Synthesis and biological evaluation of 4-(aminomethyl)-1-hydroxypyrazole analogues of muscimol as γ-aminobutyric acidA receptor agonists

Article abstract of DOI:10.1021/jm301473k

A series of bioisosteric 4-(aminomethyl)-1-hydroxypyrazole (4-AHP) analogues of muscimol, a GABA_A receptor agonist, has been synthesized and pharmacologically characterized at native and selected recombinant GABA_A receptors. The unsubstituted 4-AHP analogue (2a) (EC ₅₀ 19 μM, R_max 69%) was a moderately potent agonist at human α₁β₂γ₂ GABA_A receptors, and in SAR studies substitutions in the 3- and/or 5-position were found to be detrimental to binding affinities. Ligand-receptor docking in an α₁β₂γ₂ GABA_A receptor homology model along with the obtained SAR indicate that 2a and muscimol share a common binding mode, which deviates from the binding mode of the structurally related antagonist series based on 4-(piperidin-4-yl)-1- hydroxypyrazole (4-PHP, 1). Selectivity for α₁β ₂γ₂ over ρ₁ GABA_A receptors was observed for the 5-chloro, 5-bromo, and 5-methyl substituted analogues of 2a illustrating that even small differences in structure can give rise to subtype selectivity.

Full text of DOI:10.1021/jm301473k

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 4‑(Aminomethyl)-1-
hydroxypyrazole Analogues of Muscimol as γ‑Aminobutyric AcidA
Receptor Agonists
Jette G. Petersen,† Rikke Bergmann,† Henriette A. Møller,† Charlotte G. Jørgensen,†,‡ Birgitte Nielsen,†
Jan Kehler,§ Karla Frydenvang,† Jesper Kristensen,† Thomas Balle,# Anders A. Jensen,† Uffe Kristiansen,†
and Bente Frølund*,†
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, 2100 Copenhagen, Denmark
‡Department of Chemistry, University of Oxford, Mansfield Road, OX13TA, United Kingdom
§H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark
#Faculty of Pharmacy, The University of Sydney, Camperdown Campus, 2006 New South Wales, Australia
S
* Supporting Information
ABSTRACT: A series of bioisosteric 4-(aminomethyl)-1-
hydroxypyrazole (4-AHP) analogues of muscimol, a GABAA
receptor agonist, has been synthesized and pharmacologically
characterized at native and selected recombinant GABAA
receptors. The unsubstituted 4-AHP analogue (2a) (EC50 19
μM, Rmax 69%) was a moderately potent agonist at human
α1β2γ2 GABAA receptors, and in SAR studies substitutions in
the 3- and/or 5-position were found to be detrimental to
binding affinities. Ligand−receptor docking in an α1β2γ2
GABAA receptor homology model along with the obtained
SAR indicate that 2a and muscimol share a common binding
mode, which deviates from the binding mode of the
structurally related antagonist series based on 4-(piperidin-4-
yl)-1-hydroxypyrazole (4-PHP, 1). Selectivity for α1β2γ2 over ρ1 GABAA receptors was observed for the 5-chloro, 5-bromo, and
5-methyl substituted analogues of 2a illustrating that even small differences in structure can give rise to subtype selectivity.
subtypes have been proposed, the α1β2γ2, α2β3γ2, α3β3γ2
combinations being the predominant synaptic receptor
subtypes.4 GABAARs composed of ρ1−3 subunits assemble as
homo- or pseudohomomers and are also known as the
GABACRs.5
INTRODUCTION
■
γ-Aminobutyric acid (GABA) is the major inhibitory neuro-
transmitter in the central nervous system and hence essential
for the overall balance between neuronal excitation and
inhibition. The GABA neurotransmitter system is known to
be implicated in many disorders such as anxiety, epilepsy, mood
disorders, insomnia, cognitive disorders, and schizophrenia.1,2
Dysfunction in the GABA neurotransmitter system has major
consequences, and the restoration of the balance between
excitation and inhibition is the major aim of therapies affecting
the GABAergic system.
The effects of GABA are exerted through the ionotropic
GABAA receptors (GABAARs) and the metabotropic GABAB
receptors. The GABAARs belong to the Cys-loop receptor
superfamily also comprising the nicotinic acetylcholine
receptors (nAChRs), 5-HT3 serotonin receptors, and glycine
receptors.3 The receptors are membrane spanning ligand-gated
ion channels which, in the case of GABAARs, conduct chloride
ions. Functional GABAARs are pentamers assembled from a
pool of 19 different human subunits: α1−6, β1−3, γ1−3, δ, ε, θ, π,
and ρ1−3. At least 26 native and mainly heteromeric GABAAR
Over the years considerable insight into the GABAAR
orthosteric binding pocket has been obtained from studies of
a range of orthosteric ligands arising from the introduction of
conformational restriction and bioisosteric replacement of
functional groups in the structure of GABA. Classical ligands
include (Figure 1): muscimol,6 a potent GABAA agonist
isolated from the mushroom Amanita muscaria; 4,5,6,7-
tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP),7 a partial or
superagonist depending on subunit composition;8,9 and 5-(4-
piperidyl)-3-isoxazolol (4-PIOL),10,11 a low efficacy partial
agonist at GABAARs. These scaffolds have been extensively
explored by classical medicinal chemistry, and the results from
these studies have been compiled in pharmacophore
Received: October 11, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
AHP, 2a) and a series of 4-AHP analogues 2b−o (Figure 1)
as well as two hydroxypyrazol analogues of THIP, 16, 17, at the
GABAARs.
RESULTS
■
Chemistry. The synthesis of the 4-AHP analogues 2a−o is
illustrated in Schemes 1 and 2. The key intermediate 5 was
synthesized from pyrazole following a 5-step procedure.18−20
Directed ortho lithiation with LDA21 and subsequent treatment
with CBr4, C2Cl6 or MeI gave access to the protected 5-chloro,
5-bromo, and 5-methyl analogues (6b−d) (Scheme 1).
Compound 6d was iodinated in the 3-position using iodine
monochloride in acetic acid and water12,17 affording 7 which
was subjected to Suzuki cross coupling with methyltrifluor-
oborate22 to obtain 8. All compounds were deprotected with
aqueous HBr. The 3-position was accessed through the 3-
iodinated intermediate 9 (Scheme 2), which was selectively
obtained by refluxing 5 with iodine monochloride in acetic acid
and water. Suzuki cross coupling of 9 with arylboronic acids
readily afforded the protected phenyl, 3-biphenyl, and 4-
biphenyl analogues (10j,n,o) in good yields. The protected
vinyl analogue 10p was prepared by reaction with vinyl-
trifluoroborate salt and subsequent selective reduction of the
vinylic double bond using Wilkinson’s catalyst23 affording 10h.
The optimized conditions for the Suzuki cross coupling
developed for the formation of 8 were employed for the
preparation of 3-alkylsubstituted methyl, benzyl, and phenethyl
analogues (10g,k,l). The ethylene-4-pyridine analogue 10q was
prepared using standard Heck conditions with vinyl-4-pyridine
in excellent yield. Direct amination24 of the 3-iodinated
intermediate 9 with aqueous ammonia under copper-catalysis
afforded the 3-amino analogue 10i. All compounds except 10q
were deprotected with aqueous HBr. Deprotection and
hydrogenolysis of the exocyclic double bond of 10q was
accomplished with H2 and Pd/C.
Figure 1. Structures of GABA, muscimol, THIP, 4-PIOL, 4-PHP (1),
and a general structure of the new 1-hydroxypyrazole compounds 2a−
o.
models.12,13 In contrast, receptor homology models have had
little impact on the drug discovery process, primarily due to a
lack of structural information and insufficient sequence identity
of available template structures from acetylcholine binding
proteins (AChBPs). Progress in protein X-ray crystallography
utilizing bacterial ion channels and in particular a glutamate
gated channel from the nematode C. elegans14 has changed this
picture, and we have recently shown that reliable models of
GABAARs can be obtained using combinations of the available
templates.15,16
In our efforts to map the GABAAR orthosteric binding
pocket, we have previously established the 1-hydroxypyrazole
moiety in 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP) (1,
Figure 1) and analogues thereof as bioisosteric with the 3-
hydroxyisoxazole ring of 4-PIOL with respect to the
GABAARs.17 All published 4-PHP analogues are moderately
to highly potent GABAAR antagonists, and our study revealed
cavities in the vicinity of the 3- and 5-position of the 1-
hydroxypyrazole ring accommodating large substituents such as
phenyl and biphenyl groups.15,17 On the basis of the successful
use of the 1-hydroxypyrazole scaffold for mapping the
antagonist binding mode, we here report the synthesis and
pharmacological characterization of the comparatively smaller
muscimol analogue 4-(aminomethyl)-1-hydroxypyrazole (4-
The synthesis of the intermediate 4 from 3 (Scheme 1) by
treating 3 with iPrMgCl and reacting the resultant magnesium
species with tosylcyanide gave very variable yields (0−82%).
Therefore, we investigated palladium-catalyzed aminomethyla-
tion using a recently developed cross coupling method with
amidomethyltrifluoroborate.25 Suzuki cross coupling of potas-
sium benzamidomethyl trifluoroborate with the bromo
analogue of 3 led us directly to N-((1-(benzyloxy)-1H-
pyrazol-4-yl)methyl)benzamide, the benzamido analogue of
intermediate 5. This method was superior in yield and reduces
the number of steps (37% in two steps vs 67% in one step).
a
Scheme 1
a
Reagents and conditions: (a) i-PrMgCl, TsCN, THF, 0 °C to rt. (b) BnOCOCl, NaBH4, NiCl2, MeOH, 0 °C to rt. (c) 48% aqueous HBr, rt. (d)
LDA, THF, −78 °C, C2Cl6, CBr4 or MeI, −78 to −50 °C. (e) ICl, AcOH, H2O, 85 °C. (f) MeBF3K, Pd(OAc)2, RuPhos, Cs2CO3, toluene/H2O
10:1, 130−140 °C with MW irradiation.
B
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2
a
Reagents and conditions: (a) ICl, AcOH, H2O, 85 °C. For 10j,n,o: (b) RB(OH)2, PdCl2(PPh3)2, K2CO3, toluene/EtOH 10:1, 100 °C. For 10p:
(c) CH2CHBF3K, PdCl2(PPh3)2, K2CO3, toluene/EtOH 10:1, 100 °C. For 10g,k,l: (d) RBF3K, Pd(OAc)2, RuPhos, Cs2CO3, toluene/H2O 10:1,
100−130 °C with or without MW irradiation. For 10q: (e) CH2CH-4-pyr, Pd(OAc)2, P(o-tol)3, Et3N, MeCN, 90 °C. For 10i: (f) 35% aqueous
NH3, 2,4-pentadione, Cu(acac)2, Cs2CO3, DMF, 90 °C. For 2g−l,n−o: (g) 48% aqueous HBr, rt. For 2m: (h) H2, Pd/C, MeOH, rt. For 10h: (i)
H2, Rh(I)Cl(PPh3)3, MeOH, rt.
However, there is a difference in N-protecting groups; whereas
the amide protecting group requires harsh acidic conditions for
deprotection, the carbamate group allows for different and
milder strategies for deprotection. Recently, a cross coupling
method utilizing Boc-protected aminomethyltrifluoroborates
has been reported26 giving another option for accessing
aminomethylarenes and thereby bypassing the Grignard
reaction.
reported route.30 Compound 24 was synthesized from 18
(Scheme 4), which was prepared from hydroxyurea and
a
Scheme 4
The 1-hydroxypyrazol analogue of THIP, 16, and the
isomeric compound 17 were synthesized from the CBz-
protected piperidone 11 (Scheme 3). Treatment of 11 with
a
Scheme 3
a
Reagents and conditions: (a) i-PrBr, K2CO3, DMF, 55 °C, (b)
aqueous NH3, rt, (c) BH3, THF, rt, (d) Boc2O, Na2CO3, H2O/dioxane
3:1, 0 °C to rt, (e) n-BuLi, MeI, THF, −78 °C, (f) 33% HBr in AcOH,
60 °C.
dimethyl acetylenedicarboxylate.31 Protection of 18 as the
isopropyl ether afforded 19 which was amidated with aqueous
ammonia to form amide 2032 and reduced with borane to form
isopropyl protected muscimol 21. After Boc-protection of the
amine, the methyl group was introduced in the isoxazole 4-
position using n-butyl lithium and methyl iodide to form 23.
Deprotection with HBr in glacial acetic acid resulted in 24.
Pharmacology. The binding affinities of the compounds at
native GABAA receptors were measured by displacement of
[3H]muscimol in rat membrane preparations. Functional
characterization of selected compounds were carried out at
the human α1β2γ2S and ρ1 GABAA receptors either transiently
expressed in tsA201 cells using the FLIPR membrane potential
(FMP) blue assay or expressed in HEK 293 cells using whole-
cell patch-clamp electrophysiology.
a
Reagents and conditions: (a) DMF-DMA, 110 °C, (b) N2H4, MeOH,
reflux, (c) m-CPBA, EtOAc, rt, (d) 10% HBr in AcOH, rt.
DMF-DMA afforded 12 which cyclized upon treatment with
hydrazine to yield 13.27 Oxidation with m-CPBA28 gave rise to
two tautomers, 14 and 15, that were separated using
supercritical fluid chromatography, resulting in very low yield,
followed by deprotection using HBr in glacial acetic acid. The
configuration of the tautomer 1729 was assigned as the 2-
hydroxypyrazole using X-ray crystallography, and consequently,
tautomer 16 was the 1-hydroxypyrazole compound (see
Supporting Information for details).
The unsubstituted 4-AHP, 2a, exhibited binding affinity to
the native GABAARs in the low micromolar range (Table 1)
and a functional profile comparable to those of GABA and
muscimol, thus validating the use of the 1-hydroxypyrazole core
A new and more convergent synthetic strategy to access 4-
methylmuscimol, 24, was developed as opposed to a previously
C
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 1. Pharmacological Data for Reference Compounds GABA, Muscimol, and THIP, 2a−o, 16−17, and 24
b
compound
R1
R2
[3H]muscimol binding Ki (μM) [pKi SEM] α1β2γ2S EC50 (μM) [pEC50 SEM] ρ1 EC50 (μM) [pEC50 SEM]
c
GABA
muscimol
THIP
2a
0.049
0.007
1.8 [5.75 0.05]
0.27 [6.57 0.02]
0.70 [6.16 0.08]
d
0.54 [6.27 0.06]
e
f
0.16
14 [4.85 0.03]
100
H
H
0.22 [6.68 0.05]
0.63 [6.20 0.03]
2.8 [5.56 0.02]
15 [4.82 0.04]
>100
13 [4.89 0.06]
3.1 [5.51 0.06]
2b
H
Cl
Br
Me
Me
H
29 [4.53 0.05]
>500
nd
2c
H
nd
2d
H
>500
nd
>500
nd
2e
Me
I
2f
32 [4.50 0.04]
68 [4.16 0.08]
94 [4.03 0.01]
>100
>500
nd
>500
nd
2g
Me
Et
H
2h
H
>500
nd
>500
nd
2i
NH2
Ph
Bn
H
2j
H
>100
>500
nd
>500
nd
2k
H
>100
2l
CH2CH2Ph
CH2CH2-4-pyr
3-biPh
H
>100
nd
nd
2m
2n
H
>100
nd
nd
H
17 [4.81 0.10]
13 [4.89 0.04]
>100
>500
>300
nd
>500
>500
nd
2o
4-biPh
H
16
17
>100
nd
nd
24
54 [4.28 0.07]
nd
nd
a
GABAA receptor binding affinities at rat synaptic membranes and functional characterization at the human α1β2γ2S and ρ1 GABAA receptors
b
transiently expressed in tsA201 cells using the FLIPR membrane potential blue assay. IC50 values were calculated from inhibition curves and
c
converted to Ki values. Data are given as the mean [mean pKi SEM] of three to four independent experiments. nd: not determined. From ref 57.
d
e
f
Ref 58. Ref 59. Ref 46. Previous measurement on HEK293 cells from the antagonist assay and the value given is IC50 (μM).
Table 2. Functional Characteristics of GABA, Muscimol and 2a at the Human α1β2γ2S, α2β2γ2S, α3β2γ2S, α5β2γ2S, and ρ1 GABAAR
a
Subtypes Transiently Expressed in tsA201 Cells in the FLIPR Membrane Potential Blue Assay
α1β2γ2S EC50 (μM)
[pEC50 SEM]
α2β2γ2S EC50 (μM)
[pEC50 SEM]
α3β2γ2S EC50 (μM)
[pEC50 SEM]
α5β2γ2S EC50 (μM)
[pEC50 SEM]
ρ1 EC50 (μM)
[pEC50 SEM]
GABA
muscimol
2a
1.8 [5.75 0.05]
0.54 [6.27 0.06]
13 [4.89 0.06]
0.65 [6.18 0.06]
0.41 [6.39 0.14]
4.5 [5.35 0.11]
0.92 [6.04 0.04]
0.21 [6.67 0.10]
5.6 [5.25 0.10]
0.11 [6.97 0.12]
0.054 [7.27 0.11]
0.78 [6.10 0.09]
0.27 [6.57 0.02]
0.70 [6.16 0.08]
3.1 [5.51 0.06]
a
The EC50 values are given in μM with pEC50 SEM values in brackets.
as a bioisosteric replacement for the carboxylic acid in GABA.
However, restricting the aminomethyl side chain in a six-
membered ring with the THIP analogue 16 and the isomeric
compound 17 resulted in loss of affinity. Introduction of chloro,
bromo, and methyl substituents in the 5-position of the 1-
hydroxypyrazole ring (2b−d) also impaired binding affinity.
Substitution in the 3-position of the 1-hydroxypyrazole ring
followed the trend that larger substituents generated lower
binding affinities; there was room for iodo (2f) and methyl
(2g) substituents, but introduction of the more polar amine
group (2i) and larger ethyl (2h), phenyl (2j), benzyl (2k),
phenethyl (2l), and 2-(pyridine-4-yl)ethyl (2m) groups
resulted in loss of affinity.
Introductions of 3- or 4-biphenyl substituents in the 1-
hydroxypyrazole 5-position (2n,o) resulted in some regain of
affinity. The 5- and 3-methyl substituted analogues 2d and 2g
show some affinity (Ki 15 and 68 μM, respectively), which is
lost with the 3,5-dimethyl substituted analogue 2e (Ki > 100
μM), implying that the tight-fitting binding pocket is not able
to accommodate both substituents simultaneously. Introduc-
tion of a methyl substituent in the 5-posisiton of 4-AHP (2d)
gave rise to a significant 102 fold decrease in binding affinity
compared to the parent compound. In contrast, for the
corresponding 4-methyl-muscimol analogue 24, a more
profound 104 decrease in binding affinity relative to muscimol
was observed.
With respect to functional properties, analogues 2a and 2c
were characterized as full agonists at both α1β2γ2S and ρ1
GABAARs in the FMP assay (Rmax values not shown), whereas
the biphenyl substituted analogues 2n,o were devoid of activity
at the same receptors at concentrations up to 300−500 μM.
Analogously to muscimol, unsubstituted 2a displayed agonist
activity at all the tested GABAAR subtypes (α1β2γ2S, α2β2γ2S,
α3β2γ2S, α5β2γ2S, and ρ1) (Table 2) in the FMP assay with Rmax
values in the range 77−108% of the maximal response elicited
by GABA (data not shown). The EC50 values for 2a at the
different subtypes do not reflect any significant subtype
selectivity. Interestingly, the 5-substituted 4-AHP analogues
D
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
In the present study we used an induced fit docking protocol for
compound 2a similar to that reported for muscimol16 and obtained
essentially the same binding mode (Figure 3A, 2a shown in purple). In
the predicted binding pose of 2a, substituents in the 3- and 5-positions
would clash with either β2Tyr157 or β2Phe200 explaining the observed
affinity loss of compounds 2b−o (Figure 3B, 2a shown in purple and
2d shown in pink). The affinity decreases with increasing size of the
substituent Cl < Br < Me in the 5-position and I < Me < Et in the 3-
position supporting the identified binding pose. Finally, when the large
3-biphenyl and 4-biphenyl substituents are inserted in the 3-position
some affinity is regained. We have previously shown that there are
large hydrophobic pockets flanking the orthosteric binding site on
both sides in the antagonist conformation of the receptor.15,17
However, the 4-AHP scaffold must rotate about 45° in the binding site
to allow the biphenyl to pack in the pocket in the subunit interface
below the C-loop surrounded by β2Phe200, α1Phe45, and α1Phe64.
(2b−d) showed some degree of selectivity for α1β2γ2 over ρ1
GABARs in the electrophysiological measurements as opposed
to 24 that did not show any activity at concentrations up to 300
μM (Table 3, Figure 2A,B). While 5-chloro, 5-bromo, and 5-
Table 3. Functional Characteristics of Muscimol Analogues
2a−d and 24 at the Human α1β2γ2 and ρ1 GABAA Receptors
Transiently Expressed in HEK-293 Cells Measured Using
a
Patch-Clamp Electrophysiology
α1β2γ2
ρ1
EC50 (μM)
Rmax
EC50 (μM)
Rmax
2a
2b
2c
2d
24
19 [10; 35]
>30 μM
69 [55; 83]
>72%
55 [20; 152]
70 [42; 99]
no response at 300 μM
no response at 300 μM
no response at 300 μM
no response at 300 μM
>100 μM
>10 μM
>23%
>64%
DISCUSSION
no response at 300 μM
■
a
Values are reported as mean (n = 6) and 95% confidence interval (in
In the present study, we have expanded the use of the 1-
hydroxypyrazole scaffold as a carboxylic acid bioisosteric
replacement in the GABAAR agonist area. The 1-hydroxypyr-
azole scaffold utilized in this study has previously replaced the
3-isoxazolol ring in 4-PIOL,17 the phenol group in tamoxifen
analogues,33 and the distal carboxylic acid group in (S)-glutamic
acid34,35 and aspartic acid36 analogues. In addition to this
bioisosteric property, the 1-hydroxypyrazole scaffold allows for
introduction of substituents into the 3- and 5-positions
protruding into potentially unexplored areas of the receptor
binding pocket. In the study of 4-PIOL analogues, the
replacement of the 3-hydroxyisoxazole for 3- and 5-substituted
1-hydroxypyrazoles leads to highly potent GABAA antagonists,
indicating spacious cavities on both sides of the central binding
site where the core scaffold binds.
brackets). Rmax values are given in % of the maximal response of
GABA. Graphic concentration-response relationships on α1β2γ2
GABAARs are shown in Supporting Information.
methyl analogues showed some activity at the α1β2γ2S receptors,
the analogues were inactive at the ρ1 GABAARs at
concentrations up to 300 μM. In general, both the FMP
assay and electrophysiological measurements showed that 2a
displayed activity at α1β2γ2 and ρ1 GABAARs, whereas 2b−d
displayed reduced activity at the α1β2γ2 GABAAR and no
activity at ρ1 GABAARs.
COMPUTATIONAL MODELING
■
A recently reported full-length model of the α1β2γ2 GABAA receptor
suggesting binding modes of the GABAAR agonists GABA, muscimol,
and THIP was used as a starting point for the modeling studies.16 In
the model, GABA is predicted to bridge the β2 and α1 subunits, with
the acidic moiety forming contacts to α1Arg66 and hydrogen bonds to
β2Thr202 and α1Thr129. The ammonium group interacts with
β2Glu155 and hydrogen bonds to the backbone carbonyl of
β2Ser156 and is further stabilized by a π-cation interaction with
β2Tyr205. The hypothesis was rationalized by a large amount of
published mutational data. Supported by conformational energy
preferences, muscimol was predicted to bind in its global minimum
conformation with an aminomethyl side chain torsional angle of
around 45°. The amino group is then placed in a slightly different
position forming contacts to β2Glu155 and π-cation interactions to
two aromatic residues, β2Tyr205 and β2Phe20016 (Figure 3A,
muscimol shown in cyan).
The results of the present study of novel GABAAR agonists
show that 1-hydroxypyrazole is a valid bioisosteric replacement,
as the 1-hydroxypyrazole analogue of muscimol, 2a, displays
GABAAR affinity in the low micromolar range and pronounced
agonist activity at the receptors. We further investigated SAR
for 3- and 5-substituted 4-AHP analogues at the GABAARs. In
contrast to the findings for 4-PHP (1)17 and 4-PIOL,12,13,37,38
the SAR results for the 5-chloro, 5-bromo, and 5-methyl
analogues of 2a indicate that space is very limited in these areas
of the binding pocket. Introduction of iodo, methyl, and ethyl
substituents in the 5 position of 2a also leads to a decrease in
affinities as also seen for 16 where the aminomethyl side chain
of 2a has been incorporated into a six-membered ring.
Figure 2. Concentration−response curves for (A) 2a at α1β2γ2 and ρ1 GABAA receptors and (B) 2b at α1β2γ2 and ρ1 GABAA receptors. The
GABAARs are transiently expressed in HEK-293 cells. Responses were obtained using whole-cell patch-clamp electrophysiology and quantified using
the peak currents. The responses are given as % of the maximum response of GABA. Each point represents the mean SEM of five to seven
different cells.
E
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. The GABAA binding site at the interface between the β2 subunit (teal carbons) and the α1 subunit (green carbons). (A) The identified
docking poses of muscimol (cyan carbons) and 2a (purple carbons) are nearly identical. Similar interactions are formed between agonist and binding
site. A water molecule mediates additional contacts for muscimol and 2a to the β2 subunit. (B) The identified docking poses of 2a (purple carbons)
and 2d (pink carbons). Steric clash of the methyl substituent in the 5-position of the 4-AHP scaffold with β2Tyr157 can explain the observed
reduction of binding affinity of 2d compared to 2a.
Figure 4. The identified docking poses of 25 (wheat colored carbons) and 2a (purple carbons). The binding hypothesis of the antagonist 25 was
identified previously15 and requires an open C-loop to accommodate the bulky naphthyl substituent in the 3-position (wheat colored ribbon and
protein residues). On the contrary, 2a forms several contacts to a closed conformation of the C-loop (ribbon and residues in teal). Two different
conformations of α1Arg66 are able to interact with either 2a (green carbons) or 25 (wheat carbons).
During the progression of the present study, a receptor
homology model was developed proposing binding modes for
GABA and the GABAAR agonists muscimol and THIP.16
Docking of the 4-AHP analogues to this model suggests a
common binding mode for muscimol and 2a, which is
supported by parallel SAR of the two series. For muscimol,
as well as THIP, it has been shown that introduction of even
small aliphatic substituents are detrimental for affinity to the
GABAARs.39,40
Although the suggested binding mode for 2a corresponds
well with the SAR, which supports a common binding mode for
2a and muscimol, it does not explain the 30-fold lower affinity
of 2a compared to muscimol. An understanding of this
difference is likely to be found in a combination of
conformational energy preferences around the aminomethyl
side chain, which is the only flexible part of the molecules, and
of pKa values (4.8 for muscimol41 and 5.4 for 2a).
F
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Further SAR differences between muscimol and 2a are seen
by introduction of a methyl group in the 5-position of the 4-
AHP core (2d) and the corresponding 4-position of muscimol
(24) which leads to very different changes in binding affinities
compared to the respective parent compounds (102 vs 104
decrease). The docking studies show very similar poses for 2d
and 24 where the methyl groups clash with β2Tyr157 leading to
general affinity loss (Figure 3B). Furthermore, the methyl
substituent probably pushes the compounds toward the C-loop.
Thereby a small conformational change is enforced in these
molecules resulting in an aminomethyl torsional angle closer to
90° to avoid clashes with β2Tyr157 or the C-loop. This
conformational change could be more energetically favorable
for 2d than for 24 explaining the large discrepancy in affinity
change for the methyl substituted versus the unsubstituted
analogues.
Differing agonist and antagonist binding modes were
previously hypothesized in a common 3D-pharmacophore
model for GABAAR agonists and antagonists12,13 and are
supported by homology models.15,16 Our results indicate a
difference between agonist and antagonist binding modes when
comparing the 4-AHP analogues to the structurally related 4-
PHP analogues. The unsubstituted 4-PHP (1, Figure 1)
displayed GABAAR affinity in the low micromolar range, and
substitution in the 3- or 5-position of the 1-hydroxylpyrazole
core with bulky aromatic groups such as phenyl and biphenyl
resulted in potent antagonists at the GABAAR17 similar to the
corresponding 4-PIOL analogues. This is in sharp contrast to
the SAR observed for the 4-AHP analogues.
According to pharmacophore and receptor homology
models, two distinct low energy conformations of the
guanidinium moiety of α1Arg66 interact with the acidic moiety
of 2a and the corresponding moiety in a 4-PHP analogue as
illustrated in Figure 4 for 3,5-disubstituted 4-PHP analogue 3-
(naphthalen-2-ylmethyl)-5-phenyl-4-(piperidin-4-yl)-1H-pyra-
zol-1-ol 2515 and 2a. This leads to distinct orientations of the
heterocyclic moieties in the binding pocket and can explain the
nonparallel SAR for the 4-AHP/4-PHP (1) and muscimol/4-
PIOL series.
The series of events responsible for receptor activation upon
ligand binding is still a matter of debate. AChBPs, the structural
surrogates for the ligand binding domain of the Cys-loop
receptor superfamily, cocrystallized with different nicotinic
agonists and antagonists indicate an important role of C-loop
closure upon binding of an agonist.42 We speculate that for the
GABAAR in the antagonist binding mode there is a limited
extent of C-loop closure due to steric hindrance from the bulky
aromatic groups. For the small and relatively flat structures of
muscimol and 4-AHP full C-loop closure is allowed, which
combined with the effect of the flexible α1Arg66 can rationalize
the very limited space for the 4-AHP analogues.
CONCLUSION
■
A series of 1-hydroxypyrazole based muscimol analogues have
been synthesized and characterized pharmacologically at the
α1β2γ2 and ρ1 GABAARs. The study identified the 1-
hydroxypyrazole scaffold as a bioisosteric replacement for 3-
isoxazolol in muscimol within the GABAAR area. The
unsubstituted 4-AHP, 2a, was shown to be a moderately
potent agonist, and SAR studies showed that substitutions in
the 3- and 5-positions were detrimental to binding affinities.
The observed binding affinities have been addressed based on
ligand−receptor docking using a recently developed receptor
homology model for the GABAAR. This study and the parallel
SAR of muscimol analogues indicate that compound 2a and
muscimol share a common binding mode and network of
interactions with the binding pocket leaving no room for
substituents. The results support the hypothesized different
positions of the acidic heterocycles in the agonists, 2a and
muscimol relative to 4-PHP (1) and 4-PIOL. Altogether, these
results offer new knowledge about the architecture of the
orthosteric binding site in the restricted agonist binding mode
and illustrate that small differences in structure are crucial for
GABAAR activity and subtype selectivity, which will have to be
clarified in future mutational studies.
EXPERIMENTAL SECTION
■
Chemistry. General Procedures. Compound 4 was prepared
according to literature procedures.18 The preparation of N-((1-
benzyloxy)-1H-pyrazol-4-yl)methylbenzamide is described in Support-
ing Information. All solvents and reagents were obtained from
commercial suppliers and used without further purification. Dry THF
was obtained by distillation, and other dry solvents were obtained by
storage over 4 Å molecular sieves. All reactions involving air- and/or
moisture-sensitive reagents were performed under a nitrogen
atmosphere using syringe-septum cap techniques and/or with flame-
dried glassware. Analytical thin-layer chromatography (TLC) was
carried out using Merck silica gel 60 F254 plates, and the compounds
were visualized with UV light (254 nm), FeCl3, ninhydrin, or KMnO4
spraying agents. NMR spectra were recorded on a 300 MHz Varian
Mercury Plus spectrometer, a 300 MHz Varian Gemini 2000
spectrometer, or a 500 MHz Bruker Avance DRX500 spectrometer.
Dry column vacuum chromatography (DCVC)47 was performed using
Fischer Scientific silica gel 60 (20−45 μm). Flash chromatography
(FC) was performed using Merck silica gel 60 (40−63 μm), using
Combiflash Companion with a RediSep column with silica gel 60
(35−70 μm), or FlashMaster Personal one column (FP) (ISOLUTER
SPE Columns) apparatus. Reverse phase FC (RP-FC) was performed
using Merck LiChroprep RP-18 (40−63 μm). Details for preparative
supercritical flash chromatography (prep. SFC), preparative HPLC
(prep. HPLC), analytical HPLC (anal. HPLC) as well as details for
pKa measurement for 2a can be found in Supporting Information. All
microwave reactions were carried out in a glass reactor using a Biotage
Initiator instrument. Melting points were determined by OptiMelt
from Stanford Research Systems in open capillary tubes and are
uncorrected. HRMS was performed by Department of Organic
Chemistry, Vrije Universitet Brussel, Belgium. Elemental analysis
was performed by Mr. J. Theiner, Department of Physical Chemistry,
University of Vienna, Austria, or at the Analytical Research
Department, H. Lundbeck A/S, Denmark, and within 0.4% of the
calculated values unless otherwise stated.
A high degree of similarity exists between the orthosteric
binding pockets in the α1β2γ2 GABAA and the ρ1 GABAA
receptors, and yet the ρ GABAARs have a distinct
pharmacology in many respects compared to the other
GABAARs.43 Unnatural amino acid mutagenesis44,45 and SAR
studies in combination with receptor homology models46 have
revealed slight differences in the orthosteric binding pockets of
α1β2γ2 and ρ1 GABAARs, and the profiling of compounds from
the present study shows that receptor selectivity can be
obtained from orthosteric ligands. Future studies are needed to
clarify the underlying mechanisms of the observed selectivities.
General Procedure for Deprotection (2a,d-l,n,o). The protected
compound was dissolved in HBr (48% in H2O) and stirred at rt for 6
h. The mixture was evaporated under vacuum and re-evaporated from
H2O and toluene followed by recrystallization or column chromatog-
raphy and recrystallization.
4-(Aminomethyl)-1H-pyrazol-1-ol Hydrobromide (2a). Deprotec-
tion according to the general procedure using 5 (0.26 g, 0.77 mmol).
The product was purified by RP-FC and then HBr (48% in H2O, 1
G
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mL) was added and the mixture was evaporated under vacuum.
4-(Aminomethyl)-3-phenyl-1H-pyrazol-1-ol Hydrobromide (2j).
Deprotection according to the general procedure using 10j (0.33 g,
0.79 mmol). The product was purified by RP-FC and then HBr (48%
in H2O, 1 mL) was added and the mixture was evaporated under
vacuum. Recrystallization from MeOH/Et2O afforded the product as
white crystals (73 mg, 34%). mp >200 °C. 1H NMR (CD3OD): δ 7.93
(s, 1H), 7.60−7.53 (m, 2H), 7.52−7.37 (m, 3H), 4.19 (s, 2H). 13C
NMR (CD3OD): δ 145.0, 132.2, 129.9, 129.5, 129.2, 126.5, 109.7,
34.6. Anal. (C10H11N3O·HBr) C, H, N.
4-(Aminomethyl)-3-benzyl-1H-pyrazol-1-ol Hydrobromide (2k).
Deprotection according to the general procedure using 10k (137 mg,
0.32 mmol). Recrystallization from MeOH/Et2O afforded the product
as pale yellow crystals (50 mg, 16% over 2 steps). mp (decomp) > 143
°C. 1H NMR (D2O): δ 7.57 (s, 1H), 7.34−7.20 (m, 5H), 3.96 (s, 2H),
3.93 (s, 2H).13C NMR (D2O): δ 144.7, 139.3, 129.3, 128.8, 127.1,
125.4, 108.8, 33.0, 31.6. HRMS (ES+) calc. for C11H14N3O (MH+)
204.1131; found 204.1129. Anal. (C11H13N3O·1.3HBr) C, H, N.
4-(Aminomethyl)-3-phenethyl-1H-pyrazol-1-ol Hydrobromide
(2l). Deprotection according to the general procedure using 10l (99
mg, 0.22 mmol). Recrystallization from MeOH/Et2O afforded the
product as pale yellow crystals (23 mg, 10% over 2 steps). mp
(decomp) > 188 °C. 1H NMR (D2O): δ 7.46 (s, 1H), 7.31−7.22 (m,
3H), 7.13−7.10 (m, 2H), 3.61 (s, 2H), 2.89 (m, 4H).13C NMR
(D2O): δ 144.9, 141.7, 129.2, 129.0, 126.7, 124.6, 109.2, 35.6, 32.7,
27.4. HRMS (ES+) calc. for C12H16N3O (MH+) 218.1288; found
218.1289. Anal. (C12H15N3O·1.1HBr) C, H, N.
Recrystallization from MeOH/Et2O afforded the product as white
1
crystals (0.11 g, 73%). mp > 200 °C. H NMR (CD3OD): δ 7.88 (s,
1H), 7.56 (s, 1H), 4.11 (s, 2H). 13C NMR (CD3OD): δ 133.3, 126.0,
112.6, 34.6. Anal. (C4H7N3O·HBr·0.1H2O) C, H, N.
4-(Aminomethyl)-5-chloro-1H-pyrazol-1-ol Hydrobromide (2b).
6b (0.180 g, 0.484 mmol) was dissolved in a mixture of AcOH and
HBr (48% in H2O) and was stirred at 110 °C for 12 h. The mixture
was evaporated under vacuum and re-evaporated from H2O and
toluene followed by recrystallization from MeOH/Et2O affording the
1
product as colorless crystals (57 mg, 52%). mp >220 °C. H NMR
(D2O): δ 7.52 (d, J = 1.8 Hz, 1H), 4.09 (br s, 2H). 13C NMR
(CD3OD): δ 133.6, 122.6, 109.5, 33.8. Anal. (C4H6N3OCl·HBr) C, H,
N.
4-(Aminomethyl)-5-bromo-1H-pyrazol-1-ol Hydrobromide (2c).
Prepared as described for 2b using 6c (0.072 g, 0.172 mmol) affording
the product as colorless crystals (36 mg, 78%). mp >210 °C. 1H NMR
(CD3OD): δ 7.52 (s, 1H), 3.93 (s, 2H). 13C NMR (CDCl3): δ 135.1,
113.4, 110.9. Anal. (C4H6N3OBr·1.1HBr) C, H, N.
4-(Aminomethyl)-5-methyl-1H-pyrazol-1-ol Hydrobromide (2d).
Deprotection according to the general procedure using 6d (42 mg,
0.12 mmol). The product was purified by prep. HPLC and then HBr
(48% in H2O, 2.5 mL) was added and the mixture was evaporated
under vacuum. Recrystallization from MeOH/Et2O afforded the
1
product as white crystals (11 mg, 44%). mp (decomp) > 184 °C. H
NMR (D2O): δ 7.30 (s, 1H), 4.03 (s, 2H), 2.26 (s, 3H). 13C NMR
(CD3OD) δ 133.1, 132.8, 109.5, 34.5, 8.2. HRMS (ES+) calc. for
C5 H1 0 N3 O (MH+ ) 128.0818; found 128.0829. Anal.
(C5H9N3O·1HBr·0.2H2O) C, H, N.
4-(Aminomethyl)-3-(2-(pyridin-4-yl)ethyl)-1H-pyrazol-1-ol Hydro-
bromide (2m). 10q (248 mg, 0.56 mmol) was dissolved in MeOH (5
mL) and Pd/C (10%, 0.124 g) was added. The reaction mixture was
stirred at rt under a H2-atmosphere for 24 h. Additional Pd/C (10%,
0.124 g) was added and stirring was continued for 24 h. The reaction
mixture was filtered over Celite and evaporated under vacuum
affording the crude product as yellow solid (110 mg, 90%). The crude
product was dissolved in HBr (33% in AcOH, 4 mL) and stirred for 5
min at rt. The solvent was evaporated under vacuum affording the
product as orange solid (92 mg, 55% over two steps). mp (decomp) >
4-(Aminomethyl)-3,5-dimethyl-1H-pyrazol-1-ol Hydrobromide
(2e). Deprotection according to the general procedure using 8 (264
mg, 0.72 mmol). The product was purified by prep. HPLC and then
HBr (48% in H2O, 2.5 mL) was added and the mixture was evaporated
under vacuum followed by re-evaporation from toluene. Recrystalliza-
tion from MeOH/Et2O afforded the product as white crystals (31 mg,
9% over two steps). mp (decomp) > 181 °C. 1H NMR (D2O): δ 3.98
(s, 2H), 2.21 (s, 3H), 2.15 (s, 3H). 13C NMR (D2O): δ 141.5, 134.7,
106.4, 32.6, 10.5, 7.9. HRMS (ES+) calc. for C6H12N3O (MH+)
142.0975; found 142.0981. Anal. (C6H11N3O·1.45HBr) C, H, N.
4-(Aminomethyl)-3-iodo-1H-pyrazol-1-ol Hydrobromide (2f). De-
protection according to the general procedure using 9 (0.11 g, 0.24
mmol). The product was purified by RP-FC then HBr (48% in H2O,1
mL) was added and the mixture was evaporated under vacuum.
Recrystallization from MeOH/Et2O afforded the product as white
1
171 °C. H NMR (D2O): δ 8.53 (d, J = 5.9 Hz, 2H), 7.78 (d, J = 5.9
Hz, 2H), 7.54 (s, 1H), 3.95 (s, 2H), 3.23 (t, J = 7.3 Hz, 2H), 3.04 (t, J
= 7.3 Hz, 2H). 13C NMR (D2O): δ 163.6, 143.2, 140.6, 127.7, 125.2,
109.0, 95.0, 35.1, 32.7, 25.1. HRMS (ES+) calc. for C11H15N4O
(MH+) 219.1240; found 219.1243. Anal. HPLC purity 98% (254 nm).
Anal. (C11H14N4O·2HBr·1H2O) C, H, N.
4-(Aminomethyl)-3-(biphenyl-3-yl)-1H-pyrazol-1-ol Hydrobro-
mide (2n). Deprotection according to the general procedure using
10n (0.22 g, 0.45 mmol). The product was purified by RP-FC and
then HBr (48% in H2O, 1 mL) was added and the mixture was
evaporated under vacuum. Recrystallization from MeOH/Et2O
afforded the product as white crystals (22 mg, 13%). mp > 200 °C.
1H NMR (CD3OD): δ 7.98 (s, 1H), 7.78 (s, 1H), 7.69−7.60 (m, 3H),
7.56−7.51 (m, 2H), 7.47−7.39 (m, 2H), 7.37−7.30 (m, 1H), 4.22 (s,
2H). 13C NMR (CD3OD): δ 144.9, 142.8, 141.4, 132.8, 130.5, 129.9,
128.6, 128.2, 128.0, 127.9, 127.7, 126.5, 109.9, 34.6. Anal.
(C16H15N3O·HBr·1.5H2O): C, H, N.
4-(Aminomethyl)-3-(biphenyl-4-yl)-1H-pyrazol-1-ol Hydrobro-
mide (2o). Deprotection according to the general procedure using
10o (0.19 g, 0.38 mmol). The product was purified by RP-FC and
then HBr (48% in H2O, 1 mL) was added and the mixture was
evaporated under vacuum. Recrystallization from MeOH/Et2O
afforded the product as white crystals (41 mg, 26%). mp > 200 °C.
1H NMR (CD3OD): δ 7.82 (s, 1H), 7.76−7.70 (m, 2H), 7.68−7.62
1
crystals (44 mg, 57%). mp >200 °C. H NMR (CD3OD): δ 7.67 (s,
1H), 3.94 (s, 2H). 13C NMR (CD3OD): δ 126.2, 117.6, 92.0, 36.6.
Anal. (C4H6IN3O·HBr) C, H, N.
4-(Aminomethyl)-3-methyl-1H-pyrazol-1-ol Hydrobromide (2g).
Deprotection according to the general procedure using 10g (96 mg,
0.27 mmol). The product was purified by prep. HPLC and then HBr
(33% in AcOH, 0.8 mL) was added and the mixture was evaporated
under vacuum. Recrystallization from MeOH/Et2O afforded the
product as white crystals (16 mg, 16% over 2 steps). mp (decomp) >
190 °C. 1H NMR (D2O): δ 7.53 (s, 1H), 4.00 (s, 2H), 2.17 (s,
3H).13C NMR (D2O): δ 142.3, 125.1, 108.7, 33.01, 10.4. HRMS (ES
+) calc. for C5H10N3O (MH+) 128.0818; found 128.0824. Anal.
(C5H9N3O·1.1HBr) C, H, N.
4-(Aminomethyl)-3-ethyl-1H-pyrazol-1-ol Hydrobromide (2h).
Deprotection according to the general procedure using 10h (0.33 g,
0.79 mmol). Recrystallization from MeOH/Et2O afforded the product
as light brown crystals (13 mg, 38%). mp > 200 °C. 1H NMR
(CD3OD): δ 7.51 (s, 1H), 3.98 (s, 2H), 2.63 (q, J = 7.5 Hz, 2H), 1.24
(t, J = 7.5 Hz, 3H). 13C NMR (CD3OD): δ 147.9, 125.1, 109.2, 34.0,
20.1, 14.3. Anal. (C6H11N3O·1.45HBr): C, H, N.
(m, 4H), 7.48−7.41 (m, 2H), 7.38−7.31 (m, 1H), 4.22 (s, 2H). 13C
NMR (CD3OD): δ 144.9, 142.3, 141.5, 132.0, 129.9, 129.6, 128.6,
128.3, 127.8, 125.9, 109.9, 24.8. Anal. (C16H15N3O·1.8HBr) C, H, N.
Benzyl ((1-(benzyloxy)-1H-pyrazol-4-yl)methyl)carbamate (5). To
a stirred solution of 4 (6.0 g, 30.1 mmol) in MeOH (20 mL) at 0 °C
was added benzyl chloroformate (8.2 mL, 60.2 mmol), nickel chloride
hexahydrate (0.72 g, 3.0 mmol), and then NaBH4 (8.0 g, 210 mmol) in
small portions over 30 min. The reaction mixture was allowed to reach
rt and stirred for 18 h. Diethylenetriamine (3.3 mL, 30.1 mmol) was
added and the reaction was left to stir for 0.5 h before it was
3-Amino-4-(aminomethyl)-1H-pyrazol-1-ol Hydrobromide (2i).
Deprotection according to the general procedure using 10i (89 mg,
0.25 mmol). Recrystallization from MeOH/Et2O afforded the product
1
as light brown crystals (18 mg, 34%). mp (decomp) > 164 °C. H
NMR (D2O): δ 7.56 (s, 1H), 3.94 (s, 2H). 13C NMR (CDCl3): δ
125.9, 116.7, 91.3, 35.5. Anal. HPLC purity 97% (254 nm).
H
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
evaporated under vacuum. The remaining solid was dissolved in
EtOAc (200 mL) and washed with sat. aq. NaHCO3 (2 × 200 mL).
The organic phase was dried over MgSO4 and evaporated under
vacuum. Purification by DCVC (heptane/EtOAc 100:0 → 60:40)
afforded the product as white solid (4.6 g, 45%). mp 92.7−94.7 °C. Rf
(heptane/EtOAc 1:1) 0.48. 1H NMR (CDCl3): δ 7.28−7.17 (m,
10H), 7.12 (s, 1H), 6.90 (s, 1H), 5.17 (s, 2H), 5.02 (s, 2H), 4.75 (br s,
1H), 4.05 (d, 2H, J = 5.6 Hz). 13C NMR (CDCl3): δ 156.0, 136.3,
134.6, 132.4, 129.6, 128.6, 128.5, 128.1, 121.8, 116.1, 80.5, 66.8, 35.6.
Benzyl (1-(benzyloxy)-5-chloro-1H-pyrazol-4-yl)-
methylcarbamate (6b). A solution of 5 (0.150 mg, 0.444 mmol) in
THF (3 mL) was added dropwise to a solution of LDA (0.49 mL, 2 M
in THF) at −78 °C. A solution of C2Cl6 (210 mg, 0.89 mmol) in THF
(3 mL) was dropwise added to the reaction mixture and stirring was
continued for 1 h, and then the temperature was allowed to rise to
−50 °C. The mixture was quenched with aq. NH4Cl (3 mL) and
extracted with Et2O (3 × 5 mL). The combined organic phase was
dried over MgSO4 and evaporated under vacuum. Purification by
DCVC (heptane/EtOAc 4:1) afforded the product as colorless crystals
afforded a mixture of the product and dehalogenated starting material
(6:4) as yellow oil. Rf (heptane/EtOAc 1:1) 0.45. The mixture was
used directly for the formation of 2e.
Benzyl ((1-(benzyloxy)-3-iodo-1H-pyrazol-4-yl)methyl)carbamate
(9). Prepared as described for 7 using 5 (2.06 g, 6.1 mmol).
Purification by DCVC (heptane/EtOAc 100:0 → 80:20) afforded the
product as white solid (2.03 g, 72%). mp 57.2−59.2 °C. Rf (heptane/
EtOAc 1:1) 0.63. 1H NMR (CDCl3): δ 7.39−7.31 (m, 10 H), 6.97 (s,
1H), 5.28 (s, 2H), 5.10 (s, 2H), 4.97 (br s, 1H), 4.04 (d, 2H, J = 6.2
Hz). 13C NMR (CDCl3): δ 156.5, 136.7, 133.7, 130.1, 129.9, 129.2,
129.0, 128.7, 128.6, 124.3, 121.5, 90.9, 81.6, 67.3, 37.5.
Benzyl ((1-(benzyloxy)-3-methyl-1H-pyrazol-4-yl)methyl)-
carbamate (10g). Prepared as described for 8 using 9 (300 mg,
0.65 mmol), potassium methyltrifluoroborate (395 mg, 3.2 mmol),
Pd(OAc)2 (10.9 mg, 0.05 mmol), RuPhos (45.3 mg, 0.10 mmol),
Cs2CO3 (685 mg, 1.9 mmol) and toluene/H2O (10:1, 3.9 mL) with
heating for 2 h at 130 °C. Subsequently, extra reagents were added
followed by heating using microwave irradiation: potassium methyltri-
fluoroborate (395 mg, 3.2 mmol), Pd(OAc)2 (10.9 mg, 0.05 mmol),
RuPhos (45.3 mg, 0.10 mmol), 8 h at 130 °C. Purification by DCVC
(heptane/EtOAc 100:0 → 80:20) afforded a mixture of the product
and dehalogenated starting material (9:1). Rf (heptane/EtOAc 1:1)
0.45. The product was used directly for the formation of 2g.
1
(170 mg, 99%). H NMR (CDCl3): δ 7.29−7.40 (m, 10H), 5.24 (s,
2H), 5.10 (s, 2H), 4.12 (d, J = 5.7 Hz, 2H). 13C NMR (CDCl3): δ
156.1, 136.3, 133.5, 132.9, 130.0, 129.5, 128.6, 128.6, 128.2, 128.2,
116.4, 106.7, 81.2, 67.0, 35.7.
Benzyl (1-(benzyloxy)-3-ethyl-1H-pyrazol-4-yl)methylcarbamate
(10h). 10p (65 mg, 0.18 mmol) was dissolved in dry MeOH (5
mL) and Rh(I)Cl(PPh3)3 (Wilkinson’s catalyst) (8 mg, 9 μmol) was
added. Hydrogen was flushed through the solution, and the reaction
mixture was stirred at rt for 15 h. Extra Rh(I)Cl(PPh3)3 (8 mg, 9
μmol) was added, and hydrogen was applied once more and the
reaction mixture was stirred for another 15 h. The mixture was
evaporated under vacuum and purification by DCVC afforded the
Benzyl (1-(benzyloxy)-5-bromo-1H-pyrazol-4-yl)-
methylcarbamate (6c). Prepared as described for 6b using 5 (50
mg, 0.148 mmol) in THF (1 mL), LDA (0.165 mL, 2 M in THF) at
−78 °C and CBr4 (98 mg, 0.3 mmol) in THF (1 mL). Purification by
DCVC (heptane/EtOAc 4:1) afforded the product as colorless crystals
1
(48 mg, 79%). H NMR (CDCl3): δ 7.25−7.42 (m, 10H), 5.26 (s,
2H), 5.11 (s, 2H), 4.15 (d, J = 6.0 Hz, 2H). 13C NMR (CDCl3): δ
156.1, 136.3, 133.5, 132.9, 130.0, 129.5, 128.6, 128.2, 128.1, 116.4,
106.7, 81.2, 67.0, 35.7.
1
product as clear oil (57 mg, 87%). H NMR (CDCl3): δ 7.40−7.26
(10H, m), 6.91 (1H, s), 5.22 (2H, s), 5.10 (2H, s), 4.80 (1H, br s),
4.11 (2H, d, J = 5.3 Hz), 2.60 (2H, q, J = 7.6 Hz), 1.24 (3H, t, J = 7.6
Hz). 13C NMR (CDCl3): δ 156.0, 146.5, 136.5, 134.0, 129.7, 129.2,
128.7, 128.6, 128.2, 128.1, 122.7, 112.5, 80.5, 66.9, 34.8, 19.8, 14.1.
Benzyl ((3-amino-1-(benzyloxy)-1H-pyrazol-4-yl)methyl)-
carbamate (10i). 9 (200 mg, 432 mmol), Cu(acac)2 (11 mg, 43.2
mmol), Cs2CO3 (281 mg, 863 mmol) and DMF (2.2 mL) were added
to a vial and the solution was degassed with N2. 2,4-Pentadione and
ammonia solution (35% in H2O, 0.13 mL) were added and the
reaction mixture was stirred at 90 °C for 1 h. CH2Cl2 (10 mL) was
added and the mixture was filtered through Celite. The filtercake was
washed with CH2Cl2 and the filtrate washed with H2O (2 × 8 mL).
The aqueous phases were combined and extracted with CH2Cl2 (10
mL). The organic phases were combined, dried over MgSO4 and
evaporated under vacuum. Purification by DCVC (heptane/EtOAc
100:0 → 80:20) afforded the product as clear oil (106 mg, 70%). Rf
(heptane/EtOAc 1:1) 0.64. IR: 3419, 3321, 3032, 2948, 1701 cm−1.
1H NMR (CDCl3): δ 7.39−7.31 (m, 10H), 6.97 (s, 1H), 5.28 (s, 2H),
Benzyl (1-(benzyloxy)-5-methyl-1H-pyrazol-4-yl)-
methylcarbamate (6d). Prepared as described for 6b using 5
(0.150 mg, 0.444 mmol) in THF (3 mL), LDA (0.490 mL, 2 M in
THF) at −78 °C and methyliodide (0.060 mL, 0.977 mmol) in THF
(3 mL). Purification by DCVC (heptane/EtOAc 4:1) afforded the
1
product as colorless crystals (110 mg, 70%). H NMR (CDCl3): δ
7.14−7.32 (m, 11H), 5.19 (s, 2H), 5.07 (s, 2H), 4.07 (d, J = 5.1 Hz,
2H). 13C NMR (CDCl3): δ 156.0, 136.4, 132.1, 129.9, 129.3, 128.5,
128.4, 128.1, 128.0, 119.4, 112.9, 80.1, 66.7, 35.5. 8.0.
Benzyl ((1-(benzyloxy)-3-iodo-5-methyl-1H-pyrazol-4-yl)methyl)-
carbamate (7). To a solution of 6d (1.12 g, 3.2 mmol) in glacial
AcOH (10 mL) a solution of ICl (1.03 g, 6.4 mmol) in glacial AcOH
(10 mL) was added followed by H2O (30 mL). The reaction mixture
was stirred at 85 °C for 23 h then cooled to rt. Excess sat. aq. Na2S2O3
(30 mL) was added together with H2O (30 mL), and the mixture was
extracted with Et2O (3 × 60 mL). The organic phase was dried over
MgSO4 and evaporated under vacuum. Purification by DCVC
(heptane/EtOAc 100:0→80:20) afforded the product as white solid
1
5.10 (s, 2H), 4.97 (br s, 1H), 4.02 (d, J = 5.9 Hz, 2H). 13C NMR
(CDCl3): δ 156.0, 136.2, 133.2, 129.6, 129.4, 128.7, 128.5, 128.1,
128.0, 123.8, 121.0, 90.4, 81.1, 66.8, 37.0.
(883 mg, 49%). mp 84.3−86.1 °C. Rf (heptane/EtOAc 1:1) 0.66. H
NMR (CDCl3): δ 7.40−7.30 (m, 10H), 5.26 (s, 2H), 5.10 (s, 2H),
4.91 (br s, 1H), 4.02 (d, J = 5.7 Hz, 2H), 1.95 (s, 3H).13C NMR
(CDCl3): δ 155.9, 136.3, 133.2, 130.0, 129.5, 128.7, 128.5, 128.2,
128.1, 118.0, 91.2, 80.5, 66.8, 36.5, 8.2.
Benzyl (1-(benzyloxy)-3-phenyl-1H-pyrazol-4-yl)-
methylcarbamate (10j). 9 (0.51 g, 1.1 mmol) and phenylboronic
acid 97% (0.27 g, 2.2 mmol) were dissolved in toluene/EtOH (10:1,
22 mL). Aqueous K2CO3 (3 M, 0.72 mL) and PdCl2(PPh)2 (40 mg,
0.06 mmol) were added. The reaction mixture was stirred at 100 °C
for 20 h and then cooled to rt. Et2O (30 mL) was added and the
mixture was washed with H2O (30 mL), NaOH (1 M, 2 × 30 mL),
and H2O (30 mL). The organic phase was dried over MgSO4 and
evaporated under vacuum. Purification by DCVC (heptane/EtOAc
3:1) afforded the product as clear oil. (0.38 g, 84%). 1H NMR
(CDCl3): δ 7.62−7.56 (m, 2H), 7.46−7.30 (m, 13H), 7.09 (s, 1H),
5.33 (s, 2H), 5.10 (s, 2H), 4.81 (br s, 1H), 4.32 (d, J = 5.3 Hz, 2H).
13C NMR (CDCl3): δ 156.1, 143.7, 136.4, 133.8, 132.7, 129.7, 129.4,
Benzyl ((1-(benzyloxy)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)-
carbamate (8). 7 (300 mg, 0.65 mmol), potassium methyltriflour-
oborate (687 mg, 3.2 mmol), Pd(OAc)2 (11 mg, 0.05 mmol), RuPhos
(45 mg, 0.10 mmol), Cs2CO3 (685 mg, 1.9 mmol), and toluene/H2O
(10:1, 3.6 mL) were added to a microwave vial. The vial was purged
with N2, sealed with a cap and heated using microwave irradiation for 5
h at 130 °C. Subsequently, extra reagents were added two times
followed by heating using microwave irradiation: (1) potassium
methyltriflouroborate (687 mg, 3.2 mmol), Pd(OAc)2 (11 mg, 0.05
mmol) and RuPhos (45 mg, 0.10 mmol), 8 h at 130 °C, (2) potassium
methyltriflouroborate (275 mg, 1.3 mmol), Pd(OAc)2 (11 mg, 0.05
mmol) and RuPhos (45 mg, 0.10 mmol), 3 h at 140 °C. H2O (10 mL)
was added and the reaction mixture was extracted with EtOAc (3 × 10
mL). The organic phase was dried over MgSO4 and evaporated under
vacuum. Purification by DCVC (heptane/EtOAc 100:0 → 70:30)
128.7, 128.6, 128.2, 128.1, 127.9, 127.7, 123.8, 113.3, 80.8, 66.9, 35.7.
Benzyl ((3-benzyl-1-(benzyloxy)-1H-pyrazol-4-yl)methyl)-
carbamate (10k). Prepared as described for 8 using 9 (518 mg, 1.1
mmol), potassium benzyltrifluoroborate (554 mg, 2.8 mmol),
I
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Pd(OAc)2 (19 mg, 0.08 mmol), RuPhos (78 mg, 0.17 mmol), Cs2CO3
(1.18 g, 3.4 mmol), and toluene/H2O (10:1, 6.2 mL) with heating for
4 h at 130 °C. Subsequently, extra reagents were added followed by
heating using microwave irradiation: potassium benzyltriflouroborate
(305 mg, 1.6 mmol), Pd(OAc)2 (19 mg, 0.08 mmol) and RuPhos (78
mg, 0.17 mmol), 4 h at 130 °C. Purification by DCVC (heptane/
EtOAc 100:0 → 80:20) afforded the product as yellow oil
contaminated by phosphine ligand (∼7:3 distribution) (156 mg,
combined organic phases were washed with H2O (10 mL) and brine
(10 mL). The organic phase was dried over MgSO4 and evaporated
under vacuum. Purification by DCVC (heptane/EtOAc 100:0 →
0:100) afforded the product as yellow crystals (432 mg, 89%). Rf
1
(heptane/EtOAc 1:1) 0.08. H NMR (CDCl3): δ 8.45 (d, J = 5.9 Hz,
2H), 7.30−7.22 (m, 12 H), 7.14 (d, J = 16.1 Hz, 1H), 7.04 (d, J = 16.1
Hz, 1H), 6.88 (s, 1H), 5.22 (s, 2H), 5.03 (s, 2H), 4.83 (br s, 1H), 4.20
(d, 2H).13C NMR (CDCl3): δ 156.4, 150.5, 144.9, 141.1, 136.7, 133.9,
130.1, 129.8, 129.1, 129.0, 128.6, 128.4, 127.7, 124.1, 122.5, 121.2,
115.6, 81.2, 67.3, 35.1.
1
33%). Rf (heptane/EtOAc 1:1) 0.69. H NMR (CDCl3): δ 7.27−7.09
(m, 15H), 6.86 (s, 1H), 5.17 (s, 2H), 4.94 (s, 2H), 4.23 (br s, 1H),
3.88−3.85 (m, 4H), 1.25−1.19 (m, 6H, impurity), 0.83−0.79 (m, 4H,
impurity). 13C NMR (CDCl3): δ 155.8, 143.6, 139.4, 133.7, 129.6,
129.2, 128.5, 128.5, 128.4, 128.0, 127.9, 126.3, 123.3, 113.3, 80.4, 66.5,
34.6, 32.9, 31.8, 29.0, 22.7, 14.1.
N-Benzyloxycarbonyl-3-(dimethylaminomethylene)-4-piperidone
(12). 11 (5.0 g, 21.4 mmol) was dissolved in N,N-dimethylformamide
dimethyl acetal (20 mL) and refluxed at 110 °C for 2 h. The reaction
mixture was evaporated under vacuum and purified by FC (EtOAc/
EtOH 1:0→ 10:1) affording the product (2.7 g, 44%). Rf (EtOAc)
Benzyl ((1-(benzyloxy)-3-phenethyl-1H-pyrazol-4-yl)methyl)-
carbamate (10l). Prepared as described for 8 using 9 (350 mg, 0.76
mmol), potassium phenethyltrifluoroborate (408 mg, 1.9 mmol),
Pd(OAc)2 (13 mg, 0.06 mmol), RuPhos (53 mg, 0.11 mmol), Cs2CO3
(800 mg, 2.3 mmol), and toluene/H2O (10:1, 4.2 mL) with heating at
100 °C on oil bath for 19 h. No extra addition of reagents. Purification
by DCVC (heptane/EtOAc 100:0 → 85:15) afforded the product as
clear oil contaminated by phosphine ligand (∼75:25 distribution) (99
mg, 30%). Rf (heptane/EtOAc 1:1) 0.64. 1H NMR (CDCl3): δ 7.27−
7.02 (m, 15H), 6.76 (s, 1H), 5.13 (s, 2H), 4.96 (s, 2H), 4.17 (br s,
1H), 3.77 (d, J = 5.6 Hz, 2H), 2.87 (t, J = 6.7 Hz, 2H), 2.78 (t, J = 6.7
Hz, 2H), 1.19 (s, 5H, impurity), 0.80 (m, 3H, impurity). 13C NMR
(CDCl3): δ 155.8, 143.9, 141.3, 133.8, 133.7, 129.5, 129.1, 128.6,
128.5, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 126.1, 126.0, 122.5,
113.4, 80.4, 66.6, 36.1, 34.5, 31.9, 29.0, 29.0, 28.2, 22.6, 14.1.
Benzyl (1-(benzyloxy)-3-(biphenyl-3-yl)-1H-pyrazol-4-yl)-
methylcarbamate (10n). Prepared as described for compound 10j
using 9 (0.25 g, 0.54 mmol), 3-biphenylboronic acid (0.25 g, 1.2
mmol), toluene/EtOH (10:1, 11 mL), aqueous K2CO3 (3 M, 0.42
mL), and PdCl2(PPh)2 (22 mg, 0.03 mmol). Purification by DCVC
(heptane/EtOAc 3:1) afforded the product as clear oil (0.22 g, 82%).
1H NMR (CDCl3): δ 7.88 (s, 1H), 7.71−7.56 (m, 4H), 7.53−7.43 (m,
3H), 7.43−7.28 (m, 11H), 7.12 (s, 1H), 5.35 (s, 2H), 5.11 (s, 2H),
5.04 (br s, 1H), 4.37 (d, J = 5.3 Hz, 2H). 13C NMR (CDCl3): δ 156.1,
143.6, 141.7, 140.9, 136.4, 133.7, 133.2, 129.7, 129.4, 129.2, 128.8,
128.7, 128.6, 128.2, 128.1, 127.5, 127.3, 126.7, 126.6, 123.8, 113.4,
80.8, 66.9, 35.8.
Benzyl (1-(benzyloxy)-3-(biphenyl-4-yl)-1H-pyrazol-4-yl)-
methylcarbamate (10o). Prepared as described for compound 10j
using 9 (0.27 g, 0.58 mmol), 4-biphenylboronic acid (0.24 g, 1.2
mmol), toluene/EtOH (10:1, 11 mL), aqueous K2CO3 (3 M, 0.38
mL), and PdCl2(PPh)2 (20 mg, 0.03 mmol). Purification by DCVC
(heptane/EtOAc 3:1) afforded the product as clear oil (0.23 g, 81%).
1H NMR (CDCl3): δ 7.74−7.61 (m, 6H), 7.52−7.43 (m, 2H), 7.43−
1
0.13. H NMR (CDCl3): δ 7.49 (s, 1H), 7.29−7.36 (m, 5H), 5.16 (s,
2H), 4.61 (s, 2H), 3.69 (t, J = 7 Hz, 2H), 3.11 (br s, 6H), 2.46 (br s,
2H). 13C NMR (CDCl3): δ 194.3, 155.4, 149.3, 136.8, 128.6, 128.1,
127.9, 100.8, 67.29, 43.6, 42.3, 41.2, 37.5.
4,5,6,7-Tetrahydropyrazolo[4,5-c]pyridine-5-carboxylic Acid Ben-
zyl Ester (13). 12 (12.5 g, 43 mmol) was dissolved in MeOH (200
mL). Hydrazine hydrate (2.5 mL, 52 mmol) was added and the
reaction was refluxed for 2 h. The reaction mixture was evaporated
under vacuum and purified by FC (petroleum ether/EtOAc 1:1 →
1
0:1) affording the product (9.2 g, 82%). Rf(EtOAc) 0.4. H NMR
(CDCl3) δ 7.25−7.36 (m, 6H), 5.16 (s, 2H), 4.55 (br s, 2H), 3.76 (br
s, 2H), 2.77 (br s, 2H).
1-Hydroxy-4,5,6,7-Tetrahydropyrazolo[4,5-c]pyridine-5-carbox-
ylic Acid Benzyl Ester (14) and 1-Hydroxy-3,4,5,6-Tetrahydropyra-
zolo[4,3-c]pyridine-5-carboxylic Acid Benzyl Ester (15). 13 (9.2 g, 36
mol) was dissolved in EtOAc (100 mL) followed by the addition of m-
CPBA (70−75%, 8.1 g, 34 mol). The reaction mixture was stirred for 6
days at rt and then evaporated under vacuum. The purification was
performed over several steps, and after every purification the fractions
containing product were collected and evaporated under vacuum: The
mixture was purified by FC (1% AcOH in toluene/EtOAc 8:1 → 2:1),
then redissolved in Et2O/EtOAc (2:1 mixture) and washed with 1 N
HCl two times (which should remove starting material 13), H2O and
brine. The organic phase was dried with MgSO4 and evaporated under
vacuum giving 2.22 g (bumps very easily) of a crude mixture still
containing starting material 13. The mixture was purified again using
FC (petroleum ether/EtOAc 1:1 → 0:1) giving 1.43 g of a crude
mixture of the two products visualized by NMR (NMR: two products,
LC-MS one peak). Prep. SFC resulted in three batches with
diethylamine present (579 mg of “impure 14”, 171 mg of “impure
15”, 99 mg of “mix of 14 and 15)”). Purification by FC (EtOAc/
petroleum ether 3:1) of “impure 14” and “impure 15” gave pure 14
1
(278 mg, 3%) and pure 15 (109 mg, 1%). 14: H NMR (CDCl3): δ
11.35 (0.9H), 7.30−7.34 (m, 5H), 6.87 (br s, 1H), 5.13 (s, 2H), 4.45
7.28 (m, 11H), 7.10 (s, 1H), 5.35 (s, 2H), 5.12 (s, 2H), 4.93 (br s,
1H), 4.37 (d, J = 5.3 Hz, 2H). 13C NMR (CDCl3): δ 156.1, 143.3,
140.6, 136.4, 133.7, 131.7, 129.7, 129.4, 128.9, 128.8, 128.6, 128.3,
128.2, 128.0, 127.5, 127.4, 127.1, 123.9, 113.3, 80.8, 66.9, 35.9.
Benzyl (1-(benzyloxy)-3-vinyl-1H-pyrazol-4-yl)methylcarbamate
(10p). Prepared as described for compound 10j using 9 (200 mg,
0.42 mmol), potassium vinyl trifluoroborate (110 mg, 0.84 mmol),
toluene/EtOH (10:1, 22 mL), aqueous K2CO3 (3 M, 0.38 mL), and
PdCl2(PPh)2 (15 mg, 0.02 mmol). Purification by DCVC (heptane/
1
(s, 2H), 3.73 (br s, 2H), 2.60 (br s, 2H). 15: H NMR (CDCl3): δ
10.62 (0.3H), 7.30−7.34 (m, 5H), 7.11 (br s, 1H), 5.14 (s, 2H), 4.46
(s, 2H), 3.73 (br s, 2H), 2.60 (br s, 2H).
4,5,6,7-Tetrahydropyrazolo[4,5-c]pyridin-1-ol Hydrobromide
(16). 14 (258 mg, 0.9 mmol) was dissolved in HBr (10% in AcOH)
and stirred at rt for 15 min. The mixture was evaporated under
vacuum, triturated with Et2O, followed by recrystallization from
EtOH/Et2O affording the product as colorless crystals (118 mg, 57%).
1H NMR (DMSO-d6): δ 12.20 (s, 1H), 8.88 (br s, 2H), 7.08 (s, 1H),
1
EtOAc 3:1) afforded the product as clear oil (74 mg, 48%). H NMR
(CDCl3): δ 7.40−7.27 (m, 10H), 6.94 (s, 1H), 6.63 (dd, J = 17.6 Hz, J
= 11.1 Hz, 1H), 5.79 (d, J = 17.6 Hz, 1H), 5.31 (d, J = 11.1 Hz, 1H),
5.25 (s, 2H), 5.10 (s, 2H), 4.94 (br s, 1H), 4.20 (d, J = 5.3 Hz, 2H).
13C NMR (CDCl3): δ 156.0, 141.6, 136.4, 133.4, 129.7, 129.3, 128.7,
128.6, 128.2, 128.1, 126.7, 123.3, 116.1, 113.7, 80.7, 66.9, 35.0.
(E)-Benzyl ((1-(benzyloxy)-3-(2-(pyridin-4-yl)vinyl)-1H-pyrazol-4-
yl)methyl)carbamate (10q). 9 (500 mg, 1.1 mmol), 4-vinylpyridine
(0.35 mL, 3.2 mmol), Pd(OAc)2 (73 mg, 0.32 mmol), tri(o-
tolyl)phosphine (329 mg, 1.1 mmol), and triethylamine (0.03 mL,
21.6 mmol) were dissolved in MeCN (5 mL) and heated at 90 °C for
2.5 h. The reaction mixture was cooled to rt and added sat. aq. NH4Cl
(10 mL) followed by extraction with EtOAc (3 × 10 mL). The
4.11 (s, 2H), 3.39 (br s, 2H), 2.85 (t, J = 6 Hz, 2H). Anal.
(C6H9N3O·HBr) C, H, N.
3,4,5,6-Tetrahydro-pyrazolo[4,3-c]pyridin-1-ol Hydrobromide
(17). Prepared as described for 16 using 15 (109 mg, 0.4 mmol)
1
affording the product as colorless crystals (58 mg, 66%). H NMR
(DMSO-d6): δ 12.36 (s, 1H), 8.80 (br s, 2H), 7.49 (s, 1H), 4.09 (s,
2H), 3.39 (t, J = 7 Hz, 2H), 2.79 (t, J = 6 Hz, 2H). Anal.
(C6H9N3O·HBr) C, H, N.
3-Isopropoxy-5-(methoxycarbonyl)-isoxazol (19). A mixture of 18
(2.0 g, 13.97 mmol) and K2CO3 (2.12 g, 15.37 mmol) in dry DMF (25
mL) was stirred for 1 h at 60 °C. Isopropyl bromide (1.96 mL, 21.0
mmol) was added and the mixture was stirred overnight at 55 °C.
J
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Water (40 mL) was added and the aqueous phase was extracted with
Et2O (3 × 25 mL). The combined organic phases were dried over
MgSO4, filtered, and evaporated under vacuum. Purification by DCVC
(heptane/EtOAc 5:1) afforded the product as colorless oil (2.2 g,
88%). 1H NMR (CDCl3): δ 6.50 (s, 1H), 4.94 (hep, J = 6.0 Hz, 1H),
3.94 (s, 3H), 1.40 (d, J = 6.0 Hz, 6H). Spectral data were in agreement
with the literature.32
[3H]muscimol (5 nM final concentration), and 25 μL of test substance
in various concentrations, for 60 min at 0 °C. The reaction was
terminated by rapid filtration through GF/C filters (Perkin-Elmer Life
Sciences), using a 96-well Packard FilterMate cell-harvester, followed
by washing with 3 × 250 μL of ice-cold buffer. The dried filters were
added Microscint scintillation fluid (Perkin-Elmer Life Sciences), and
the amount of filterbound radioactivity was quantified in a Packard
TopCount microplate scintillator counter. The experiments were
performed in triplicate at least three times for each compound.
Nonspecific binding was determined using 1.0 mM GABA. The
binding data were analyzed by a nonlinear regression curve-fitting
procedure using GraphPad Prism v. 5.04 (GrapPad Software, CA,
USA).
5-Carbamoyl-3-isopropoxyisoxazole (20). A mixture of 19 (3.5 g,
18.9 mmol) and aqueous ammonia (25%) was stirred overnight at rt.
The reaction mixture was evaporated under vacuum and evaporated
twice with water and twice with toluene affording the product as
1
colorless crystals (3.0 g, 94%). H NMR (CD3OD): δ 6.57 (s, 1H),
4.88−4.93 (m, 1H), 1.42 (d, J = 6.6 Hz, 6H). Spectral data were in
agreement with the literature.32
Functional Characterization in the FMP Assay. The functional
characterization of compounds 2a,b,d,f,h,j,n,o at GABAARs in the
FLIPR membrane potential blue assay was performed essentially as
described previously.49 8 × 105 tsA-201 cells were split into a 6 cm
tissue culture plate and transfected the following day with a total of 5
μg cDNA using Polyfect (Qiagen, Hilden, Germany). Cells were
cotransfected with 1 μg of α1,2,3,5-pcDNA3.1, 1 μg of β2-pcDNA3.1 and
3 μg of γ2S-pCDNA3.1, or transfected with 5 μg of ρ1-pcDNA3. The
following day, cells were split into poly-D-lysine-coated black 96-well
plates with clear bottom (BD Biosciences, Bedford, MA). 16−24 h
later the medium was aspirated, and the cells were washed with 100 μL
of Krebs buffer [140 mM NaCl/4.7 mM KCl/2.5 mM CaCl2/1.2 mM
MgCl2/11 mM HEPES/10 mM D-glucose, pH 7.4]. 50 μL of Krebs
buffer was added to the wells (in the antagonist experiments, various
concentrations of the antagonist were dissolved in the buffer), and
then an additional 50 μL of Krebs buffer supplemented with assay dye
(1 mg/mL) was added to each well. Then the plate was incubated at
37 °C in a humidified 5% CO2 incubator for 30 min and assayed in a
NOVOstar plate reader (BMG Labtechnologies, Offenburg, Germany)
measuring emission [in fluorescence units (FU)] at 560 nm caused by
excitation at 530 nm before and up to 1 min after addition of 33 μL of
agonist solution. The experiments were performed in duplicate at least
three times for each compound at each receptor. EC70−EC90
concentrations of GABA were used as agonist in the antagonist
experiments. Concentration−response curves for agonists and
concentration-inhibition curves for antagonists were constructed
based on the difference in the fluorescence units (ΔFU) between
the maximal fluorescence recording made before and after addition of
agonist obtained for different concentrations of the respective ligands.
The curves were generated by nonweighted least-squares fits using the
program KaleidaGraph 3.6 (Synergy Software).
Functional Characterization Using Whole-Cell Patch-Clamp
Electrophysiology. The electrophysiological characterization of
compounds 2a,b,c,d at GABAARs was performed essentially as
described previously.46 HEK 293 cells (ECACC, UK) were split into
35 mm Petri dishes. 18−24 h later, the cells were transfected with
either a combination of rat α1-pCis, β2-pCis, and γ2S-pCis (1:1:2 ratio)
or human ρ1-pCDNA3, and cotransfected with plasmid coding for
green fluorescent protein (pGreen Lantern, Life technologies, Paisley,
UK) in order to visualize transfected cells. Targefect 293 was used as a
DNA carrier according to the manufacturer’s protocol (Targeting
Systems, CA, USA). The cells were used for whole-cell patch-clamp
experiments 40−72 h after transfection. Petri dishes with cells were
transferred to the stage of an Axiovert 10 microscope (Zeiss,
Germany), and the culture medium was exchanged for extracellular
recording solution at room temperature (20−22 °C). The extracellular
solution contained (in mM): NaCl 140, KCl 3.5, Na2HPO4 1.25,
MgSO4 2, CaCl2 2, glucose 10, and HEPES 10; pH 7.35. Cells
containing green fluorescent protein were visualized with UV light
from a HBO 50 lamp (Zeiss, Germany). The cells were approached
with micropipets of 1.7−3 MΩ resistance manufactured from 1.5 mm
o.d. glass (World Precision Instruments, FA, USA). The intrapipette
solution contained (in mM): KCl 140, MgCl2 1, CaCl2 1, EGTA 10,
MgATP 2, and HEPES 10; pH 7.3. Standard patch-clamp techniques50
in voltage clamp mode were used to record from neurons in the
whole-cell configuration using an EPC-9 amplifier (HEKA, Germany).
A clamping potential of −60 mV was used. Series resistance was 65−
5-Aminomethyl-3-isopropoxyisoxazole (21). A solution of 20 (3.0
g, 17.6 mmol) in dry THF (50 mL) was slowly added a solution of
borane in THF (26.4 mL, 1 M). The reaction mixture was stirred
overnight at rt. 4N HCl (aq) was added to pH 1 followed by stirring
for 1 h and evaporation under vacuum. The remaining solid was
suspended in water and 5 N NaOH (aq) was added to pH 12. The
aqueous mixture was extracted with Et2O (3 × 50 mL) and the
combined organic phase was dried over MgSO4 and evaporated under
1
vacuum affording the product as yellow oil (2.0 g, 72%). H NMR
(CDCl3/CD3OD): δ 5.78 (1H, s), 4.80 (hep, J = 6.0 Hz, 1H), 3.82 (s,
2H), 3.62 (br s, 2H), 1.37 (d, J = 6.0 Hz, 6H).
5-(tert-Butyloxycarbonyl)aminomethyl-3-isopropoxyisoxazole
(22). To an ice cold solution of Na2CO3 (0.679 g, 6.4 mmol) in water
(2 mL) was slowly added a solution of 21 (0.5 g, 3.2 mmol) in
dioxane/H2O (3:1, 8 mL) over 1 h at 0 °C. A solution of di-tert-butyl
dicarbonate (0.77 g, 3.5 mmol) in dioxane (2 mL) was added
dropwise, and the reaction mixture was stirred overnight at rt. Dioxane
was evaporated under vacuum and the aqueous phase was extracted
with Et2O (3 × 20 mL). The combined organic phases were dried over
MgSO4 and evaporated under vacuum. Purification by DCVC
(heptane/EtOAc 4:1) afforded the product as viscous oil (0.67 g,
1
82%). H NMR (CDCl3/CD3OD): δ 5.76 (s, 1H), 5.36 (br s, 1H),
4.85 (hep, J = 6.0 Hz, 1H), 4.29 (d, J = 6.3 Hz, 2H), 1.45 (s, 9H), 1.36
(d, J = 6.3 Hz, 6H). 13C NMR (CDCl3): δ 170.9, 170.2, 155.5, 93.8,
80.1, 73.2, 37.1, 28.4, 22.8, 22.0, 14.2.
5-(tert-butyloxycarbonyl)aminomethyl-4-methyl-3-isopropoxyi-
soxazole (23). A solution of 22 (0.610 g, 2.38 mmol) in dry THF (10
mL) at −78 °C was dropwise added n-BuLi (3.3 mL, 1.6 M in THF,
5.23 mmol) and stirring was continued for 45 min at −78 °C. Methyl
iodide (0.355 mL, 5.71 mmol) was added and stirring was continued
for 2 h at −78 °C. The mixture was quenched with aqueous NH4Cl
(10 mL) and extracted with CH2Cl2 (3 × 5 mL). The combined
organic phase was dried over MgSO4 and evaporated under vacuum.
Purification by DCVC (heptane/EtOAc 4:1) afforded the product as
colorless oil (380 mg, 59%). 1H NMR (CDCl3/CD3OD): δ 4.94 (br s,
1H), 4.88 (hep, J = 6.3 Hz, 1H), 4.25 (d, J = 6.0 Hz, 2H), 1.86 (s, 3H),
1.44 (s, 9H), 1.37 (d, J = 6.3 Hz, 6H). 13C NMR (CDCl3): δ 170.2,
164.0, 155.4, 102.4, 80.0, 73.1, 35.7, 28.5, 22.1, 5.5.
4-(Aminomethyl)-5-methyl-isoxazol-1-ol hydrobromide (24). A
suspension of 23 (0.300 g, 1.109 mmol) in 33% HBr/AcOH (10 mL)
was stirred at 60 °C overnight. The reaction mixture was evaporated
under vacuum and purified using prep. HPLC. The purified product
was stirred in 33% HBr/AcOH at rt for 5 min and evaporated under
vacuum. Recrystallization from MeOH/Et2O afforded the product as
1
colorless crystals (131 mg, 56%): mp (decomp) > 212 °C. H NMR
(D2O): δ 4.23 (s, 2H), 1.91 (s, 3H). Anal. (C5H8N2O2·HBr) C, H, N.
Pharmacology. Characterization of 2a-o,16,17,24 in Muscimol
Binding. The binding assay was performed using rat brain synaptic
membranes of cortex and the central hemispheres from male SPRD
rats with tissue preparation as described in the literature.48 On the day
of the experiment, the membrane preparation was quickly thawed,
homogenized in 50 vol of ice-cold buffer (50 mM Tris-HCl buffer, pH
7.4), and centrifuged at 48000g for 10 min at 4 °C. This washing step
was repeated four times and the final pellet was resuspended in buffer.
The assay was carried out in 96-well plates, by incubation of
membranes (70−80 μg protein) in 200 μL of buffer, 25 μL of
K
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
80% compensated. Whole-cell currents were analyzed using Pulse
software (HEKA, Germany). Extracellular solution containing the
agonists was applied using a gravity fed 7-barrelled perfusion pipet
(List, Germany), which allowed exchange of the extracellular solution
surrounding the recorded neuron with a time constant of ∼50 ms. The
agonists were applied for 5 s every 1 min. Between the agonist
applications, drug-free ABSS was applied from one of the barrels. For
the agonist concentration−response relationship the equation:
ABBREVIATIONS:
■
4-AHP, 4-(aminomethyl)-1-hydroxypyrazole; 4-PHP, 4-(piper-
idin-4-yl)-1-hydroxypyrazole; 4-PIOL, 5-(4-piperidyl)-3-isoxa-
zol; DCVC, dry column vacuum chromatography; ELIC,
Erwinia ligand-gated ion channel; FC, flash chromatography;
FLIPR, fluorescent imaging plate reader; FMP, FLIPR
membrane potential; GABA, γ-aminobutyric acid; GluCl,
glutamate-gated chloride channel; IFD, induced fit docking;
MW, microwave; RP-FC, reverse phase flash chromatography;
SFC, supercritical flash chromatography; THIP, 4,5,6,7-
tetrahydroisoxazolo[5,4-c]pyridine-3-ol
I = Imax/(1 + antilog((log EC50 − A)nH))
was fitted to the experimental data (Graphpad Prism Graphpad
Software, CA, USA), where I is the peak membrane current, A the
logarithm of the agonist concentration, Imax is the maximum peak
current that the agonist can induce, EC50 is the agonist concentration
eliciting 50% of Imax, and nH is the Hill coefficient. Data were described
using mean and standard error (S.E.).
REFERENCES
■
(1) Foster, A. C.; Kemp, J. A. Glutamate- and GABA-based CNS
therapeutics. Curr. Opin. Pharmacol. 2006, 6, 7−17.
(2) Johnston, G. A. R. GABAA Receptor Channel Pharmacology.
Curr. Pharm. Des. 2005, 11, 1867.
(3) Dougherty, D. A. Cys-Loop Neuroreceptors: Structure to the
Rescue? Chem. Rev. 2008, 108, 1642−1653.
(4) Paul, J, W. GABAA receptor subtypes in the brain: a paradigm for
CNS drug discovery? Drug Discovery Today 2003, 8, 445−450.
(5) Sieghart, W. Structure, Pharmacology, and Function of GABAA
Receptor Subtypes. In Adv. Pharmacol.; Enna, S. J., Ed.; Academic
Press: New York, 2006; Vol. 54, pp 231−263.
(6) Johnston, G. A. R.; Curtis, D. R.; de Groat, W. C.; Duggan, A. W.
Central actions of ibotenic acid and muscimol. Biochem. Pharmacol.
1968, 17, 2488−2489.
(7) Krogsgaard-Larsen, P.; Johnston, G. A. R.; Lodge, D.; Curtis, D.
R. A new class of GABA agonist. Nature 1977, 268, 53−55.
(8) Ebert, B.; Wafford, K. A.; Whiting, P. J.; Krogsgaard-Larsen, P.;
Kemp, J. A. Molecular pharmacology of gamma-aminobutyric acid
type A receptor agonists and partial agonists in oocytes injected with
different alpha, beta, and gamma receptor subunit combinations. Mol.
Pharmacol. 1994, 46, 957−963.
(9) Brown, N.; Kerby, J.; Bonnert, T. P.; Whiting, P. J.; Wafford, K.
A. Pharmacological characterization of a novel cell line expressing
human α4β3δ GABAA receptors. Br. J. Pharmacol. 2002, 136, 965−974.
(10) Kristiansen, U.; Lambert, J. D. C.; Falch, E.; Krogsgaard-Larsen,
P. Electrophysiological studies of the GABAA receptor ligand, 4-PIOL,
on cultured hippocampal neurones. Br. J. Pharmacol. 1991, 104, 85−
90.
(11) Byberg, J. R.; Labouta, I. M.; Falch, E.; Hjeds, H.; Krogsgaard-
Larsen, P. Synthesis and biological activity of a GABAA agonist which
has no effect on benzodiazepine binding and of structurally related
glycine antagonists. Drug Des. Delivery 1987, 1, 261−274.
(12) 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 GABAA Antagonists: Synthesis, Pharmacol-
ogy, and Molecular Modeling. J. Med. Chem. 2002, 45, 2454−2468.
(13) Frølund, B.; Tagmose, L.; Liljefors, T.; Stensbøll, T. B.;
Engblom, C.; Kristiansen, U.; Krogsgaard-Larsen, P. A Novel Class of
Potent 3-Isoxazolol GABAA Antagonists: Design, Synthesis, and
Pharmacology. J. Med. Chem. 2000, 43, 4930−4933.
(14) Hibbs, R. E.; Gouaux, E. Principles of activation and permeation
in an anion-selective Cys-loop receptor. Nature 2011, 474, 54−60.
(15) Sander, T.; Frølund, B.; Bruun, A. T.; Ivanov, I.; McCammon, J.
A.; Balle, T. New insights into the GABAA receptor structure and
orthosteric ligand binding: Receptor modeling guided by experimental
data. Proteins: Struct., Funct., Bioinf. 2011, 79, 1458−1477.
(16) Bergmann, R.; Kongsbak, K.; Sørensen, P. L.; Sander, T.; Balle,
T. A Unified model of the GABAA receptor comprising agonist and
benzodiazepine binding sites. PLoS ONE 8, e52323, doi: 10.1371.
(17) Møller, H. A.; Sander, T.; Kristensen, J. L.; Nielsen, B.; Krall, J.;
Bergmann, M. L.; Christiansen, B.; Balle, T.; Jensen, A. A.; Frølund, B.
Novel 4-(Piperidin-4-yl)-1-hydroxypyrazoles as γ-Aminobutyric AcidA
Computational Modeling. A novel homology model of the full-
length GABAα1β2γ2 receptor was used for all modeling.16 In short, the
receptor model was generated as follows: a structural alignment was
generated with the glutamate gated chloride channel (GluCl) from C.
elegans, (PDB: 3RIF) and the ligand gated ion channel from E.
chrysanthemi (ELIC), (PDB: 2VL0).14,51 All sequences for the human
glycine receptor α subunits and GABA receptor subunits were
subsequently aligned to the sequences of GluCl and ELIC as a profile
in Clustal X v. 2.0.12.52 Model building was performed using Modeler
v. 9.7.53 Ligands for docking were created in their ionized states in
Maestro 9.2 followed by conformational searches with MacroModel
9.9 (default settings).54 The global energy minimum conformations
were identified for docking. Ligand docking was accomplished with the
induced fit docking (IFD)54,55 workflow in Maestro v. 9.2 and the XP
scoring function.56 By default the IFD procedure allows residues to
adapt to the docked ligand in a 5 Å sphere. Docking was performed
both with and without a water molecule in the binding site. The water
molecule was placed between the β2Ser156 and β2Tyr157 backbone
and the aromatic ring of β2Tyr157 and was able to move during IFD.
Docking poses were selected based on mutational data and
identification of common interaction patterns in the binding site.
Finally, selected ligand poses including an 8 Å sphere were energy
minimized to convergence using MacroModel 9.9. This allowed the C-
loop to open very slightly and gave rise to less strained poses with
better geometry in the binding pocket.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis details, elemental analysis data of new target
compounds, HPLC and SFC details, pKa measurement, X-ray
crystal structure analysis, and electrophysiological concentra-
tion−response relationship details. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: bfr@sund.ku.dk. Phone (+45)35336495.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Flemming Hansen for technical assistance with
collection of X-ray data, Ulla Geneser for technical laboratory
assistance, Peter Brøsen for SFC purification and Erling B.
Olsen for pKa determination. J.G.P. was supported by the
Lundbeck Foundation, R.B. was supported by the Velux
Foundation, U.K. was supported by the Carlsberg Foundation,
and A.A.J. was supported by the Novo Nordisk and Carlsberg
Foundations.
L
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Receptor Ligands: Synthesis, Pharmacology, and Structure-Activity
Relationships. J. Med. Chem. 2010, 53, 3417−3421.
(18) Felding, J.; Kristensen, 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.
Brauner-Osborne, H.; Traynelis, S. F.; Kristensen, J. L. N-
̈
Hydroxypyrazolyl Glycine Derivatives as Selective N-Methyl-d-aspartic
Acid Receptor Ligands. J. Med. Chem. 2008, 51, 4179−4187.
(37) 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,
Pharmacology, and Molecular Modeling. J. Med. Chem. 2004, 48,
427−439.
(38) Frølund, B.; Jensen, L. S.; Storustovu, S. I.; Stensbøl, T. B.;
Ebert, B.; Kehler, J.; Krogsgaard-Larsen, P.; Liljefors, T. 4-Aryl-5-(4-
Piperidyl)-3-Isoxazolol GABAA Antagonists: Synthesis, Pharmacology,
and Structure-Activity Relationships. J. Med. Chem. 2007, 50, 1988−
1992.
(19) Caddick, S.; Judd, D. B.; Lewis, A. K. d. K.; Reich, M. T.;
Williams, M. R. V. A generic approach for the catalytic reduction of
nitriles. Tetrahedron 2003, 59, 5417−5423.
(20) Caddick, S.; de K. Haynes, A. K.; Judd, D. B.; Williams, M. R. V.
Convenient synthesis of protected primary amines from nitriles.
Tetrahedron Lett. 2000, 41, 3513−3516.
(21) Vedsø, P.; Begtrup, M. Synthesis of 5-Substituted 1-
Hydroxypyrazoles through Directed Lithiation of 1-(Benzyloxy)-
pyrazole. J. Org. Chem. 1995, 60, 4995−4998.
(22) Dreher, S. D.; Lim, S.-E.; Sandrock, D. L.; Molander, G. A.
Suzuki-Miyaura Cross-Coupling Reactions of Primary Alkyltrifluor-
oborates with Aryl Chlorides. J. Org. Chem. 2009, 74, 3626−3631.
(23) Young, J. F.; Osborn, J. A.; Jardine, F. H.; Wilkinson, G. Hydride
intermediates in homogeneous hydrogenation reactions of olefins and
acetylenes using rhodium catalysts. Chem. Commun. (London) 1965,
131−132.
(24) Xia, N.; Taillefer, M. A Very Simple Copper-Catalyzed Synthesis
of Anilines by Employing Aqueous Ammonia. Angew. Chem., Int. Ed.
2009, 48, 337−339.
(39) Krogsgaard-Larsen, P.; Johnston, G. A. R. Structure-Activity
Studies on the Inhibition of GABA Binding to Rat Brain Membranes
by Muscimol and Related Compounds. J. Neurochem. 1978, 30, 1377−
1382.
(40) Haefliger, W.; Revesz, L.; Maurer, R.; Romer, D.; Buscher, H.-H.
́
́
̈
̈
Synthesis and structure-activity studies on analogues and derivatives of
muscimol and THIP. Chim. Ther 1984, 19, 149−156.
(41) 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.
(25) Molander, G. A.; Hiebel, M.-A. Synthesis of Amidomethyltri-
fluoroborates and Their Use in Cross-Coupling Reactions. Org. Lett.
2011, 12, 4876−4879.
(42) Hansen, S. B.; Sulzenbacher, G.; Huxford, T.; Marchot, P.;
Taylor, P.; Bourne, Y. Structures of Aplysia AChBP complexes with
nicotinic agonists and antagonists reveal distinctive binding interfaces
and conformations. EMBO J. 2005, 24, 3635−3646.
(43) Graham, A. R. J.; Mary, C.; Jane, R. H.; Kenneth, N. M. GABAC
Receptors as Drug Targets. Curr. Drug Targets: CNS Neurol. Disord.
2003, 2, 260−268.
(44) Lummis, S. C. R.; L. Beene, D.; Harrison, N. J.; Lester, H. A.;
Dougherty, D. A. A Cation-π Binding Interaction with a Tyrosine in
the Binding Site of the GABAC Receptor. Chem. Biol. 2005, 12, 993−
997.
(45) Padgett, C. L.; Hanek, A. P.; Lester, H. A.; Dougherty, D. A.;
Lummis, S. C. R. Unnatural Amino Acid Mutagenesis of the GABAA
Receptor Binding Site Residues Reveals a Novel Cation−π Interaction
between GABA and β2Tyr97. J. Neurosci. 2007, 27, 886−892.
(46) 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 Character-
ization of a Series of Novel γ-Aminobutyric AcidC Receptor Agonists. J.
Med. Chem. 2007, 50, 4147−4161.
(47) Pedersen, D. S.; Rosenbohm, C. Dry Column Vacuum
Chromatography. Synthesis 2001, 2431−2434.
(48) Ransom, R. W.; Stec, N. L. Cooperative Modulation of
[3H]MK-801 Binding to the N-Methyl-d-Aspartate Receptor-Ion
Channel Complex by l-Glutamate, Glycine, and Polyamines. J.
Neurochem. 1988, 51, 830−836.
(26) Molander, G. A.; Shin, I. Suzuki-Miyaura Cross-Coupling
Reactions of Potassium Boc-Protected Aminomethyltrifluoroborate
with Aryl and Hetaryl Mesylates. Org. Lett. 2012, 14, 3138−3141.
(27) Fukui, H.; Inoguchi, K.; Nakano, J. Synthesis of the Bicyclic
Secondary Amines via Dimethylaminomethylene Ketones from 3-
Pyrrolidone and 4-Piperidone. Heterocycles 2002, 56, 257−264.
(28) Begtrup, M.; Vedsø, P. Preparation of N-hydroxyazoles by
oxidation of azoles. J. Chem. Soc., Perkin Trans. 1 1995, 243−247.
(29) CCDC 901038 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge via www.
ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.
cam.ac.uk, or by contacting The Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223
336033.
(30) Krogsgaard-Larsen, P.; Larsen, A. L. N.; Thyssen, K. GABA
Receptor Agonists. Synthesis of Muscimol Analogues Including (R)-
and (S)-5-(1-Aminoethyl)-3-isoxazolol and (RS)-5-Aminomethyl-2-
isoxazolin-3-ol. Acta Chem. Scand. 1978, B32, 469−477.
(31) Frey, M.; Jager, V. Synthesis of N-Substituted Muscimol
̈
Derivatives Including N-Glycylmuscimol. Synthesis 1985, 1100, 1104.
(32) Vogensen, S. B.; Clausen, R. P.; Greenwood, J. R.; Johansen, T.
N.; Pickering, D. S.; Nielsen, B.; Ebert, B.; Krogsgaard-Larsen, P.
Convergent Synthesis and Pharmacology of Substituted Tetrazolyl-2-
amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid Analogues.
J. Med. Chem. 2005, 48, 3438−3442.
(49) Jensen, A. A.; Bergmann, M. L.; Sander, T.; Balle, T. Ginkgolide
X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique
Selectivity Profile at Glycine Receptors. J. Biol. Chem. 2010, 285,
10141−10153.
(50) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J.
Improved patch-clamp techniques for high-resolution current record-
(33) Wenckens, M.; Jakobsen, P.; Vedsø, P.; Huusfeldt, P. O.; Gissel,
B.; Barfoed, M.; Lundin Brockdorff, B.; Lykkesfeldt, A. E.; Begtrup, M.
N-Alkoxypyrazoles as biomimetics for the alkoxyphenyl group in
tamoxifen. Bioorg. Med. Chem. 2003, 11, 1883−1899.
(34) Jørgensen, C. G.; Brauner-Osborne, H.; Nielsen, B.; Kehler, J.;
̈
Clausen, R. P.; Krogsgaard-Larsen, P.; Madsen, U. Novel 5-substituted
1-pyrazolol analogues of ibotenic acid: Synthesis and pharmacology at
glutamate receptors. Bioorg. Med. Chem. 2007, 15, 3524−3538.
(35) Stensbøl, T. B.; Uhlmann, P.; Morel, S.; Eriksen, B. L.; Felding,
ing from cells and cell-free membrane patches. Pflug. Arch. Eur. J. Phys.
̈
1981, 391, 85−100.
(51) Hilf, R. J. C.; Dutzler, R. X-ray structure of a prokaryotic
pentameric ligand-gated ion channel. Nature 2008, 452, 375−379.
(52) Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.;
McGettigan, P. A.; McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm,
A.; Lopez, R.; Thompson, J. D.; Gibson, T. J.; Higgins, D. G. Clustal
W and Clustal X version 2.0. Bioinformatics 2007, 23, 2947−2948.
(53) Sali, A.; Blundell, T. L. Comparative Protein Modelling by
Satisfaction of Spatial Restraints. J. Mol. Biol. 1993, 234, 779−815.
J.; Kromann, H.; Hermit, M. B.; Greenwood, J. R.; Brauner-Osborne,
̈
H.; Madsen, U.; Junager, F.; Krogsgaard-Larsen, P.; Begtrup, M.;
Vedsø, P. Novel 1-Hydroxyazole Bioisosteres of Glutamic Acid.
Synthesis, Protolytic Properties, and Pharmacology. J. Med. Chem.
2001, 45, 19−31.
(36) Clausen, R. P.; Christensen, C.; Hansen, K. B.; Greenwood, J.
R.; Jørgensen, L.; Micale, N.; Madsen, J. C.; Nielsen, B.; Egebjerg, J.;
M
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(54) MacroModel, version 9.9, Schrodinger, LLC, New York, NY,
̈
2011.
(55) Jaguar, version 7.8, Schrodinger, LLC, New York, NY, 2011.
̈
(56) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T.
Extra Precision Glide: Docking and Scoring Incorporating a Model of
Hydrophobic Enclosure for Protein-Ligand Complexes. J. Med. Chem.
2006, 49, 6177−6196.
(57) Lolli, M. L.; Hansen, S. L.; Rolando, B.; Nielsen, B.;
Wellendorph, P.; Madsen, K.; Larsen, O. M.; Kristiansen, U.;
Fruttero, R.; Gasco, A.; Johansen, T. N. Hydroxy-1,2,5-oxadiazolyl
Moiety as Bioisoster of the Carboxy Function. Synthesis, Ionization
Constants, and Pharmacological Characterization of γ-Aminobutyric
Acid (GABA) Related Compounds. J. Med. Chem. 2006, 49, 4442−
4446.
(58) Ebert, B.; Frølund, B.; Diemer, N. H.; Krogsgaard-Larsen, P.
Equilibrium binding characteristics of [3H]thiomuscimol. Neurochem.
Int. 1999, 34, 427−434.
(59) Krehan, D.; Frølund, B.; Ebert, B.; Nielsen, B.; Krogsgaard-
Larsen, P.; Johnston, G. A. R.; Chebib, M. Aza-THIP and related
analogues of THIP as GABAC antagonists. Bioorg. Med. Chem. 2003,
11, 4891−4896.
N
dx.doi.org/10.1021/jm301473k | J. Med. Chem. XXXX, XXX, XXX−XXX