Exploring the orthosteric binding site of the γ-aminobutyric acid type a receptor using 4-(Piperidin-4-yl)-1-hydroxypyrazoles 3- or 5-imidazolyl substituted: Design, synthesis, and pharmacological evaluation

Article abstract of DOI:10.1021/jm4006466

A series of 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP) 3- or 5-imidazolyl substituted analogues have been designed, synthesized, and characterized pharmacologically. All analogues showed binding affinities in the low micro- to low nanomolar range at nat

Full text of DOI:10.1021/jm4006466

Brief Article
pubs.acs.org/jmc
Exploring the Orthosteric Binding Site of the γ‑Aminobutyric Acid
Type A Receptor Using 4‑(Piperidin-4-yl)-1-hydroxypyrazoles 3- or
5‑Imidazolyl Substituted: Design, Synthesis, and Pharmacological
Evaluation
Jacob Krall,† Claus H. Jensen,† Troels E. Sørensen,†,§ Birgitte Nielsen,† Anders A. Jensen,†
Tommy Sander,‡ Thomas Balle,§ and Bente Frølund*,†
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark
‡Novo Nordisk A/S, Novo Alle,
́
DK-2880 Bagsværd, Denmark
§Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales, Australia
S
* Supporting Information
ABSTRACT: A series of 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP) 3- or 5-imidazolyl substituted analogues have been
designed, synthesized, and characterized pharmacologically. All analogues showed binding affinities in the low micro- to low
nanomolar range at native rat GABAA receptors and were found to be antagonists at the human α1β2γ2s receptor. The structure−
activity relationship of the compound series demonstrates distinct differences in size and architecture of previously discovered
cavities in the vicinity of the 4-PHP scaffold in the orthosteric binding site.
serotonin type 3 (5-HT3) receptors, and a zinc-activated ion-
channel.2 The GABAARs are transmembrane proteins
assembled by five subunits to form a chloride-selective
pentameric channel. To date, 19 different human GABAAR
subunits have been identified (α1−6, β1−3,γ1−3, δ, ε, π, θ, ρ1−3).
So far, the existence of 26 native GABAARs have been
proposed,3,4 major combinations assembled being the α1β2γ2,
α3β3γ2, and α2β3γ2 subtypes.5 GABA binds at the interface of an
α and a β subunit in the receptor complex.6
INTRODUCTION
γ-Aminobutyric acid (GABA, Figure 1) is one of the major
neurotransmitters in the mammalian central nervous system
■
The three-dimensional structure of the GABAAR is yet to be
elucidated. Consequently, structure−activity relationship
(SAR) studies and pharmacophore models have played a
major role and are still essential in probing the structural basis
for receptor−ligand interactions and the molecular determi-
nants of ligand affinity, potency, efficacy, and subtype selectivity
toward the GABAARs. However, in recent years, valuable
insight into the architecture of LGICs has been obtained.
Several X-ray structures represent excellent templates for
homology modeling of LGICs, including the GABAARs.
These templates comprises acetylcholine-binding proteins
from various snails,7−9 distantly related bacterial ion-chan-
nels,10,11 and a recently published glutamate gated ion-
channel.12
On the basis of multiple templates, we have previously
presented homology models of the extracellular domain of the
GABAAR reflecting both the agonist- and antagonist-bound
receptors.13,14 Extensive SAR data, made available using the low
efficacy partial agonists 5-(piperidin-4-yl)-3-isoxazolol (4-
PIOL)15−17 and 4-(piperidin-4-yl)-1-hydroxypyrazole (4-
Figure 1. Structures of GABA, 4-PIOL, 4-PHP, 1, and general
structures of new 4-PHP analogues (2a−e and 3a−d).
(CNS) and is responsible for the majority of the overall
neuronal inhibition. The physiological effects of GABA are
mediated through the ionotropic GABAA and the metabotropic
GABAB receptors. The ionotropic GABAA receptors (GA-
BAARs) are widely distributed throughout the CNS where they
play an essential role in numerous physiological processes.
Furthermore, the GABAARs have been linked to a variety of
neurological and psychiatric disorders, e.g., Alzheimer’s,
Parkinson’s, and Huntington’s disease, epilepsy, anxiety,
depression, schizophrenia, and cognitive disorders.1
The GABAARs belong to the Cys-loop superfamily of
pentameric ligand-gated ion-channels (LGICs) including the
nicotinic acetylcholine receptors, the glycine receptors, the
Received: May 1, 2013
© XXXX American Chemical Society
A
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Brief Article
Figure 2. 4-PHP analogues 1 (A, brown), 2d (B, white), and 3c (C, green), respectively, docked into a homology model of the α1β2γ2 GABAAR.
Contours of favorable van der Waals interactions calculated with the program GRID are depicted with gray mesh (C3 probe, isolevel = −2 kcal/
mol). Contours of favorable hydrogen bond acceptor sites are depicted with red surfaces (COO− probe, isolevel = −10 kcal/mol). Contours of
favorable hydrogen bond donor sites are depicted with blue surfaces (N2+ probe, isolevel = −10 kcal/mol).
PHP) as scaffolds for a series of potent antagonists, including
compound 1 (Figure 1),18 has been part of the development of
the above-mentioned models. On the basis of these studies,
cavities near the 3- and the 5-position of the 4-PHP scaffold in
the orthosteric binding site were identified.14 The spaciousness
of the cavities along with the ambiguity of binding modes led us
to further challenge their boundaries with a series of imidazole
substituted 4-PHP analogues (2a−e and 3a−d, Figure 1). Here
we report the design, synthesis, and pharmacological character-
ization of the compounds at native and recombinant GABAARs
Figure 3. Overlay of 2d (white) with both possible poses of 1
(brown).
and discuss the findings in the context of a homology model.
RESULTS AND DISCUSSION
■
Computational Modeling. Docking of compound 1 into
the homology model14 revealed two possible orientations with
the 3-biphenyl substituent, either pointing “left” or “right”, as
illustrated in Figure 2A. In both orientations, GLIDE XP
docking scores19−22 are highly favorable, −10.6 (“left”) and
−9.8 (“right”) kcal/mol, and in both orientations the core 4-
PHP scaffold is placed with charged moieties within areas
predicted by GRID23,24 calculations to be favorable for a
positively charged amine and a negatively charged carboxylic
acid, respectively. GRID calculations of the binding site using a
methyl probe (−2 kcal/mol), revealed a cavity below the 4-
PHP scaffold toward the membrane region, denoted the “lower
cavity”. The more favorable docking score along with a better
fit of the 3-biphenyl moiety within the favorable van der Waals
contact area of the lower cavity suggests the “left” orientation to
be the most likely binding mode in agreement with previous
results.14 Nonetheless, the alternative binding mode suggests
ample space in the receptor to accommodate substituents even
larger than the 3-biphenyl substituent in 1. This led to the
design of the imidazole analogue 2d, which, as illustrated in
Figure 3, addresses both predicted orientations simultaneously
and when docked into the model (Figure 2B) the 1-benzyl-2-
phenyl-4-imidazolyl substituent of 2d overlaps almost perfectly
with the unified binding modes for 1.
near perfect fit and furthermore predicts a hydrogen bond from
the imidazole moiety to β2-Gly158 (Figure 2C). However,
unlike the lower cavity, the 4-PHP scaffolds bigger substituents
are not predicted to contribute to increased affinity.
A complete list of docking scores is listed in Supporting
Information (SI).
Chemistry. Compounds 2a−e and 3a−d were all
synthesized using the imidazole building blocks 4a−d (please
refer to SI for synthetic details) and core scaffolds 10 and 11
(Scheme 1), which were prepared as described previously.18
i
Metal−halogen exchange of 4a,b,d using PrMgCl or direct
deprotonation of the 4(5)-position of compound 4c followed
by quenching with tributyltin chloride formed the Stille reagent
intermediates (not shown). These were used without
purification in a Stille cross-coupling reaction using 10 or 11,
tetrakis(triphenylphosphine)Pd(0), copper(I) iodide, and
cesium fluoride in DMF to form 12a−d and 13a−d. Acidic
deprotection of 12a−d and 13a−d resulted in 2a−d and 3a−d,
while compound 2e was synthesized from 12b by catalytic
hydrogenation using 10% palladium on carbon followed by
acidic deprotection.
Pharmacology. The synthesized compounds were charac-
terized pharmacologically in binding studies using rat brain
membrane preparations, where the binding affinities of 2a−e
and 3a−d at native rat GABAARs were measured by
displacement of [3H]muscimol. Functional characterization of
2a−e and 3a−d at the human ρ1 (data not shown) and α1β2γ2S
GABAARs transiently expressed in tsA201 cells was performed
using the FLIPR Membrane Potential (FMP) Blue assay as
The GRID calculations using the methyl probe also revealed
a cavity above the 4-PHP scaffold away from the membrane
region denoted the “upper cavity”. The upper cavity is
narrower, and docking studies suggests that it is capable of
accommodating the 5-substituted 4-PHP analogue 3c with a
B
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a
Scheme 1
Table 1. Pharmacological Data for Gabazine, 4-PHP, 1, 2a−
e, and 3a−d: GABAAR Binding Affinities at Rat Synaptic
Membranes and Functional Characterization at the Human
α1β2γ2S GABAAR Transiently Expressed in tsA201 Cells in
the FMP Blue Assay
[3H]muscimol
binding Ki (μM)
[pKi SEM]
α1β2γ2S tsA201 cell line
a
b
IC50 (μM) [pIC50
R1
R2
SEM]
c
c
Gabazine
0.074
0.24
d
d
4-PHP
1
10
>500
d
d
0.030
0.21
2a
H
H
5.1 [5.31 0.08]
0.74 [6.14 0.02]
2.5 [5.61 0.05]
0.28 [6.57 0.05]
0.75 [6.13 0.03]
7.5 [5.14 0.08]
0.83 [6.09 0.04]
0.014 [7.86 0.04]
0.23 [6.64 0.04]
∼30 [∼4.5]
2b
2c
Bn
H
Cl
Ph
Ph
H
nd
9.7 [5.01 0.06]
0.21 [6.67 0.05]
nd
2d
2e
Bn
Bn
H
3a
H
nd
3b
3c
Bn
H
Cl
Ph
Ph
nd
0.69 [6.16 0.04]
0.78 [6.10 0.08]
3d
Bn
a
a
i
n
IC50 values were calculated from inhibition curves and converted to
Ki values. Data is given as the mean [mean pKi
independent experiments. For the characterization of the antagonists,
Reagents and conditions: (i) 4a,b,d, PrMgCl, THF, rt, or 4c, BuLi,
SEM] of 3−4
THF, −78 °C; (ii) Bu3SnCl, THF, rt, or −78 °C to rt; (iii) 10 or 11,
Pd(PPh3)4, CuI, CsF, DMF, 45−110 °C; (iv) 48% aq. HBr, reflux; (v)
10% Pd/C, CH2Cl2, rt.
b
assay concentrations of GABA of 8 μM (EC85−EC95) were used.
c
d
From ref 15. From ref 18. nd: not determined.
described previously25 and in SI. The pharmacological
characteristics of 4-PHP, 1, 2a−e, and 3a−d in the radioligand
binding and FMP assays are listed in Table 1.
the binding affinity compared to 4-PHP substantially.
Introduction of the larger 1-benzyl-2-chloro-4-imidazolyl
substituent (2b and 3b, respectively) and 1-benzyl-4-imidazolyl
(2e) led to one order of magnitude enhancement in binding
affinity compared to compound 2a and 3a, respectively. The
identical binding affinity of 2b and 2e indicates that the
chlorine in the 2-position of the 1-benzyl substituted imidazole
has no influence on the binding affinity. Interestingly,
introduction of a 2-phenyl-4-imidazolyl in 2a (2c) did not
affect the binding affinity compared to 4-PHP and 2a, whereas
a similar structural change in 3a (3c) led to a 180-fold
improvement in binding affinity compared to 3a. This marked
difference in binding affinity suggests that the introduced
substituents in the 3- and 5-position of the 4-PHP scaffold
project into different areas in the binding site. Docking studies
(Figure 2C) suggest a possible interaction partner for the
imidazole N−H hydrogen in 3c, namely a hydrogen bond to
β2-Gly158. Furthermore, the more narrow upper cavity
suggests a better complementarity of the substituent to the
surrounding protein in the cavity compared to 2c (please refer
to SI for docking results of 2c).
Combining the 1-benzyl and 2-phenyl imidazolyl substituents
in compounds 2d and 3d leads to equivalent binding affinities
for the two series of compounds. However, the introduction of
the 1-benzyl group in 3d led to a 16-fold loss in binding affinity
compared to 3c, whereas a similar introduction of a benzyl
group in 2d resulted in a 9-fold increase in binding affinity
compared to 2c. Again, the opposing effect in binding affinity
for the two series of compounds, illustrated by 2d and 3d,
supports our previous findings that the cavity above the 4-PHP
The inhibitory potencies exhibited by the compounds in the
FMP Blue assay are given as IC50 instead of Ki because of the
mixed competitive/noncompetitive antagonistic profile we
observed in the assay in a previous study.26 The GABA
concentrations used for the characterization of the antagonists
in the assay were 2-fold higher than its EC50 at the α1β2γ2s
receptor. Thus, if the compounds were indeed completely
competitive antagonists, as we expect them to be, the respective
functional Ki for the compounds would be approximately 3
times lower than their IC50.
Compounds 2a,c,d, and 3c,d were found to be antagonists
with moderate to high potencies (0.21−30 μM) with 2d
showing antagonistic potency comparable to the standard
GABAAR antagonist Gabazine.15 The functional data are, in
general, in good agreement with the binding affinity data. In
contrast to the α1β2γ2s data, none of the compounds displayed
agonist or antagonist effects at the human ρ1 GABAAR when
tested at concentrations up to 300 μM.
Receptor Binding and Structure−Affinity Relation-
ships. The 4-PHP analogues 2a−e and 3a−d all show binding
affinities in the low micro- to low nanomolar range. The fact
that 2a−e and 3a−d bind to the orthosteric binding site of the
GABAARs supports the presence of cavities capable of
accommodating relatively large substituents, as previously
suggested, in the vicinity of the 4-PHP scaffold in the
orthosteric binding site.
The introduction of an unsubstituted imidazole in the 3- or
5-position of 4-PHP (2a and 3a, respectively) does not affect
C
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Journal of Medicinal Chemistry
Brief Article
scaffold is more narrow than the cavity below, which has been
shown to be able to accommodate more bulky substituents.14
The binding data and molecular modeling of 2d (Figure 2B)
indicate that the binding pocket is able to accommodate the
disubstituted 1-benzyl-2-phenyl-4-imidazolyl introduced in the
3-position of 4-PHP, an area corresponding to the combined
space occupied by the two possible orientations of the 3-
biphenyl group of 1. The similar high binding affinity shown for
the corresponding analogue 3d does not seem to fit with the
homology model, where the limited space in the upper cavity of
the binding site (Figure 2C) would hinder binding of the
disubstituted 1-benzyl-2-phenyl-4-imidazolyl at the 5-position
of 4-PHP. However, the SAR could be explained by the ligand
turning 180° around the bond connecting the piperidine and 1-
hydroxypyrazole moieties, thereby placing the bulky substituent
in the more spacious lower cavity, which is also what is
observed in docking studies (please refer to SI). This would not
necessarily interfere with the salt-bridge formed between the 1-
hydroxypyrazole moiety and α1-Arg66 because the negative
charge is distributed between the oxygen and the pyrazole N2.
AUTHOR INFORMATION
Corresponding Author
*Phone: +45 35336495. Fax: +45 35336040. E-mail: bfr@sund.
ku.dk.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
J.K. and T.E.S. were supported by The Danish Medical
Research Council and A.A.J. was supported by the Novo
Nordisk and Carlsberg Foundations.
ABBREVIATIONS USED
■
DCVC, dry column vacuum chromatography; FLIPR,
fluorescent imaging plate reader; FMP, FLIPR membrane
potential; GABA, γ-aminobutyric acid; GABAARs, γ-amino-
butyric acid type A receptors; 5-HT, 5-hydroxytryptamine; 4-
PIOL, 5-(piperidin-4-yl)-3-isoxazolol; 4-PHP, 4-(piperidin-4-
yl)-1-hydroxypyrazole; SAR, structure−activity relationship
CONCLUSION
REFERENCES
■
■
On the basis of a previously reported homology model of the
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substituted analogues (2a−e and 3a−d) has been designed,
synthesized, and characterized pharmacologically at GABAARs.
All analogues showed low micro- to low nanomolar binding
affinities. Ligand−receptor docking suggests a common binding
mode for the core 4-PHP scaffold with the 3- (2a−e) and 5-
substituents (3a−c), addressing two different cavities in the
vicinity of the 4-PHP scaffold. The SAR data from the present
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substituent in the larger lower cavity. Altogether, these new
results offer a more detailed insight into the architecture of the
orthosteric binding site in the antagonist binding mode of
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EXPERIMENTAL SECTION
■
Chemistry. The syntheses of selected compounds are described
below as representative. The purity of all tested compounds was
analyzed using combustion analysis or HPLC. Elementary analyses
calculated are within 0.4% of found values, and HPLC purity is ≥95%
unless otherwise stated.
General Procedure for Stille Cross-Coupling (12a−d, 13a−
d). The imidazole 4a,b,d (1 equiv) in CH2Cl2 or THF was added to
iPrMgCl (1.2−2.3 equiv), while 4c in THF at −78 °C was added to
nBuLi (1.1 equiv). The solutions were stirred for 30 min before
addition of Bu3SnCl (1.1−1.4 equiv). The resulting mixtures were
stirred overnight at rt before removal of solvent. The crude Stille
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis details, 1H NMR and 13C NMR of synthesized
compounds, elementary analyses of all new target compounds,
pharmacological methods, and molecular modeling methods.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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