Positional isomers of bispyridine benzene derivatives induce efficacy changes on mGlu5negative allosteric modulation

Article abstract of DOI:10.1016/j.ejmech.2017.01.013

Modulation of metabotropic glutamate receptor 5 (mGlu₅) with partial allosteric antagonists has received increased interest due to their favourable in?vivo activity profiles compared to the unfavourable side-effects of full inverse agonists. He

Full text of DOI:10.1016/j.ejmech.2017.01.013

Accepted Manuscript
Positional isomers of bispyridine benzene derivatives induce efficacy changes on
mGlu negative allosteric modulation
5
Xavier Gómez-Santacana, James A.R. Dalton, Xavier Rovira, Jean Philippe Pin, Cyril
Goudet, Pau Gorostiza, Jesús Giraldo, Amadeu Llebaria
PII:
S0223-5234(17)30021-1
10.1016/j.ejmech.2017.01.013
EJMECH 9164
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 November 2016
Revised Date: 6 January 2017
Accepted Date: 9 January 2017
Please cite this article as: X. Gómez-Santacana, J.A.R. Dalton, X. Rovira, J.P. Pin, C. Goudet, P.
Gorostiza, J. Giraldo, A. Llebaria, Positional isomers of bispyridine benzene derivatives induce efficacy
changes on mGlu negative allosteric modulation, European Journal of Medicinal Chemistry (2017), doi:
5
10.1016/j.ejmech.2017.01.013.
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ACCEPTED MANUSCRIPT
Positional isomers of bispyridine benzene derivatives
induce efficacy changes on mGlu5 negative allosteric
modulation
Xavier Gómez-Santacanaa,b,c,§, James A.R. Daltonb,d, Xavier Rovirae,f, Jean Philippe
Pine,f, Cyril Goudete,f, Pau Gorostizac,g,h, Jesús Giraldob,d and Amadeu Llebariaa*
(a) MCS, Laboratory of Medicinal Chemistry & Synthesis, Institute for Advanced Chemistry of Catalonia
(IQAC−CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain. (b) Laboratory of Molecular
Neuropharmacology and Bioinformatics, Institut de Neurociències and Unitat de Bioestadística, Universitat
Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain. (c) Nanoprobes and Nanoswitches, Institute for
Bioengineering of Catalonia (IBEC), Baldiri Reixac, 08028, Barcelona, Spain. (d) Network Biomedical
Research Center on Mental Health (CIBERSAM), Spain (e) Department of Neurosciences, Institute of
Functional Genomics, Université de Montpellier, Unité Mixte de Recherche 5302 CNRS, Montpellier,
France; (f) Unité de recherche U1191, INSERM, Montpellier, France (g) Network Biomedical Research
Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. (h) Catalan Institution for
Research and Advanced Studies (ICREA), Barcelona, Spain. (§) Present address: Division of Medicinal
Chemistry, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), VU University
Amsterdam, The Netherlands. * Corresponding author: amadeu.llebaria@iqac.csic.es
Keywords: mGlu5, isomers, partial efficacy, NAM, antagonist, inverse agonist
Graphical Abstract
Highlights
•
Efficacy of bispyridine benzene mGlu5 negative allosteric modulators
depends on molecule geometry.
•
•
1,4-disubstituted linear compounds have mGlu5 partial antagonist activity.
1,3-disubstituted angular compounds have mGlu5 full inverse agonist
activity.
•
1,4- and 1,3-isomers bind preferentially to different receptor
conformations according to computational docking.
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Abstract
Modulation of metabotropic glutamate receptor 5 (mGlu5) with partial allosteric
antagonists has received increased interest due to their favourable in vivo activity
profiles compared to the unfavourable side-effects of full inverse agonists. Here we
report on a series of bispyridine benzene derivatives with a functional molecular switch
affecting antagonistic efficacy, shifting from inverse agonism to partial antagonism with
only a single change in the substitution pattern of the benzene ring. These efficacy
changes are explained through computational docking, revealing two different receptor
conformations of different energetic stability and different positional isomer binding
preferences.
Introduction
Metabotropic glutamate receptors (mGluRs) are class C G Protein-Coupled Receptors
(GPCRs) activated by glutamate, the major excitatory neurotransmitter in the central
nervous system (CNS). They are widely distributed throughout the CNS, regulating
several neuronal and glial functions [1], and are considered potential targets for the
treatment of several neurological disorders [2]. The mGlu receptor family comprises
eight subtypes classified in three groups based upon sequence homology and
molecular function [3]. In particular, subtype 5 (mGlu5) belongs to group I mGluRs and
is mainly postsynaptic. Current research indicates that inactivating mGlu5 may be an
effective treatment for neuropathic and inflammatory pain [4, 5] and may exert a
neuroprotective effect on dopaminergic neurons, particularly in animal models of
Parkinson's disease and L-DOPA-induced dyskinesia [6-9]. Furthermore, preclinical
and clinical trials have revealed a possible treatment for other diseases such as
Fragile-X syndrome [10, 11], depression [12] and gastro-oesophageal reflux [13].
The classical approach in controlling the pharmacological activity of mGlu5 involves the
use of orthosteric agonists and antagonists, which compete with glutamate, the
endogenous agonist. These orthosteric ligands, e.g. Quisqualate (1) [14] or (S)-MCPG
(2) [15], bind to the mGlu5 receptor extracellular domain but achieving suitable
selectivity for one subtype over another is difficult because of the highly conserved
orthosteric binding-site exhibited amongst mGlu-family members [16].
Allosteric modulators may solve this selectivity issue, since they bind to a less
conserved allosteric site in the transmembrane domain, which shows more variation
across subtypes [17]. Moreover, allosteric ligands, with lower polarity due to the higher
hydrophobicity of the allosteric site, generally allow for better absorption [18] and more
efficient crossing of the blood-brain barrier (BBB) [19]. Examples of such ligands
include positive allosteric modulators (PAMs) CDPPB (3) [20] and VU0409551/JNJ-
46778212 (4) [21]; and negative allosteric modulators (NAMs) fenobam (5) [22], MPEP
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(6) [23], raseglurant (7) [24] and mavoglurant (8) [25], with the last having been
recently co-crystallized in the allosteric pocket of the mGlu5 transmembrane domain
[26].
Figure 1. Examples of mGlu5 ligands. Compound 1 is an orthosteric agonist [14], compound 2 is an
orthosteric antagonist [15], compounds 3 and 4 are positive allosteric modulators (PAMs) and compounds
5-11 are negative allosteric modulators (NAMs) [22-25, 27-30]. Compounds 6, 7, 11, 12a and 12b are 2-
ethynylpyridine derivatives and compounds 9 and 10 are phenylazopyridine derivatives [20, 21].
Interestingly, many NAMs of mGlu5, which usually display an inverse agonist activity,
include a terminal pyridine or thiazole with an ethynyl unit at position 2 or 4,
respectively [24]. According to recent publications [26, 31], this ethynyl moiety
traverses a narrow channel in the allosteric site of the transmembrane domain, which
might explain the predominance of the diaryl alkyne scaffold among mGlu5 allosteric
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modulators. However, the presence of a triple bond is not imperative for potent mGlu5
PAMs and NAMs, since azo-bridged compounds such as SIB-1757 (9) are also mGlu5
NAMs with potencies in the nanomolar range [27].
Recently we reported that alloswitch-1 (10), a photoswitchable mGlu5 NAM with
potency in the nanomolar range (IC50 = 25 nM) and containing a phenylazopyridine,
displays reversible photoisomerization from trans- to cis-isomer [28], which can be
translated to reversible activation-deactivation of mGlu5 receptors in transfected cells,
native cell cultures and in vivo [32].
In fact, trans-alloswitch-1 (10) most likely binds to the mGlu5 receptor in a very similar
fashion to mavoglurant (8) or MPEP (6), as we recently reported [33], whereas the
binding of the cis-isomer is unstable or absent, in agreement with its lower observed
activity [28]. In our computational simulations, the position of the azo-bond of the trans
isomer occupies exactly the same narrow channel described by Doré et al. [26],
sharing a common binding mode. In addition, alloswitch-1 (10) possesses a high
structural resemblance to XGS-RC-009 (11), a recently reported potent (IC50 = 24 nM)
mGlu5 NAM [29], and only differs in the spacer between the western aromatic rings: an
azo-bond for alloswitch-1 (10) and an ethynyl-bond for XGS-RC-009 (11). This further
indicates the suitability of compounds containing either spacer type for achieving
proper NAM activity in the mGlu5 receptor.
In the present study, we confirm this correlation between azo-bonds and ethynyl-bonds
for mGlu5 NAMs from the functional and binding points of view. For that reason, we
selected 1,3-(2-bisPEB) (12a) and 1,4-(2-bisPEB) (12b) [30], having considered their
nanomolar NAM potency and their geometrically defined non-complex structure (Figure
1). Thus, we synthesised and characterised the corresponding azo-derivatives with the
aim of obtaining new photoswitchable compounds for precise spatiotemporal control of
the mGlu5 receptor.
Results and Discussion
Chemistry
Previously reported compounds 12a and 12b [30] were prepared from 2-
ethynylpyridine (13) and the corresponding diiodobenzene 14a-b using a palladium
catalysed Sonogashira coupling in moderate to good yields (Scheme 1). A two-step
synthetic route yielded the azocompounds 19a and 19b, starting with a Sonogashira
coupling of 2-chloropyridine 16 and the corresponding ethynyl aniline 15a-b to give the
intermediates 17a and 17b in moderate yields. The following Mills reaction of 17a and
17b with 2-nitrosopyridine 18 in acidic conditions gave compounds 19a and 19b in
good yields. Finally, compounds 21a and 21b were also obtained by a single-step
synthesis from the Mills reaction between 2-nitrosopyridine 18 and the corresponding
diaminobenze 20a-b in moderate to good yields.
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Scheme 1. Synthetic procedures for 12, 17 and 19. a) PdCl2(PPh3)2, CuI, Et3N, DMF, 50ºC, 12a (87%), 12b (47%), 15a
(30%), 15b (23%); b) AcOH, DCM, r.t., 17a (76%), 17b (78%), 19a (5%), 19b (98%)
All the compounds were screened with high performance liquid chromatography
(HPLC) coupled to a photodiode array detector (PDA) and a mass spectrometer (MS)
to measure the UV-Vis absorption and mass spectra of the compounds (supplementary
material). In addition, we were able to chromatographically separate and identify the cis
and trans isomers of compounds 19a, 19b, 21a and 21b, since cis-isomers have a
characteristic n-π* band at 420-440 nm and a displacement of the characteristic π-π*
at shorter wavelengths (supplementary material).
Next, we tested the photoisomerization properties of azocompounds 19a-b and 21a-b
with UV-Vis spectroscopy. In the absence of illumination we detected only the trans-
isomer in all cases. To generate the cis-isomers we exposed the azocompound
solution in acetonitrile to violet 380-nm-light since this wavelength is optimally located
between the π-π* transition of the trans isomers and the minimum between π-π* and
n-π* transitions of the cis isomers. Finally, 430-nm-light was used to recover the trans
isomers since it is located in the centre of the n-π* transition of the cis-isomers. We
were able to detect a nearly complete photoisomerisation of compound 19b, since the
profile of the UV-Vis spectrum obtained after violet illumination was the same as that
obtained for the cis isomer peak detected by HPLC-PDA-MS (supplementary material).
However, for compounds 19a and 21a-b we did not detect satisfactory
photoisomerisation after illumination with violet light, in a similar manner to other
previously reported bisazocompounds [34] (supplementary material). This indicates
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that not all azo-compounds display a suitable photoisomerisation response for
obtaining a high population of cis isomers under illumination, which is a necessary
condition for efficient on/off receptor switching.
Pharmacology
We tested the compounds using an inositol phosphate (IP) accumulation assay in
transiently transfected HEK293 cells with mGlu5. Dose-response curves were
generated to evaluate the antagonistic activity using the orthosteric agonist quisqualate
and fenobam (5) as an mGlu5 allosteric inverse agonist reference compound. Whilst
compounds 12a and 12b were only tested in dark conditions, the corresponding
incubations of compounds 19a, 19b, 21a and 21b were performed in parallel in both
dark conditions and under 380-nm-illumination, to evaluate the photoswitching effect of
these compounds on mGlu5 receptor activity.
Figure 2. Dose-response curves of compounds 12a,b, 19a,b, and 21a,b with fenobam (5) as control,
obtained from IP accumulation assays with a constant concentration of quisqualate (100 nM) in dark
conditions. The curves are normalised with respect to the response of fenobam, between 0% and 100%.
Every point represents the mean of a minimum of three independent experiments with corresponding SEM
as error bars.
The 1,3-disubstituted compounds 19a and 21a were found to be mGlu5 NAMs. In
comparison with fenobam (5), compound 19a displayed better potency and efficacy
whereas compound 21a displayed higher efficacy but 2-fold lower potency. Both 19a
and 21a showed similar efficacies to the structurally related compound 12a (1,3-(2-
BisPEB)), whose pharmacological properties were previously reported [30, 35]. The
replacement of a triple bond in 12a by an azo group in 19a resulted in a 2-fold loss of
potency in mGlu5, a result which was also observed but to a greater extent (a 13-fold
loss) when replacing both ethynyl spacers by N=N in 21a.
The 1,4 substituted compounds 12b, 19b and 21b also displayed NAM activity in
mGlu5, with lower potency than the corresponding 1,3-disubstituted compounds and,
interestingly, with a much lower efficacy, signifying a partial antagonistic efficacy, which
we have attributed to the different substitution pattern at the central benzene ring in the
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2-ethynylpyridines or the 2-azopyridines, respectively (Figure 2 and Table 1).
Interestingly, 1,3-disubstituted compounds display a similar efficacy, or even a bit
higher than fenobam (5), a classic allosteric inverse agonist [22], while 1,4-
disubstituted compounds only partially antagonized the effect of quisqualate in mGlu5.
Moreover, the potencies (IC50) of the 1,4-isomers were found to be within a similar
range as the 1,3-isomers.
IC50 ± SEM
(µM)
Efficacya ± SEM
(%)
PPSb
NTd
NTd
1.3
PDEc (%)
12a
0.43±0.08
1.21±0.40
0.96±0.21
3.09±0.27
4.92±0.95
6.13±0.16
2.30±0.25
114±5
42±4
NTd
NTd
9
12b
19a
115±4
35±4
19b
2.9
15
15
-1
21a
114±3
19±4
2.2
21b
1.2
Fenobam
100±0
1.0
0
Table 1. Potency (IC50) and efficacy of the series of compounds in mGlu5, including photoinduced potency
shift (PPS) of the IC50 and photoinduced difference of efficacy (PDE) between dark and illuminated
conditions. Data extracted from dose-response curves obtained with an IP accumulation and constant 100
nM concentration of quisqualate. (a) Efficacy measured from bottom of the dose-response curve,
normalised with respect to the response of fenobam between 0% and 100%. (b) PPS corresponds to the
ratio of the IC50 in dark conditions and under violet illumination, (c) PDE corresponds to the difference in
efficacy (%) between dark conditions and violet illumination, (d) NT: non-tested.
Figure 3. Dose-response curves of compounds 19a,b, and 21a,b obtained from IP accumulation assays
with a constant concentration of quisqualate (100 nM) in dark conditions (plain line) and under illumination
at 380 nm (dotted line). Every point represents the mean of a minimum of three independent replicates
with corresponding SEM as error bars.
As anticipated from the photoisomerisation experiments, the response of the
compounds tested under different illumination conditions was not efficient enough to
provide a useful light-dependent control of mGlu5 activity in cell assays. Upon
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illumination at 380 nm, we detected successful receptor photoswitching behaviour for
compound 19b, which was a partial antagonist as a trans-isomer but experiences a 3-
fold loss in potency and 15% loss in efficacy under violet illumination (mainly cis-
isomer) (Figure 3 and Table 1). The photoinduced potency shifts (PPS) [36] of the dose
response curves with the azo-compounds 19a and 21a,b were not sufficient to achieve
useful on/off receptor photoswitching. This result was probably due to the compounds
intrinsic molecular photochemical properties, which do not allow for a clear-cut
difference in the induction of different populations of the cis/trans isomers at the
wavelengths used. Another possible explanation for this lack of photoactivity could be
an insufficient difference in the affinity of the cis and trans isomers for the receptor,
which would result in low stability of the on/off states upon binding. This reflects the
necessary combination of photochemical and pharmacological properties of the azo
compounds for achieving an effective photoswitching of receptor activity in cell assays
or in vivo applications [36].
After examination of the results obtained, we were particularly interested in the reasons
behind the mGlu5 efficacy differences observed for the 1,4 and 1,3 positional isomers
(measured by the asymptotic minimum effects of the dose-response curves on the y-
axis), which occur without significantly altering their location along the x-axis (IC50
values). We hypothesized that this could be due to different binding modes in the
transmembrane domain of the mGlu5 receptor. The high homology between isomers
and similarity of the trans-azo and ethynyl bonds, both from the structural and
geometrical points of view, result in compounds with very rigid scaffolds where the
aromatic rings are either in angular or linear arrangements. Therefore, the binding
modes of each geometrical type may be significantly different, since these molecules
do not have the flexibility needed to rearrange their scaffold in order to bind in the
same pose in the same receptor pocket. To verify this hypothesis, we performed some
computational docking.
In silico binding model
To explore the possible binding modes of compounds 12a-b, 19a-b and 21a-b, each
compound was docked into the crystal structure of mGlu5 (PDB id 4OO9, minus its
mavoglurant co-crystallized ligand). For each respective docking, the top-ranked
protein-ligand complex was energy-minimized to optimize intermolecular interactions.
From the results shown in Figure 4, the 1,3-compounds are able to bind at the bottom
of the allosteric pocket because their structures are easily accommodated within the
distinctive angular binding-site, displaying a similar binding pose to the previously co-
crystallized mGlu5 NAMs: mavoglurant (in PDB id 4OO9 [26]) and HTL14242 (in PDB
id 5CGD [37]). In doing so, each 1,3-compound achieves a favourable docking score
(Table 2), making an H-bond with S809 on TM7 (after energy minimization), and in the
case of 19a and 21a, an additional H-bond with N747 on TM5. However, the 1,4-
compounds are unable to bind at the bottom of the allosteric pocket (at least in the
mGlu5 crystal structure of PDB id 4OO9), as they are unable to accommodate their
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structures within the angular binding-site. Instead, they bind higher in the pocket with a
pose similar to that observed with the co-crystallized ligand FITM in the crystal
structure of mGlu1 (PDB id: 4OR2 [38]). As a result of this higher binding pose, the 1,4-
compounds achieve much less favourable docking scores (Table 3) and are unable to
make an H-bond with S809 on TM7 or any other residue in the allosteric pocket.
Although this higher binding pose is apparently functional in terms of mGlu1 allosterism,
it is unlikely to be functional in mGlu5, as reflected by the low docking scores observed
here (indicating a poor fit) and by experimental data (including current mGlu5 crystal
structures) [26, 37], which suggests S809 interaction is critical for correct mGlu5 NAM
functionality [35].
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Figure 4. Six compounds docked into crystal structure of mGlu5 receptor (PDB id 4OO9) using
AutoDock4.2: (A) 12a, (B) 12b, (C) 19a, (D) 19b, (E) 21a, (F) 21b and, for comparison, co-crystallized: (G)
mavoglurant (PDB id 4OO9) and (H) HTL14242 (PDB id 5CGC). Ligands are shown in cyan, receptor in
beige, and protein-ligand H-bonds as black lines. The allosteric binding pocket is shown from the side with
selected residues labelled (TM5 front-left, TM7 front-right, TM3 central, TM4 background-left, TM2
background-right, TM6 invisible).
A
B
C
Figure 5. Linear-shaped 1,4-compounds: (A) 12b, (B) 19b, (C) 21b re-docked into an alternative receptor
conformation of mGlu5, derived from original crystal structure (PDB id 4OO9). Ligands are shown in cyan,
receptor in grey, and protein-ligand H-bonds as black lines. The allosteric binding pocket is shown from the
side with selected residues labelled (TM5 front-left, TM7 front-right, TM3 central, TM4 background-left,
TM2 background-right, TM6 invisible).
Docking Score in mGlu5
crystal 4OO9
Docking Score in alternative
mGlu5 conformation
(Predicted Ki, nM)
(Predicted Ki, nM)
12a
12b
17a
17b
19a
19b
437.77
9000.15
124.13
817.74
140.54
768.31
-
1000.27
-
405.60
-
373.97
Table 2. Docking scores for six compounds in two conformations of mGlu5 (original crystal structure and a
computationally-generated alternative). Three angular-shaped 1,3-compounds: 12a, 19a and 21a, and
three linear 1,4-compounds: 12b, 19b and 21b.
In recognition of the fact that GPCRs are flexible dynamic receptors, an alternative
conformation of mGlu5 was utilised to re-dock the 1,4-compounds in an attempt to
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identify better binding poses at the bottom of the allosteric pocket (more similar in
nature to current co-crystallized mGlu5 NAMs). This alternative receptor conformation
was derived from the original mGlu5 crystal structure (PDB id 4OO9) through
computational conformational sampling performed previously [33]. This process results
in a relatively subtle adjustment of side-chain packing between TM6 and TM7 but,
more significantly, an enlargement and alteration of the binding pocket from an angular
shape to a wider linear shape. Using this alternative receptor conformation, the 1,4-
compounds instead dock at the bottom of the allosteric pocket, making an H-bond with
S809 on TM7 (Figure 5), the residue designated as important for interaction with mGlu5
NAMs [35], and packing against hydrophobic residues on TM5 (e.g. L744) and TM6. In
accordance with this changed binding-mode, the docking scores of the 1,4-compounds
are improved compared to the previous dockings in the original crystal structure (PDB
id 4OO9) (Table 2). However, it should be noted that this alternative receptor
conformation is perhaps not as readily accessible in energetic terms as the original
crystal structure conformation, potentially affecting the pharmacological activity of the
1,4-compounds.
Concluding remarks
We synthesised four derivatives of the mGlu5 NAMs 1,3-(2-BisPEB) and 1,4-(2-
BisPEB) via azologisation [39] of one or two ethynyl groups, which maintain similar
pharmacological properties to the previous reported isosteres [30], including similar
potencies.
Despite the similar potencies among the compounds tested, we found substantial
differences in their antagonistic efficacies on the mGlu5 receptor: compounds with 1,3-
substitution displayed an inverse agonist efficacy similar to the reference compound
fenobam, whereas compounds with 1,4-substitution displayed partial antagonistic
effects.
Computational docking in the mGlu5 crystal structure [26] revealed the angular-shaped
1,3-substituted compounds bind in a very similar pose to the co-crystallised inverse
agonist mavoglurant, whereas the linear 1,4-substituted compounds bind in a different
pose, unlikely to be functional and with a concomitantly worse docking score. However,
an alternative conformation of the mGlu5 transmembrane domain might adapt to these
linear-shaped compounds in a functional-like pose, which may be responsible for their
partial antagonism behaviour.
The discovery of a molecular switch from full allosteric inverse agonists to partial
allosteric antagonists is of high interest, since recent publications have reported the
advantageous properties of partial mGlu5 NAMs [40, 41], which might avoid on-target
side-effects of full mGlu5 inhibition by classical allosteric inverse agonists [42, 43].
Therefore, our approach based on changing substituent positions of molecularly rigid
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mGlu5 NAMs may be useful for discovering new partial mGlu5 NAMs with safer
pharmacological profiles and better drug design outcomes.
Experimental Section
Chemistry
All the chemicals and solvents were provided from commercial suppliers and used
without purification, except the anhydrous solvents, which were treated previously
through a system of solvent purification (PureSolv), degasified with inert gases and
dried over alumina or molecular sieve (DMF). Dimethylisopropylamine (DMAP) and
triethylamine (TEA) were also dried over alumina. Compounds 12a-b, 19a-b, and 21a-
b were synthesized according to the protocols described below. Reactions were
monitored by thin layer chromatography (60F, 0.2 mm, Macherey-Nagel) by
visualisation under 254 and/or 365 nm lamp. Flash column chromatography was
performed with SNAP KP-Sil 50 µm (Biotage) or SNAP KP-C18-HS 50 µm (Biotage)
columns, automated with Isolera One with UV-Vis detection (Biotage). Melting points
(mp) were determined with a Büchi B-545, ramp 2 ºC/min. Nuclear magnetic resonance
(NMR) spectra were determined with Variant-Mercury 400 MHz (Agilent Technologies).
Chemical shifts are reported in parts per million (ppm) against the reference compound
using the signal of the residual non-deuterated solvent (Chloroform δ = 7.26 ppm (1H),
δ = 77.16 ppm (13C), DMSO δ = 2.50 ppm (1H)). Purity determination and absorption
UV-Vis spectra were determined with High-Performance Liquid Chromatography
Thermo Ultimate 3000SD (Thermo Scientific Dionex) coupled to a photodiode array
detector and a mass spectrometer LTQ XL ESI-ion trap (Thermo Scientific) (HPLC-
PDA-MS); 5 µL of sample 0.25 mM in a mixture DMSO/AcN 1:9 were injected, using an
Acquity UPLC BEH C18 column (Waters) 1.7 µm 2.1x100 mm (SN 021532354157 76)
at 30ºC. The mobile phase used was a mixture of A = NH4HCO3 aqueous buffer 10 mM
pH=7.4 and B = acetonitrile (AcN) with the method described as follows: flow 0.3
mL/min, 5%B-100%B 3 min, 100%B 5 min, total runtime 15 min; purity is given as % of
absorbance of the cis and trans isomers at 254 nm; UV-Vis spectra were collected
every 0.2 s between 650 and 275 nm and bands are % of maximal absorbance; data
from mass spectra were analysed by electrospray ionization in positive and negative
mode every 0.3 s between 50 and 1000 Da and peaks are given m/z (% of basis peak).
High-resolution mass spectra (HRMS) and elemental composition were analysed by
FIA (flux injected analysis) with ultra-high performance liquid chromatography (UPLC)
Aquity (Waters) coupled to LCT Premier orthogonal accelerated time of flight mass
spectrometer (TOF) (Waters). Data from mass spectra were analysed by electrospray
ionization in positive and negative mode. Spectra were scanned between 50 and 1500
Da with values every 0.2 s and peaks are given m/z (% of basis peak).
1,3-bis(pyridin-2-ylethynyl)benzene 12a: General procedure A: 2-ethylpyridine 13
(35 µL, 0.35 mmol) and dry diisopropylethylamine (DIPEA) (0.1 ml, 0.57 mmol) were
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added to a dispersion of 1,3-diiodobenzene 14a (46 mg, 0.14 mmol), copper(I) iodide
(2 mg, 0.01 mmol) and bis(triphenylphosphine) palladium (II) chloride (5.4 mg, 0.01
mmol) in dry dimethyl formamide (DMF) (1 mL) in a Schlenk flask with argon
atmosphere at room temperature. The mixture was stirred at 50ºC for 5 hours. The
reaction mixture was diluted in EtOAc (15 mL) and washed with aqueous Na2CO3 1 N
(10 mL), saturated NaHCO3 (10 mL), water (10 mL) and brine (10 mL), dried over
anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified
through flash chromatography using Biotage Isolera equipment (SNAP KP-C18-HS
12g, A=H2O, B=MeCN; 0%B 5CV, 0%B-1%B 2CV, 1%B-4%B 2.5 CV, 4%B 1.5CV,
4%B-7%B 6CV, 7%B-9%B 0.5CV, 9%B-100%B 2 CV, 100%B 5CV). The title product
12a was obtained as brown colour solid (34 mg) with an 87% yield, mp 89.9-91.3ºC. 1H
NMR (400 MHz, CDCl3) δ 8.65 (br, 2H), 7.82 (t, J = 1.6 Hz, 1H), 7.70 (td, J = 7.7, 1.6
Hz, 2H), 7.60 (dd, J = 7.8, 1.6 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.37 (t, J = 7.7 Hz, 1H),
7.31 – 7.22 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 150.12, 143.22, 136.43, 135.42,
132.58, 128.76, 127.51, 123.16, 122.86, 89.33, 88.37. HPLC-PDA-MS: RT = 5.91 min,
99.87% (254 nm), PDA λmax = 296 nm, MS (m/z) [M+H]+ 280.94.
1,4-bis(pyridin-2-ylethynyl)benzene 12b: General procedure A was used with 13 (36
µL, 0.36 mmol) and dry DIPEA (0.1 ml, 0.57 mmol) and 1,4-diiodobenzene 14b (47 mg,
0.14 mmol), CuI (2 mg, 0.01 mmol) and PdCl2(PPh3)2 (5.4 mg, 0.01 mmol) in DMF (1
mL). The residue was purified through flash chromatography using Biotage Isolera
equipment (SNAP KP-C18-HS 12g, A= H2O B=MeCN; 0%B 5CV, 0%B-1%B 2CV,
1%B-4%B 2.5 CV, 4%B 1.5CV, 4%B-7%B 6CV, 7%B-9%B 0.5CV, 9%B-100%B 2 CV,
100%B 5CV). The title product 12b was obtained as cream colour solid (18 mg) with a
45% yield, mp 187.9-188.1ºC. 1H NMR (400 MHz, CDCl3) δ 8.65 (br, 2H), 7.72 (td, J =
7.7, 1.6 Hz, 2H), 7.60 (s, 4H), 7.56 (d, J = 8.0 Hz, 2H), 7.28 (dd, J = 6.9, 5.3 Hz, 2H).
13C NMR (101 MHz, CDCl3) δ 150.00, 143.08, 136.62, 132.18, 127.56, 123.23, 122.96,
90.55, 89.24. HPLC-PDA-MS: RT = 5.89 min, 99.72% (254 nm), PDA λmax = 319 nm,
MS (m/z) [M+H]+ 280.91.
3-(pyridin-2-ylethynyl)aniline 17a: General procedure B: 2-chloropyridine 16 (0.13
mL, 1.37 mmol), 3-ethynylaniline 15a (0.24 mL, 2.13 mmol) and dry triethylamine (TEA)
(0.76 ml, 5.46 mmol) were added to a dispersion of copper(I) iodide (13 mg, 0.07
mmol) and bis(triphenylphosphine) palladium (II) chloride (48 mg, 0.07 mmol) in dry
dimethyl formamide (DMF) (8 mL) in a Schlenk flask with argon atmosphere at room
temperature. The mixture was stirred at 60ºC for 4 hours. The reaction mixture was
diluted in EtOAc (100 mL) and washed with aqueous Na2CO3 1 N (50 mL), saturated
NaHCO3 (50 mL), water (50 mL) and brine (50 mL), dried over anhydrous MgSO4,
filtered and concentrated in vacuo. The residue was purified through silica flash
chromatography (DCM and DCM + 1% MeOH) to afford the title compound 17a as
brown thick oil (80 mg, 30% yield). 1H NMR (400 MHz, CDCl3) δ 8.61 (ddd, J = 4.9, 1.8,
1.0 Hz, 1H), 7.67 (td, J = 7.7, 1.8 Hz, 1H), 7.50 (dt, J = 7.9, 1.1 Hz, 1H), 7.23 (ddd, J =
7.6, 4.9, 1.2 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.00 (dt, J = 7.6, 1.2 Hz, 1H), 6.91 (dd, J
= 2.4, 1.5 Hz, 1H), 6.69 (ddd, J = 8.1, 2.4, 1.0 Hz, 1H), 3.13 (br, 2H). 13C NMR (101
13

ACCEPTED MANUSCRIPT
MHz, CDCl3) δ 150.11, 146.42, 143.65, 136.31, 129.45, 127.30, 123.02, 122.79,
122.62, 118.30, 116.17, 89.80, 88.09. HRMS (m/z): [M+H]+ calculated for C13H11N2,
195.0922; found, 195.0922.
2-((3-(pyridin-2-yldiazenyl)phenyl)ethynyl)pyridine 19a: General procedure C: One
drop of acetic acid was added to a solution of 2-nitrosopyridne 18 (15 mg, 0.14 mmol)
and aniline 17a (18 mg, 0.09 mmol) in DCM (1 mL) and the resulting solution was left
stirring at room temperature for 3 days. The reaction mixture was concentrated in
vacuo and the remains of acetic acid were co-evaporated with toluene twice. The
obtained residue was purified through flash chromatography using Biotage Isolera
equipment (SNAP KP-C18-HS 12g, A= H2O B=MeOH; 0%B 3CV, 0%B-60%B 5CV,
60%B-85%B 8CV, 85%B-100%B 1CV, 100%B 4CV). The title product 19a was
1
obtained (20 mg, orange solid) with a 76% yield, mp 79.1-79.8 ºC. H NMR (400 MHz,
CDCl3) δ 8.76 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 8.65 (ddd, J = 5.0, 1.8, 0.9 Hz, 1H), 8.31
– 8.25 (m, 1H), 8.08 (ddd, J = 8.1, 2.0, 1.2 Hz, 1H), 7.93 (ddd, J = 8.0, 7.3, 1.8 Hz, 1H),
7.85 (dt, J = 8.0, 1.1 Hz, 1H), 7.77 (dt, J = 7.7, 1.3 Hz, 1H), 7.75 (td, J = 7.7, 1.8 Hz,
1H), 7.58 (dt, J = 7.6, 0.9 Hz, 1H), 7.54 (t, J = 7.9 Hz, 1H), 7.43 (ddd, J = 7.3, 4.8, 1.2
Hz, 1H), 7.31 (ddd, J = 7.6, 4.9, 1.2 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 162.73,
152.36, 149.74, 149.67, 142.76, 138.60, 136.99, 135.45, 129.51, 127.65, 127.02,
125.70, 124.59, 123.38, 123.27, 116.09, 89.32, 88.91. HPLC-PDA-MS: RT = 5.50 min,
21.53% (254 nm), PDA λmax = 290, 428 nm, MS (m/z) [M+H]+ 284.96 (cis-isomer); RT =
5.76 min, 78.00% (254 nm), PDA λmax = 302. 446 nm, MS (m/z) [M+H]+ 284.95 (trans-
isomer). HRMS (m/z): [M+H]+ calculated for C18H13N4, 285.1145; found, 285.1140.
4-(pyridin-2-ylethynyl)aniline 17b: General procedure B was used with 16 (0.13 mL,
1.37 mmol), dry TEA (0.76 ml, 5.46 mmol), 4-ethynylaniline 15b (249 mg, 2.05 mmol),
CuI (13 mg, 0.07 mmol) and PdCl2(PPh3)2 (48 mg, 0.07 mmol) in DMF (8 mL). The
residue was purified through silica flash chromatography (DCM/hexanes 9:1, DCM,
DCM + 0.5% MeOH and DCM + 1% MeOH) to afford the title compound 17b as brown
1
thick oil (60 mg, 23% yield). H NMR (400 MHz, CDCl3) δ 8.58 (ddd, J = 4.9, 1.9, 1.0
Hz, 1H), 7.64 (td, J = 7.7, 1.8 Hz, 1H), 7.47 (dt, J = 7.9, 1.1 Hz, 1H), 7.40 (d, J = 8.7 Hz,
2H), 7.18 (ddd, J = 7.6, 4.9, 1.2 Hz, 1H), 6.63 (d, J = 8.7 Hz, 2H), 3.48 (br, 2H). 13C
NMR (101 MHz, CDCl3) δ 150.02, 147.46, 144.18, 136.20, 133.67, 126.88, 122.26,
114.78, 111.51, 90.69, 87.10. HRMS (m/z): [M+H]+ calculated for C13H11N2, 195.0922;
found, 195.0923.
2-((4-(pyridin-2-yldiazenyl)phenyl)ethynyl)pyridine 19b: General procedure C was
used with one drop of AcOH, 18 (11 mg, 0.10 mmol) and 17b (13 mg, 0.07 mmol) in
DCM (1 mL). The obtained residue was purified through flash chromatography using
Biotage Isolera equipment (SNAP KP-C18-HS 12g, A= H2O B=MeOH; 0%B 3CV,
0%B-60%B 5CV, 60%B-85%B 8CV, 85%B-100%B 1CV, 100%B 8CV). The title
product 19b was obtained (15 mg, orange solid) with a 78% yield, mp 185.9-186.6ºC.
1H NMR (400 MHz, CDCl3 NMR) δ 8.76 (ddd, J = 4.8, 1.8, 0.8 Hz, 1H), 8.65 (ddd, J =
4.9, 1.8, 0.9 Hz, 1H), 8.06 (d, J = 8.6 Hz, 2H), 7.93 (ddd, J = 8.0, 7.3, 1.9 Hz, 1H), 7.85
14

ACCEPTED MANUSCRIPT
(dt, J = 8.0, 1.1 Hz, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.73 (dd, J = 7.7, 1.8 Hz, 1H), 7.58
(dt, J = 7.9, 1.1 Hz, 1H), 7.43 (ddd, J = 7.3, 4.7, 1.2 Hz, 1H), 7.30 (ddd, J = 7.7, 4.9, 1.2
Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 162.86, 152.17, 149.94, 149.76, 142.86, 138.57,
136.79, 133.14, 127.62, 126.08, 125.65, 123.79, 123.33, 116.16, 91.06, 89.43. HPLC-
PDA-MS: RT = 5.48 min, 19.34% (254 nm), PDA λmax = 307, 435 nm, MS (m/z) [M+H]+
284.95 (cis-isomer); RT = 5.75 min, 80.09% (254 nm), PDA λmax = 349, 443 nm, MS
(m/z) [M+H]+ 284.96 (trans-isomer). HRMS (m/z): [M+H]+ calculated for C18H13N4,
285.1140; found, 285.1140.
1,3-bis(pyridin-2-yldiazenyl)benzene 21a: General procedure C was used with two
drops of AcOH added to 16 (120 mg, 1.11 mmol) and benzene-1,3-diamine 20a (40 mg,
0.37 mmol) in DCM (10 mL). The obtained residue was purified through flash
chromatography using Biotage Isolera equipment (SNAP KP-C18-HS 12g, A= H2O
B=MeOH; 5%B 4CV, 5%B-50%B 3CV, 50%B-10%B 8CV, 100%B 6CV). The title
product 21a was obtained as a dark solid (5 mg) with a 5% yield, mp 101.3-101.9ºC.
1H NMR (400 MHz, CDCl3) δ 8.77 (ddd, J = 4.8, 1.8, 0.9 Hz, 2H), 8.70 (t, J = 2.0 Hz,
1H), 8.25 (dd, J = 7.9, 2.0 Hz, 2H), 7.95 (ddd, J = 8.0, 7.2, 1.8 Hz, 2H), 7.89 (dt, J = 8.0,
1.1 Hz, 2H), 7.73 (t, J = 7.9 Hz, 1H), 7.45 (ddd, J = 7.2, 4.8, 1.3 Hz, 2H). 13C NMR (101
MHz, CDCl3) δ 162.79, 153.33, 149.80, 138.61, 130.01, 127.09, 125.74, 118.01,
116.37. HPLC-PDA-MS: RT = 5.11 min, 3.65% (254 nm), PDA λmax = 278, 428 nm, MS
(m/z) [M+H]+ 288.93 (cis-cis-isomer); RT = 5.34 min, 24.86% (254 nm), PDA λmax = 314,
432 nm, MS (m/z) [M+H]+ 288.95 (cis-trans-isomer); RT = 5.60 min, 70.71% (254 nm),
PDA λmax = 319, 443 nm, MS (m/z) [M+H]+ 288.96 (trans-trans-isomer). HRMS (m/z):
[M+H]+ calculated for C16H13N6, 289.1202; found, 289.1216.
1,4-bis(pyridin-2-yldiazenyl)benzene 21b: General procedure C was used with two
drops of AcOH, 18 (33 mg, 0.31 mmol) and benzene-1,4-diamine 20b (15 mg, 0.14
mmol) in DCM (1 mL). The title product 21b (39 mg, orange solid) was afforded with a
1
98% yield, mp 211.6-212.7ºC. H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 3.4 Hz, 2H),
8.22 (s, 4H), 7.96 (td, J = 7.7, 1.8 Hz, 2H), 7.89 (d, J = 7.9 Hz, 2H), 7.46 (ddd, J = 7.4,
4.8, 1.3 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 162.72, 154.18, 149.71, 138.73, 125.85,
124.70, 116.27. HPLC-PDA-MS: RT = 5.34 min, 5.33% (254 nm), PDA λmax = 336, 437
nm, MS (m/z) [M+H]+ 288.89 (cis-trans-isomer); RT = 5.59 min, 90.61% (254 nm), PDA
λmax = 353, 456 nm, MS (m/z) [M+H]+ 288.90 (trans-trans-isomer), HRMS (m/z): [M+H]+
calculated for C15H13N6, 289.1202; found, 289.1216.
Pharmacology
HEK 293 cells were cultured and transfected by electroporation as previously
described for expression of all rat mGlu receptors [44]. To maintain ambient glutamate
at minimal concentrations, the excitatory amino acid transporter 1 (EAAC1) was co-
transfected. All receptors contained an HA tag in their N-terminus to monitor their cell
surface expression by ELISA. Once transfected, cells were seeded in black clear-
bottom 96-well plates at a concentration of 1.5 × 105 cells/well. At least 4h before the
experiment, we changed the medium to glutamate-free DMEM GlutaMAX-I (Thermo
15

ACCEPTED MANUSCRIPT
Fisher Scientific). The day after, we estimated IP accumulation using the IP-One HTRF
kit (Cisbio Bioassays) according to the manufacturer’s instructions. Cells were
stimulated to induce IP accumulation while being treated with test compounds for 30
min, at 37°C and 5% CO2, and placed over an LED plate [28] (FCTecnics) at a
distance of 2–3 cm for continuous illumination. To allow stimulation in both dark
conditions and violet light illumination, two-thirds of the 96-well plate bottom was
covered with aluminium foil. To avoid effects derived from the fast relaxation of azo-
compounds in aqueous solution after the 30-min stimulation, we washed cells once in
stimulation buffer alone before continuing with the lysis step of the assay protocol. For
fluorescence readings with a RUBYstar HTRF HTS microplate reader (BMG Labtech),
we transferred cell lysates to a black opaque-bottom 96-well plate. Dose-response data
from IP accumulation assays were first normalized by the dose-response to reference
allosteric modulator fenobam in both dark and illuminated conditions and then fitted
with a standard Hill equation using GraphPad Prism version 6.0.
Computational
The molecular structures of six compounds: 12a-b, 19a-b, 21a-b in trans-isomer state
were constructed with Maestro version 9.8 (Schrӧdinger Release 2014). Each
compound was docked into the transmembrane domain crystal structure of mGlu5
(PDB id 4OO9, minus co-crystallized mavoglurant) using AutoDock4.2 with default
parameters [45]. For each respective docked compound, the top-ranked complex
(according to AutoDock docking score) was energy-minimized in the Amber14SB
force-field [46] using CHIMERA [47] to optimize protein-ligand interactions. The three
linear 1,4-compounds in trans-isomer state: 12b, 19b and 21b were also docked (using
same parameters) into an alternative mGlu5 receptor conformation, previously derived
from the mGlu5 crystal structure (PDB id 4OO9) by using a conformational sampling
technique [33].
Acknowledgments
This research has been supported by RecerCaixa foundation (2010ACUP00378 to
P.G., J.G., and A.L.), the Marató de TV3 Foundation (110230 to J.G., 110231 to A.L.,
110232 to C.G., and 111531 to P.G.), the Catalan government (2010 BP-A00194 to
X.R., 2012 CTP 00033 and 2012 BE1 00597 to X.G.-S., 2014SGR-1251 to P.G., and
2014SGR-00109 to A.L.), the Spanish Government (CTQ2014-57020-R and PCIN-
2013-017-C03-01 to A.L., and SAF2014-58396-R and PCIN-2013-018-C03-02 to J.G.),
and the ERANET Neuron LIGHTPAINProject (to A.L., J.G., and J.-P.P.); SynBio
MODULIGHTOR, Human Brain Project WAVESCALES and Ramón Areces foundation
grants (to P.G.); the Agence Nationale de la Recherche (ANR-16-CE16-0010 to A.L.
and C.G.).
Contributions
A.L. conceived and supervised the project. A.L., J.G., X.G.-S, X.R., J.D., P.G., C.G.,
and J.-P.P. designed experiments. X.G.-S., J.D., J.G. and A.L. designed compounds.
16

ACCEPTED MANUSCRIPT
X.G.-S. synthesized the compounds, characterized their photoisomerisation and
performed pharmacological assays. X.R., C.G. and J.-P.P analysed pharmacological
results. J.D. performed the computational dockings. X.G.-S., J.D., J.G. and A.L. wrote
the paper. All authors made corrections and approved the final manuscript.
References
[1] P.J. Conn, Physiological roles and therapeutic potential of metabotropic glutamate
receptors, Ann. N. Y. Acad. Sci., 1003 (2003) 12-21.
[2] F. Nicoletti, J. Bockaert, G.L. Collingridge, P.J. Conn, F. Ferraguti, D.D. Schoepp,
J.T. Wroblewski, J.P. Pin, Metabotropic glutamate receptors: from the workbench to the
bedside, Neuropharmacol., 60 (2011) 1017-1041.
[3] J.P. Pin, F. Acher, The metabotropic glutamate receptors: structure, activation
mechanism and pharmacology, Curr. Drug Targets CNS Neurol. Disord., 1 (2002) 297-
317.
[4] C. Ghelardini, C. Menicacci, D. Cerretani, E. Bianchi, Spinal administration of
mGluR5 antagonist prevents the onset of bortezomib induced neuropathic pain in rat,
Neuropharmacol., 86 (2014) 294-300.
[5] M.C. Montana, L.F. Cavallone, K.K. Stubbert, A.D. Stefanescu, E.D. Kharasch,
R.W.t. Gereau, The metabotropic glutamate receptor subtype 5 antagonist fenobam is
analgesic and has improved in vivo selectivity compared with the prototypical
antagonist 2-methyl-6-(phenylethynyl)-pyridine, J. Pharmacol. Exp. Ther., 330 (2009)
834-843.
[6] F. Caraci, G. Battaglia, M.A. Sortino, S. Spampinato, G. Molinaro, A. Copani, F.
Nicoletti,
V.
Bruno,
Metabotropic
glutamate
receptors
in
neurodegeneration/neuroprotection: still a hot topic?, Neurochem. Int., 61 (2012) 559-
565.
[7] A. Vallano, V. Fernandez-Duenas, G. Garcia-Negredo, M.A. Quijada, C.P. Simon,
M.L. Cuffi, L. Carbonell, S. Sanchez, J.M. Arnau, F. Ciruela, Targeting striatal
metabotropic glutamate receptor type 5 in Parkinson's disease: bridging molecular
studies and clinical trials, CNS Neurol. Disord. Drug Targets, 12 (2013) 1128-1142.
[8] D. Petrov, I. Pedros, M.L. de Lemos, M. Pallas, A.M. Canudas, A. Lazarowski, C.
Beas-Zarate, C. Auladell, J. Folch, A. Camins, Mavoglurant as a treatment for
Parkinson's disease, Expert Opin. Investig. Drugs, 23 (2014) 1165-1179.
[9] C. Finlay, S. Duty, Therapeutic potential of targeting glutamate receptors in
Parkinson's disease, J. Neural. Transm., 121 (2014) 861-880.
[10] A.S. Pop, B. Gomez-Mancilla, G. Neri, R. Willemsen, F. Gasparini, Fragile X
syndrome: a preclinical review on metabotropic glutamate receptor 5 (mGluR5)
antagonists and drug development, Psychopharmacology (Berl.), 231 (2014) 1217-
1226.
[11] S. Jacquemont, E. Berry-Kravis, R. Hagerman, F. von Raison, F. Gasparini, G.
Apostol, M. Ufer, V. Des Portes, B. Gomez-Mancilla, The challenges of clinical trials in
fragile X syndrome, Psychopharmacology (Berl.), 231 (2014) 1237-1250.
[12] J.A. Quiroz, P. Tamburri, D. Deptula, L. Banken, U. Beyer, M. Rabbia, N. Parkar, P.
Fontoura, L. Santarelli, Efficacy and Safety of Basimglurant as Adjunctive Therapy for
Major Depression: A Randomized Clinical Trial, JAMA Psychiatry, 73 (2016) 675-684.
[13] W.O. Rohof, A. Lei, D.P. Hirsch, L. Ny, M. Astrand, M.B. Hansen, G.E.
Boeckxstaens, The effects of a novel metabotropic glutamate receptor 5 antagonist
(AZD2066) on transient lower oesophageal sphincter relaxations and reflux episodes in
healthy volunteers, Aliment. Pharmacol. Ther., 35 (2012) 1231-1242.
[14] R.H. Porter, P.J. Roberts, D.E. Jane, J.C. Watkins, (S)-homoquisqualate: a potent
agonist at the glutamate metabotropic receptor, Br. J. Pharmacol., 106 (1992) 509-510.
[15] D.E. Jane, P.L. Jones, P.C. Pook, T.E. Salt, D.C. Sunter, J.C. Watkins,
Stereospecific antagonism by (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) of
17

ACCEPTED MANUSCRIPT
(1S,3R)-ACPD-induced effects in neonatal rat motoneurones and rat thalamic
neurones, Neuropharmacol., 32 (1993) 725-727.
[16] P. Wellendorph, H. Brauner-Osborne, Molecular basis for amino acid sensing by
family C G-protein-coupled receptors, Br. J. Pharmacol., 156 (2009) 869-884.
[17] P.J. Flor, F.C. Acher, Orthosteric versus allosteric GPCR activation: the great
challenge of group-III mGluRs, Biochem. Pharmacol., 84 (2012) 414-424.
[18] F. Kesisoglou, S. Panmai, Y. Wu, Nanosizing--oral formulation development and
biopharmaceutical evaluation, Adv. Drug. Deliv. Rev., 59 (2007) 631-644.
[19] W.M. Pardridge, Drug transport across the blood-brain barrier, J. Cereb. Blood.
Flow. Metab., 32 (2012) 1959-1972.
[20] T. de Paulis, K. Hemstapat, Y. Chen, Y. Zhang, S. Saleh, D. Alagille, R.M. Baldwin,
G.D. Tamagnan, P.J. Conn, Substituent effects of N-(1,3-diphenyl-1H-pyrazol-5-
yl)benzamides on positive allosteric modulation of the metabotropic glutamate-5
receptor in rat cortical astrocytes, J. Med. Chem., 49 (2006) 3332-3344.
[21] S. Conde-Ceide, C.M. Martinez-Viturro, J. Alcazar, P.M. Garcia-Barrantes, H.
Lavreysen, C. Mackie, P.N. Vinson, J.M. Rook, T.M. Bridges, J.S. Daniels, A. Megens,
X. Langlois, W.H. Drinkenburg, A. Ahnaou, C.M. Niswender, C.K. Jones, G.J.
Macdonald, T. Steckler, P.J. Conn, S.R. Stauffer, J.M. Bartolome-Nebreda, C.W.
Lindsley, Discovery of VU0409551/JNJ-46778212: An mGlu5 Positive Allosteric
Modulator Clinical Candidate Targeting Schizophrenia, ACS Med. Chem. Lett., 6
(2015) 716-720.
[22] R.H. Porter, G. Jaeschke, W. Spooren, T.M. Ballard, B. Buttelmann, S. Kolczewski,
J.U. Peters, E. Prinssen, J. Wichmann, E. Vieira, A. Muhlemann, S. Gatti, V. Mutel, P.
Malherbe, Fenobam: a clinically validated nonbenzodiazepine anxiolytic is a potent,
selective, and noncompetitive mGlu5 receptor antagonist with inverse agonist activity,
J. Pharmacol. Exp. Ther., 315 (2005) 711-721.
[23] F. Gasparini, K. Lingenhohl, N. Stoehr, P.J. Flor, M. Heinrich, I. Vranesic, M.
Biollaz, H. Allgeier, R. Heckendorn, S. Urwyler, M.A. Varney, E.C. Johnson, S.D. Hess,
S.P. Rao, A.I. Sacaan, E.M. Santori, G. Velicelebi, R. Kuhn, 2-Methyl-6-
(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5
receptor antagonist, Neuropharmacol., 38 (1999) 1493-1503.
[24] C. Keywood, M. Wakefield, J. Tack, A proof-of-concept study evaluating the effect
of ADX10059, a metabotropic glutamate receptor-5 negative allosteric modulator, on
acid exposure and symptoms in gastro-oesophageal reflux disease, Gut, 58 (2009)
1192-1199.
[25] I. Vranesic, S. Ofner, P.J. Flor, G. Bilbe, R. Bouhelal, A. Enz, S. Desrayaud, K.
McAllister, R. Kuhn, F. Gasparini, AFQ056/mavoglurant, a novel clinically effective
mGluR5 antagonist: identification, SAR and pharmacological characterization, Bioorg.
Med. Chem., 22 (2014) 5790-5803.
[26] A.S. Dore, K. Okrasa, J.C. Patel, M. Serrano-Vega, K. Bennett, R.M. Cooke, J.C.
Errey, A. Jazayeri, S. Khan, B. Tehan, M. Weir, G.R. Wiggin, F.H. Marshall, Structure
of class C GPCR metabotropic glutamate receptor 5 transmembrane domain, Nature,
511 (2014) 557-562.
[27] M.A. Varney, N.D. Cosford, C. Jachec, S.P. Rao, A. Sacaan, F.F. Lin, L. Bleicher,
E.M. Santori, P.J. Flor, H. Allgeier, F. Gasparini, R. Kuhn, S.D. Hess, G. Velicelebi, E.C.
Johnson, SIB-1757 and SIB-1893: selective, noncompetitive antagonists of
metabotropic glutamate receptor type 5, J. Pharmacol. Exp. Ther., 290 (1999) 170-181.
[28] S. Pittolo, X. Gomez-Santacana, K. Eckelt, X. Rovira, J. Dalton, C. Goudet, J.P.
Pin, A. Llobet, J. Giraldo, A. Llebaria, P. Gorostiza, An allosteric modulator to control
endogenous G protein-coupled receptors with light, Nat. Chem. Biol., 10 (2014) 813-
815.
[29] X. Gómez-Santacana, X. Rovira, J.A. Dalton, C. Goudet, J.P. Pin, P. Gorostiza, J.
Giraldo, A. Llebaria, A double effect molecular switch leads to a novel potent negative
allosteric modulator of metabotropic glutamate receptor 5, MedChemComm, 5 (2014)
1548-1554.
18

ACCEPTED MANUSCRIPT
[30] B.H. Kaae, K. Harpsoe, T. Kvist, J.M. Mathiesen, C. Molck, D. Gloriam, H.N.
Jimenez, M.A. Uberti, S.M. Nielsen, B. Nielsen, H. Brauner-Osborne, P. Sauerberg,
R.P. Clausen, U. Madsen, Structure-activity relationships for negative allosteric
mGluR5 modulators, ChemMedChem, 7 (2012) 440-451.
[31] K.A. Bennett, A.S. Dore, J.A. Christopher, D.R. Weiss, F.H. Marshall, Structures of
mGluRs shed light on the challenges of drug development of allosteric modulators,
Curr. Opin. Pharmacol., 20 (2015) 1-7.
[32] X. Rovira, A. Trapero, S. Pittolo, C. Zussy, A. Faucherre, C. Jopling, J. Giraldo, J.P.
Pin, P. Gorostiza, C. Goudet, A. Llebaria, OptoGluNAM4.1, a Photoswitchable
Allosteric Antagonist for Real-Time Control of mGlu4 Receptor Activity, Cell Chem.
Biol., 23 (2016) 929-934.
[33] J.A. Dalton, I. Lans, X. Rovira, F. Malhaire, X. Gómez-Santacana, S. Pittolo, P.
Gorostiza, A. Llebaria, C. Goudet, J.P. Pin, J. Giraldo, Shining Light On An mGlu5
Photoswitchable NAM: A Theoretical Perspective, Curr. Neuropharmacol., 14 (2016)
441-454.
[34] S. Samanta, H.I. Qureshi, G.A. Woolley, A bisazobenzene crosslinker that
isomerizes with visible light, Beilstein J. Org. Chem., 8 (2012) 2184-2190.
[35] C. Molck, K. Harpsoe, D.E. Gloriam, R.P. Clausen, U. Madsen, L.O. Pedersen,
H.N. Jimenez, S.M. Nielsen, J.M. Mathiesen, H. Brauner-Osborne, Pharmacological
characterization and modeling of the binding sites of novel 1,3-
bis(pyridinylethynyl)benzenes as metabotropic glutamate receptor 5-selective negative
allosteric modulators, Mol. Pharmacol., 82 (2012) 929-937.
[36] X. Gómez-Santacana, S. Pittolo, X. Rovira, M. Lopez, C. Zussy, J.A.R. Dalton, A.
Faucherre, C. Jopling, J.-P. Pin, F. Ciruela, C. Goudet, J. Giraldo, P. Gorostiza, A.
Llebaria, Illuminating Phenylazopyridines To Photoswitch Metabotropic Glutamate
Receptors: From the Flask to the Animals, ACS Cent. Sci., in press (2016) DOI:
10.1021/acscentsci.1026b00353.
[37] J.A. Christopher, S.J. Aves, K.A. Bennett, A.S. Dore, J.C. Errey, A. Jazayeri, F.H.
Marshall, K. Okrasa, M.J. Serrano-Vega, B.G. Tehan, G.R. Wiggin, M. Congreve,
Fragment and Structure-Based Drug Discovery for a Class C GPCR: Discovery of the
mGlu5 Negative Allosteric Modulator HTL14242 (3-Chloro-5-[6-(5-fluoropyridin-2-
yl)pyrimidin-4-yl]benzonitrile), J. Med. Chem., 58 (2015) 6653-6664.
[38] H. Wu, C. Wang, K.J. Gregory, G.W. Han, H.P. Cho, Y. Xia, C.M. Niswender, V.
Katritch, J. Meiler, V. Cherezov, P.J. Conn, R.C. Stevens, Structure of a class C GPCR
metabotropic glutamate receptor 1 bound to an allosteric modulator, Science, 344
(2014) 58-64.
[39] J. Broichhagen, J.A. Frank, D. Trauner, A roadmap to success in
photopharmacology, Acc. Chem. Res., 48 (2015) 1947-1960.
[40] R.W. Gould, R.J. Amato, M. Bubser, M.E. Joffe, M.T. Nedelcovych, A.D.
Thompson, H.H. Nickols, J.P. Yuh, X. Zhan, A.S. Felts, A.L. Rodriguez, R.D. Morrison,
F.W. Byers, J.M. Rook, J.S. Daniels, C.M. Niswender, P.J. Conn, K.A. Emmitte, C.W.
Lindsley, C.K. Jones, Partial mGlu Negative Allosteric Modulators Attenuate Cocaine-
Mediated
Behaviors
and
Lack
Psychotomimetic-Like
Effects,
Neuropsychopharmacology, (2015).
[41] H.H. Nickols, J.P. Yuh, K.J. Gregory, R.D. Morrison, B.S. Bates, S.R. Stauffer, K.A.
Emmitte, M. Bubser, W. Peng, M.T. Nedelcovych, A. Thompson, X. Lv, Z. Xiang, J.S.
Daniels, C.M. Niswender, C.W. Lindsley, C.K. Jones, P.J. Conn, VU0477573: Partial
negative allosteric modulator of the subtype 5 metabotropic glutamate receptor with in
vivo efficacy, J. Pharmacol. Exp. Ther., (2015).
[42] K. Abou Farha, R. Bruggeman, C. Balje-Volkers, Metabotropic glutamate receptor
5 negative modulation in phase I clinical trial: potential impact of circadian rhythm on
the neuropsychiatric adverse reactions-do hallucinations matter?, ISRN Psychiatry,
2014 (2014) 652750.
19

ACCEPTED MANUSCRIPT
[43] U.C. Campbell, K. Lalwani, L. Hernandez, G.G. Kinney, P.J. Conn, L.J. Bristow,
The mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) potentiates PCP-
induced cognitive deficits in rats, Psychopharmacology (Berl.), 175 (2004) 310-318.
[44] I. Brabet, M.L. Parmentier, C. De Colle, J. Bockaert, F. Acher, J.P. Pin,
Comparative effect of L-CCG-I, DCG-IV and gamma-carboxy-L-glutamate on all cloned
metabotropic glutamate receptor subtypes, Neuropharmacol., 37 (1998) 1043-1051.
[45] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J.
Olson, AutoDock4 and AutoDockTools4: Automated docking with selective receptor
flexibility, J. Comput. Chem., 30 (2009) 2785-2791.
[46] D.A. Case, T.E. Cheatham, 3rd, T. Darden, H. Gohlke, R. Luo, K.M. Merz, Jr., A.
Onufriev, C. Simmerling, B. Wang, R.J. Woods, The Amber biomolecular simulation
programs, J. Comput. Chem., 26 (2005) 1668-1688.
[47] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C.
Meng, T.E. Ferrin, UCSF Chimera--a visualization system for exploratory research and
analysis, J. Comput. Chem., 25 (2004) 1605-1612.
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Highlights
•
•
•
•
Efficacy of bispyridine benzene mGlu5 negative allosteric modulators
depends on molecule geometry.
1,4-disubstituted linear compounds have mGlu5 partial antagonist
activity.
1,3-disubstituted angular compounds have mGlu5 full inverse agonist
activity.
1,4- and 1,3-isomers bind preferentially to different receptor
conformations according to computational docking.

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Supplementary Material
Positional isomers of bispyridine benzene derivatives induce efficacy changes on mGlu5
negative allosteric modulation
Xavier Gómez-Santacanaa,b,c,§, James A. R. Daltonb,d, Xavier Rovira e,f, Jean Philippe Pin e,f, Cyril Goudet e,f
Pau Gorostizac,g,h, Jesús Giraldob,d and Amadeu Llebariaa*
,
(a) MCS, Laboratory of Medicinal Chemistry & Synthesis, Institute for Advanced Chemistry of Catalonia (IQAC−CSIC), Jordi Girona 18-26,
08034 Barcelona, Spain.
(b) Laboratory of Molecular Neuropharmacology and Bioinformatics, Institut de Neurociències and Unitat de Bioestadística, Universitat
Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain.
(c) Nanoprobes and Nanoswitches, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac, 08028, Barcelona, Spain.
(d) Network Biomedical Research Center on Mental Health (CIBERSAM), Spain
(e) Department of Neurosciences, Institute of Functional Genomics, Université de Montpellier, Unité Mixte de Recherche 5302 CNRS,
Montpellier, France; (f) Unité de recherche U1191, INSERM, Montpellier, France (g) Network Biomedical Research Center on
Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain.
(h) Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain.
(§) Present address: Division of Medicinal Chemistry, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), VU University
Amsterdam, The Netherlands.
* Corresponding author: amadeu.llebaria@iqac.csic.es
1) Nuclear Magnetic Resonance Spectra
2) High Performance Liquid Chromatography coupled to a Photodiode Array and Mass
Spectrometer
3) UV/Vis spectra under illumination
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