were added and the mixture was poured into a separating funnel
and extracted with 3 ꢀ Et2O. The combined organic phase was
dried over MgSO4 and concentrated in vacuo.
General Procedures for Deprotection. Procedure E (2a-e,
2g-o). A mixture of the protected compound in concentrated
aqueous HCl was stirred vigorously while heated at 130 °C for
1 h followed by evaporation and recrystallization.
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late (5a). 1-(Benzyloxy)-4-iodopyrazole 416 (5.35 g, 17.8 mmol)
was dissolved in THF (50 mL) and cooled to 0 °C where
iPrMgCl (16.5 mL, 21.4 mmol) was added. The reaction was
monitored by TLC. After 1 h, 1-ethoxycarbonyl-4-piperidone 3
(4.0 mL, 26.7 mmol) was added upon which the mixture was
removed from the ice bath. The water phase was further
extracted with diethyl ether (3 ꢀ 50 mL). The combined organic
phase was dried over MgSO4 and concentrated in vacuo. The
raw product was dissolved in CH2Cl2 (50 mL) where Et3SiH
(5.7 mL, 35.6 mmol) and trifluoroacetic acid (TFA)
(41 mL, 0.53 mol) were added. The mixture was heated for 2 h
at 50 °C. When the mixture was cooled, water (50 mL) was
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colorless oil (3.72 g, 63% over two steps).
Ethyl 4-(1-(Benzyloxy)-5-iodo-1H-pyrazol-4-yl)piperidine-1-
carboxylate (7). Diisopropylamine (0.84 mL, 6.0 mmol) was
dissolved in THF (5 mL) and cooled to -30 °C where n-BuLi
(4.3 mL, 6.0 mmol) was added. The mixture was cooled to
-78 °C where 5a (1.66 g, 5.0 mmol) was added. After 10 min, I2
(3.8 g, 15.0 mmol) in THF (10 mL) was added and the mixture
was allowed to slowly reach room temperature overnight. The
reaction was quenched with Na2SO3 and extracted with EtOAc
(3 ꢀ 50 mL). DCVC was applied, and product was isolated as
white crystals (1.25 g, 54%): mp 66-68 °C.
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Ethyl 4-(1-(Benzyloxy)-3-iodo-1H-pyrazol-4-yl)piperidine-1-
carboxylate (8a). To a solution of 5a (0.99 g, 3.0 mmol) in AcOH
(5 mL) a solution of ICl (0.59 g, 3.6 mmol) in AcOH (5 mL) was
added followed by water (15 mL). The mixture was stirred for
18 h at 85 °C, and upon cooling the reaction was quenched with
Na2S2O3. Water (30 mL) was added, and the mixture was
extracted with Et2O (3 ꢀ 30 mL). The combined organic phase
was dried over MgSO4 and concentrated in vacuo. DCVC
resulted in pure product as white crystals (0.95 g, 70%): mp
78-81 °C.
(15) 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.
(16) Felding, J. K., J.; Bjerregaard, T.; Sander, L.; Vedsø, P.; Begtrup,
M. Synthesis of 4-substituted 1-(benzyloxy)pyrazoles via iodine-
magnesium exchange of 1-(benzyloxy)-4-iodopyrazole. J. Org.
Chem. 1999, 64, 4196–4198.
(17) Kursanov, D. N.; Parnes, Z. N.; Loim, N. M. Application of ionic
hydrogenation to organic synthesis. Synthesis 1974, 633–651.
(18) Jensen, A. A.; Kristiansen, U. Functional characterisation of the
human alpha1 glycine receptor in a fluorescence-based membrane
potential assay. Biochem. Pharmacol. 2004, 67, 1789–1799.
€
(19) Jensen, A. A.; Mikkelsen, I.; Frølund, B.; Brauner-Osborne, H.;
Acknowledgment. H.A.M. and A.A.J. were supported by
the Danish Medical Research Council. A.A.J. was also sup-
ported by the Lundbeck Foundation. T.B. was supported by
the Carlsberg Foundation. Dr. Whiting and Merck Sharp &
Dohme are thanked for their generous gifts of the human
GABAAR cDNAs.
Falch, E.; Krogsgaard-Larsen, P. Carbamoylcholine homologs:
novel and potent agonists at neuronal nicotinic acetylcholine
receptors. Mol. Pharmacol. 2003, 64, 865–875.
€
(20) Krzywkowski, K.; Davies, P. A.; Feinberg-Zadek, P. L.; Brauner-
Osborne, H.; Jensen, A. A. High-frequency HTR3B variant asso-
ciated with major depression dramatically augments the signaling
of the human 5-HT3AB receptor. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 722–727.
Synthesis details, 1H
(21) Madsen, C.; Jensen, A. A.; Liljefors, T.; Kristiansen, U.; Nielsen,
B.; Hansen, C. P.; Larsen, M.; Ebert, B.; Bang-Andersen, B.;
Krogsgaard-Larsen, P.; Frølund, B. 5-Substituted imidazole-
4-acetic acid analogues: synthesis, modeling, and pharmacological
characterization of a series of novel gamma-aminobutyric acid(C)
receptor agonists. J. Med. Chem. 2007, 50, 4147–4161.
Supporting Information Available:
NMR and 13C NMR of all synthesized compounds, elemental
analyses data of all new target compounds, and pharmacologi-
cal methods. This material is available free of charge via the
Internet at http://pubs.acs.org.
(22) Joesch, C.; Guevarra, E.; Parel, S. P.; Bergner, A.; Zbinden, P.;
Konrad, D.; Albrecht, H. Use of FLIPR membrane potential dyes
for validation of high-throughput screening with the FLIPR and
microARCS technologies: identification of ion channel modula-
tors acting on the GABA(A) receptor. J. Biomol. Screening 2008,
13, 218–228.
(23) Liu, J.; Chen, T.; Norris, T.; Knappenberger, K.; Huston, J.;
Wood, M.; Bostwick, R. A high-throughput functional assay for
characterization of gamma-aminobutyric acid(A) channel modu-
lators using cryopreserved transiently transfected cells. Assay Drug
Dev. Technol. 2008, 6, 781–786.
References
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