Development of Thiochroman Dioxide Analogues of Benzothiadiazine Dioxides as New Positive Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors

Article abstract of DOI:10.1021/acschemneuro.1c00255

On the basis of the activity of 1,2,4-benzothiadiazine 1,1-dioxides as positive allosteric modulators of AMPA receptors, thiochroman 1,1-dioxides were designed applying the isosteric replacement concept. The new compounds expressed strong modulatory activ

Full text of DOI:10.1021/acschemneuro.1c00255

pubs.acs.org/chemneuro
Research Article
Development of Thiochroman Dioxide Analogues of
Benzothiadiazine Dioxides as New Positive Allosteric Modulators of
α‑Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)
Receptors
̀
Koffi Sénam Etse, Jerzy Dorosz, Katrine McLain Christensen, Jean-Yves Thomas, Iuliana Botez Pop,
Eric Goffin, Thomas Colson, Pierre Lestage, Laurence Danober, Bernard Pirotte,
Jette Sandholm Kastrup,* and Pierre Francotte*
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ABSTRACT: On the basis of the activity of 1,2,4-benzothiadia-
zine 1,1-dioxides as positive allosteric modulators of AMPA
receptors, thiochroman 1,1-dioxides were designed applying the
isosteric replacement concept. The new compounds expressed
strong modulatory activity on AMPA receptors in vitro, although
lower than their corresponding benzothiadiazine analogues. The
pharmacokinetic profile of three thiochroman 1,1-dioxides (12a,
12b, 12e) was examined in vivo after oral administration, showing
that these compounds freely cross the blood−brain barrier.
Structural analysis was achieved using X-ray crystallography after
cocrystallization of the racemic compound 12b in complex with
the ligand-binding domain of GluA2 (L504Y/N775S). Interestingly, both enantiomers of 12b were found to interact with the GluA2
dimer interface, almost identically to its benzothiadiazine analogue, BPAM344 (4). The interactions of the two enantiomers in the
cocrystal were further analyzed (mapping Hirshfeld surfaces and 2D fingerprint) and compared to those of 4. Taken together, these
data explain the lower affinity on AMPA receptors of thiochroman 1,1-dioxides compared to their corresponding 1,2,4-
benzothiadiazine 1,1-dioxides.
KEYWORDS: Ionotropic glutamate receptors, Thiochroman 1,1-dioxide, 1,2,4-Benzothiadiazine 1,1-dioxide, Positive allosteric modulator,
AMPA receptor, X-ray crystallography, Hirshfeld surface analysis
scaffold proteins regulating the building and trafficking of
AMPARs in neurons. To date, three distinct families of
auxiliary proteins have been identified for AMPARs: trans-
membrane AMPAR regulatory proteins, cornichon homo-
logues 2 and 3, and the GSG1L protein.4 Each subunit is
composed of an extracellular part with a N-terminal domain
and a ligand-binding domain (LBD), a membrane-spanning
region, and an intracellular C-terminal domain. Besides the
orthosteric site for L-glutamate in the LBD, allosteric sites have
been identified.5
INTRODUCTION
■
L-Glutamate is the main excitatory neurotransmitter in the
central nervous system. After its release into the synaptic cleft,
this amino acid may bind either to ionotropic glutamate
receptors (iGluRs, which are ligand-gated ion channels) or
metabotropic receptors, which are coupled to G-proteins. The
iGluR family is composed of three major classes of receptors
that were differentiated upon their responses to nonendogenic
compounds: N-methyl-D-aspartate receptors, α-amino-3-hy-
droxy-5-methyl-4-isoxazolepropionate receptors (AMPARs),
and kainate receptors.1
AMPARs are homo- or heterotetrameric structures made
from four subunits named GluA1−4, in various combinations.
Subunit composition varies upon development and across
brain regions. Subunit diversity emerges from post-transcrip-
tional and post-translational maturation processes, leading to a
great heterogeneity of function, kinetics, divalent ion
permeability, and conductance properties.2,3 The diversity of
AMPARs is reinforced by their association with various
Modulation of AMPARs appeared as an attractive
therapeutic approach. While development of AMPAR
Received: April 21, 2021
Accepted: June 15, 2021
Published: July 9, 2021
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© 2021 American Chemical Society
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antagonists was achieved to design neuroprotectant drugs,
many efforts were also consented to design AMPAR
potentiators. This strategy, thanks to positive allosteric
modulation, was followed with the idea of developing cognitive
enhancers.6 Surprisingly, positive allosteric modulators were
also found to act as neuroprotectants, in particular through
stimulation of brain-derived neurotrophic factor (BDNF)
release.7,8 This discovery reinforced the interest in AMPARs
and stimulated the interest of AMPAR positive allosteric
modulators (AMPAR PAMs).
Table 1. Effects of 2H-Thiochromene Dioxides and
Thiochroman Dioxides on the Fluorescence Induced by 300
μM AMPA on Primary Cultures of Neurons from Rat
Embryonic Cortex
From the discovery of the potentiator effects on AMPARs of
two 1,2,4-benzothiadiazine 1,1-dioxides (BTDs), namely,
cyclothiazide (1) and IDRA-21 (2; Figure 1), different
a
b
c
Cpd.
R1
R2
EC2x
≥50 (4)
18.0 [4.9; 65.6] (4) 3.1 0.4 (4)
≥25 (5) ≤2.3 (5)
Emax
cLogP
11a
11b
11c
11d
11g
12a
12b
12d
12e
12f
H
F
Cl
H
H
H
H
F
H
H
H
H
H
F
≤2.0 (4)
2.46
2.77
2.99
2.45
2.73
2.59
2.90
2.59
2.17
4.19
2.87
2.08
2.30
1.47
0.73
OCH3
21.1 [7.4; 60.5] (4) 2.5 0.3 (4)
23.4 [7.9; 69.0] (4) 2.9 0.4 (4)
11.9 [4.1; 33.8] (3) 4.8 0.7 (3)
2.2 [0.5; 10.5] (4)
41.6 [8.6; 201.1] (3) 2.2 0.2 (3)
1.2 [0.4; 3.5] (3) 8.8 1.2 (3)
18.2 [4.6; 72.2] (3) 3.3 0.7 (3)
H
H
F
8.5 1.5 (4)
OCH3
OH
4-FC6H4O
12g
13b
13c
417
H
F
Cl
F
OH
9.1 [3.8; 21.4] (4)
>100 (4)
6.4 1.1 (4)
<2.0 (4)
H
H
H
H
>100 (4)
<2.0 (4)
0.26 [0.15;0.47](3)
0.5 [0.3;0.7] (4)
15.0 2.7 (3)
6.2 0.1 (4)
1418
a
EC2x = modulator concentration giving a 2-fold increase of
Figure 1. Selection of 1,2,4-benzothiadiazine 1,1-dioxides acting as
AMPAR PAMs.
fluorescence induced by AMPA (300 μM) expressed in micromolarity
as a geometric mean, and 95% geometric confidence interval (CI95)
low and upper bounds in brackets. Numbers in parentheses refer to
pharmacomodulations were achieved.9 Our group was
particularly involved for two decades in the development of
BTDs as putative AMPAR PAMs. From the pharmacomodu-
lations emerged successively BPAM121 (3)10 and BPAM344
(4)11 and more recently BPAM538 (5).12 Interestingly, this
latter compound was the first example of BTDs able to occupy
two sites of the LBD dimer interface. Finally, a structure-based
approach led to the preparation of dimers 6 and 7. These
compounds are active in vitro as AMPAR PAMs with
nanomolar potency at GluA2, and they were also found to
interact on both sites of the GluA2-LBD dimer interface.13,14
In the present paper, we disclose the results of our studies on
a new series of AMPAR PAMs designed using the classical
isosteric replacement concept.15,16 We have here focused our
efforts on the replacement of the two nitrogen atoms of the
thiadiazine ring by carbon atoms, leading to the preparation of
thiochroman 1,1-dioxides. The idea beyond this approach was
to explore a scaffold closely related to the 3,4-dihydro-2H-
benzothiadiazine 1,1-dioxide core, with a higher expected
lipophilicity (clogP) that could be responsible for a better
distribution into the CNS.
Considering that the best substitution on the nitrogen atom
at the 4 position of BTD-type AMPAR PAMs to date is the
cyclopropyl moiety,11 the present work privileges the synthesis
of 4-cyclopropyl-substituted thiochroman 1,1-dioxides with
selected substituents at the 7 and 8 positions (Table 1) known
to positively impact the activity on AMPARs in accordance
with established structure−activity relationships on BTD-type
modulators.9−12,18
After the preparation of the thiochromans and their in vitro
pharmacological evaluation, a preliminary pharmacokinetic
evaluation was performed to determine the brain/plasma ratio
after oral administration in mice.
b
the number of experiments. Emax = maximal effect normalized to
unity for AMPA-evoked response expressed as arithmetic mean
SEM. Numbers in parentheses refer to the number of experiments.
c
Consensus cLogP obtained using swissadme.ch.20
Moreover, we present the crystallographic structure and
analysis of the cocrystallized thiochroman dioxide 12b with the
flip-like isoform of the ligand-binding domain protein of GluA2
(GluA2-LBD (L504Y/N775S)), forming a preformed dimer
due to the L504Y mutation.19
The interactions of 12b with GluA2-LBD were further
analyzed by the mapping of Hirshfeld surfaces and 2D
fingerprint of the modulator molecule at the dimer interface
of the protein crystal structure. We combined short-contact
descriptors like di, de and dnorm and the shape with surface
properties such as the Shape index and the electrostatic
potential mapped on the Hirshfeld surface to describe the
molecular environment of the ligand in the LBD. Finally, the
Hirshfeld surfaces and the 2D fingerprint of 12b/GluA2-LBD
were compared to those of 4/GluA2-LBD to explore the
relationship between structure and activity of these two
modulators.
RESULTS AND DISCUSSION
■
Synthesis. The synthetic pathway to prepare the target
compounds is illustrated in Scheme 1. The thiochroman series
12 was prepared starting from the commercially available
thiochroman-4-one 8. The cyclopropyl ring was introduced
using cyclopropyl magnesium bromide with Grignard reaction
conditions. The resulting tertiary alcohol 9 was then
dehydrated in diluted sulfuric acid for 2 h to give 10. Then,
oxidation of the sulfur atom was achieved using metachlor-
operbenzoic acid to provide the corresponding thiochromene
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analogues (see 11a, 11b, and 11g). Such observation supports
the view that a subtle modification of the orientation of the
cyclopropyl ring at the 4 position of the thiochroman/
thiochromene ring significantly influences the recognition of
the drug at the level of the AMPAR allosteric binding site.
The presence of a hydroxy group at the 4-position (see 13b
and 13c) was responsible for a complete loss of activity on the
AMPARs.
Scheme 1. Synthetic Pathway to the 7- and 8-Substituted
Thiochroman 1,1-Dioxides
a
Although the introduction of a phenoxy group at the 7
position of BTDs was found to improve the potency on
AMPARs, this is not the case for thiochroman 1,1-dioxides
(see 12f). However, more examples of 7-phenoxy-substituted
thiochroman 1,1-dioxides appropriately substituted on the
phenyl ring have to be examined before establishing a
definitive conclusion.
For representative compounds found in Table 1, it seemed
interesting to estimate druglikeness through the lipophilic
efficiency, which links activity and lipophilicity. In our case, it
was calculated using the following equation: pEC2x-cLogP.
This parameter is around 5.1−5.6 for prototypes 4 and 14,
while their corresponding thiochroman analogues 12b and 12e
display a lipophilic efficiency value of 2.8 and 3.8, respectively.
Although the isosteric replacement envisaged here leads to
compounds with a cLogP expected to be optimal in order to
target the brain, this pharmacomodulation does not lead to an
increase of the lipophilicity efficiency. However, this result has
to be taken with caution as we compare racemates with
nonchiral compounds.
a
Conditions: (i) cyclopropyl bromide, magnesium, I2, THF, 20 min;
(ii) H2SO4 2,5 M, 60 °C, 2 h.; (iii) mCPBA, CH2Cl2, 0 °C; (iv) H2 10
atm, Pd/C, EtOH; (v) BBr3, CH2Cl2; (vi) 4-fluorophenylboronic
acid, Cu(OAc)2, CH2Cl2.
dioxide series 11. The double bond of 11 was saturated by
hydrogenation using palladium on carbon as a catalyst. As
could be expected, two issues were encountered during this
step. The reduction led to the removal of the chlorine atom at
the 7-position of compound 11c. Another issue was the partial
hydrogenolysis of the cyclopropane ring, yielding n-propyl
adducts. From 12d, demethylation of the methoxy group using
boron tribromide led to the 7-hydroxy-substituted compound
12e. Finally, this compound was converted into the para-
fluoro-substituted 7-phenoxythiochroman 1,1-dioxide 12f
using a Chan-Lam coupling reaction. The oxidation of the
sulfur atom was also performed on 9b and 9c, resulting in the
preparation of 13b and 13c.
Improvement of Lipophilicity. The replacement of the
two nitrogen atoms of the thiadiazine ring of BTDs by two
carbon atoms is expected to increase the logP value of the
corresponding thiochromans, reflecting an increase of lip-
ophilicity. In fact, the calculated logP value (consensus
logPo/w20) of the BTD compounds 4 and 14 was found to
be 1.47 and 0.73, respectively, whereas the value for the
corresponding thiochroman dioxides 12b and 12e was 2.90
and 2.17, respectively, corresponding to an increase of 1.4 logP
units.
Biological Evaluation. The newly synthesized compound
series 11, 12, and 13 were evaluated in vitro as AMPAR
potentiators by means of a fluorescence assay, performed on
primary cultures of neurons from rat embryonic cortex,
measuring the effect of the modulators on AMPA-evoked
membrane depolarization. For each compound, the EC2x value
was determined, which corresponds to the concentration of
modulator giving a 2-fold increase of the fluorescence induced
by AMPA (300 μM; Table 1).
Comparing the in vitro activity exhibited by thiochroman
1,1-dioxides and the corresponding previously reported 1,2,4-
benzothiadiazine 1,1-dioxides, 12e and 14 were found to
express a similar positive modulatory activity (EC2x) on
AMPARs (1.2 μM and 0.5 μM, respectively). When comparing
this isosteric switch between 12b and 4, 12b was seen to
possess a weaker potency (2.2 μM) compared to 4 (0.26 μM).
Except in the case of the 7-methoxy-substituted compounds
(see 11d and 12d), the saturated thiochroman 1,1-dioxides
(see 12a, 12b, and 12g) were found to be more potent than
their corresponding unsaturated thiochromene 1,1-dioxide
Considering the in vitro results obtained after screening,
three compounds were selected for in vivo testing in order to
characterize their drug transport across the blood−brain
barrier. After oral administration of 3 mg/kg in mice, the
plasma and brain concentrations were measured and a brain/
plasma ratio was determined. The results are shown in Table 2.
Table 2. In Vivo Evaluation of the Drug Transport Across
the Blood−Brain Barrier
Time after oral
administration
[brain]
(ng/g)
[plasma]
(ng/mL)
ratio brain/
plasma (mL/g)
Cpd
12a
20 min
60 min
20 min
60 min
20 min
15
23
77
18
72
10
21
58
14
81
1.5
1.1
1.3
1.3
0.9
12b
12e
These data suggest that the compounds readily cross the
blood−brain barrier and show an absence of a concentration
effect inside the brain, as the ratio of brain/plasma was found
close to 1. Plasma concentrations obtained 20 min after the
oral administration are higher for 12b and 12e than 12a (58
and 81 ng/mL vs 10 ng/mL, respectively) suggesting that
compounds may have different biopharmaceutical properties in
mice compared to that shown by compound 12a with probably
a better absorption and/or a lower clearance.
X-ray Structure of GluA2 with 12b. Among the 13 new
compounds, the thiochroman 1,1-dioxides 12b and 12e
possess the greatest potencies and EC2x. Compound 12b was
crystallized in complex with GluA2-LBD (L504Y/N775S) and
glutamate to determine the exact binding mode at the dimer
interface between two GluA2 molecules. Compound 12b is a
racemic mixture, and determining the structure should
potentially allow us to address whether only one enantiomer
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of 12b binds to GluA2 or if both enantiomers are capable of
binding to the receptor.
GluA2-LBD (L504Y/N775S) with glutamate and 12b
crystallized with three protein molecules in the asymmetric
unit of the crystal (chains A−C). The structure was
determined at a high-resolution of 1.9 Å (Table 3). Chain B
(chain A);21 Figure 2A). This domain closure is slightly
smaller than that introduced by 4 (20.7° (chain A), 19.5°
(chain B), 20.2° (chain C)).17 This observation might be part
of the explanation for the lower Emax of 12b at GluA2 (8.5)
compared to the Emax of 4 (15.0).
Compound 12b forms one weak hydrogen bond only to
protein residues, i.e. from one of the oxygen atoms in the sulfur
dioxide group of 12b to the hydrogen atom attached to the
backbone nitrogen atom of Gly752 (length 3.2−3.3 Å; angle
∼120°; Figure 2B,C). In contrast, the N−H in the sulfonamide
group of 4 makes a hydrogen bond to the backbone oxygen
atom of Pro515, whereas the contact to Gly752 is longer (3.6
Å; Figure 2C). Thus, the weaker hydrogen-bonding network of
12b compared to 4 most likely contributes to the ∼8 fold
lower positive modulatory activity (EC2x) of 12b. However,
the presence of the S-enantiomer in the racemate might also
contribute to the lower potency of 12b. It is noteworthy that
hydrogen bonding does not seem to be required for the
potency of these BPAMs, whereas steric complementary may
play a major role. On the other hand, the most likely
explanation for the similar positive modulatory activity (EC2x)
of 12e and 14 containing a hydroxyl group in the R1 position is
that this group can form a hydrogen bond to Ser518 as
previously observed for 14.18 The structure also helps explain
the loss of activity on AMPARs when introducing a hydroxy
group at the 4 position (13b and 13c). In the R-form of 13b
and 13c, the 4-hydroxy group will clash with the backbone
carbonyl oxygen of Pro515, whereas in the S-form of 13b and
13c, the 4-hydroxy group will clash with the backbone
carbonyl oxygen of Ser750.
Table 3. Statistics of Data Collection and Refinements
PDB code
6ZYU
crystal data
space group
P21212
unit cell (a, b, c; Å)
molecules in asymmetric unit
114.32, 163.08, 47.45
3
data collection
resolution (Å)
no. unique reflections
completeness (%)
average multiplicity
I/σI
49.09−1.90 (2.00−1.90)
71 004 (10 193)
100 (100)
6.7 (6.9)
6.4 (1.2)
Rmerge (%)
CC1/2
Wilson B factor (Å2)
8.7 (60.3)
1.00 (0.85)
21.2
refinement
R
work/Rfree (%)
16.2/18.7
no. residues (chains A/B/C)
glutamate/12b
263/262/262
3/3
glycerol/cacodylate/acetate/zinc/
chloride/water
6/1/5/8/5/534
average B values (Å2) for:
chains A/B/C
26.7/32.2/35.9
Hirshfeld Surface Analysis. The interactions between the
thiochroman 1,1-dioxide 12b and the GluA2-LBD were
analyzed using Hirshfeld surface (HS) and 2D fingerprint
plots. HSs are well recognized as a powerful tool to highlight
and identify the various contacts between a selected molecule
and the other close neighbors in a crystal.22 Furthermore, a
comparison of interaction surfaces and shapes of target drugs
in experimental crystal structures can serve as interesting tools
for prodrug identification and understanding or prediction of
potency.23 The HS for a given molecule is unique because its
generation is not only based on the structure of the compound
but also on the molecular environment defined by the atomic
electron densities.24
In the present work, the short-contact descriptor dnorm of the
two enantiomers of 12b has been mapped on their HS and
color-coded using a red−white−blue color scheme. The red
color highlights short close contacts. White is used for contacts
around the van der Waals separation, and blue is for longer
contacts. In order to get a precise view of the HS, we
considered all the residues surrounding the modulator at a
distance of 8 Å (Figures S2−S4). Even if some residues and
water molecules do not show direct contact with the
modulator, they were still considered to respect at best the
molecular environment and thereby obtain the most precise
HS for the analysis.
glutamate/12b
glycerol/cacodylate/acetate/zinc/
chloride/water
RMSD bond length (Å)/angles (deg)
Ramachandran outliers/favored (%)
22.6/24.7
72.4/26.2/49.6/39.5/56.9/32.3
0.008/0.87
0/99.1
and chain C form a dimer (Figure 2A), whereas chain A forms
a dimer with a symmetry-related chain A. Well-defined
electron density was observed for glutamate and 12b (Figure
S1), allowing these molecules to be unambiguously modeled
into the structure. Glutamate was bound at the orthosteric
binding site in all three chains and formed the same contacts to
protein residues as previously reported.19 Two molecules of
12b were located at each dimer interface, forming similar
contacts to protein residues (Figure 2A,B). Interestingly, the
electron densities for 12b clearly suggested that both
enantiomers of 12b can bind to GluA2-LBD (L504Y/
N775S; Figure 2B, Figure S1). The occupancy of the R-
enantiomer of 12b was refined to 63% (chain A), 82% (chain
B), and 62% (chain C) and for the S-enantiomer to 37%
(chain A), 18% (chain B), and 38% (chain C). This shows that
both enantiomers of 12b are likely to bind to GluA2, but that
the R-enantiomer is preferred. R-12b adopts both a similar
conformation and binding mode to that of 4 (PDB code
4N0717), positioning the cyclopropyl group in 12b and 4 in
the same manner (Figure 2C). This observation is in line with
a higher occupancy of the R-12b being observed in the
structure.
The HS mapped over dnorm of 12b was first analyzed.
Various GluA2 residues and water molecules were found in
close interactions with the modulator. Although some water
molecules interact with the modulator, these polar, nonpolar,
and weak contacts are not hydrogen bonds, confirming the
crystallographic analysis. The results also highlight that the
residues Pro515, Phe516, and Ser518 form weak hydrogen
bonds with R-12b (Figure 3). Indeed, the Pro515 carbonyl
When glutamate binds to the GluA2-LBD, the conformation
of the clamshell-like structure changes. In the presence of 12b,
glutamate introduces a lobe D1−D2 closure of 19.6° (chain
A), 18.5° (chain B), and 18.9° (chain C), relative to the
unbound (apo) structure of GluA2-LBD (PDB code 1FTO
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Figure 2. X-ray structure of GluA2-LBD (L504Y/N775S) in complex with glutamate and 12b. (A) Cartoon representation of the GluA2 dimer
(chain B: brown/chain C: light brown). A side view is shown to the left and a bottom view to the right. Glutamate (cyan carbon atoms) and 12b
(R-enantiomer, green carbon atoms; S-enantiomer, magenta carbon atoms) are shown in sticks representation. Fluorine atoms are light blue,
nitrogen atoms, blue; oxygen atoms, red; and sulfur atoms, yellow. The lobe D1−D2 closure is indicated by an arrow. (B) Zoom on the modulator
binding site at the dimer interface (chain B/chain C). Potential hydrogen bonds are shown as red dotted lines (within 3.5 Å). (C) Superimposition
of the structures of GluA2 with 12b and 4 (gray carbon atoms). Zoom on the modulator binding site. Potential hydrogen bonds are shown as red
dotted lines for 12b and black dotted lines for 4 (within 3.5 Å).
oxygen atom makes a polar contact with the hydrogen atom at
the 4 position on the thiochroman heterocycle. Furthermore,
the same residue participates in a π-stacking interaction with
the aromatic ring of 12b through the Cα hydrogen atom in the
Pro515 backbone (Figure 3B). As shown in Figure 3A, the
white regions on the HS around the Ser750 and Lys751
backbone reveal an interaction at the van der Waals distance,
suggesting a π−π stacking contact. One oxygen atom of the
sulfone group of 12b is engaged in weak van der Waals
interactions with Lys751 and Gly752 (Figure S5). The
interaction with Lys751 was previously described by Nørholm
and co-workers17 in the case of 4 crystallized in complex with
GluA2-LBD.
Furthermore, as observed in the structure of GluA2-LBD
with 4, the π character of the cyclopropyl moiety of 12b allows
formation of short contacts with the Phe516 backbone atoms.
The deep red spot observed on the HS for such interaction
reveals a distance lower than the van der Waals radii. In
addition, water molecules located close to the cyclopropyl
group are also engaged in short interaction contacts. Ser518
contact to R-12b is demonstrated by a red spot around the
fluorine atom on the surface mapped over dnorm. All of these
interactions depicted herein are observed for both enantiomers
of 12b (Figure S3). On the contrary, the H−F hydrogen bond
of 12b seems to be stronger than that of 4 in GluA2-LBD as
Ser518 is located almost at the van der Waals distance.
The maps of di, de, and the shape index on molecular HS of
R-12b and S-12b are shown in Figure 4 and compared to those
of 4. To allow better visualization and description, neighboring
molecules surrounding the inhibitors are removed for clarity.
The different red spots on the surface mapped over di reveal
that various atoms of the two thiochroman 1,1-dioxides are
close to the surface contrary to those of the benzothiadiazine
1,1-dioxide 4. Although the shape of the HS is very similar for
the two enantiomers of 12b and that of 4, a slight difference is
observed due to the close contact near the fluorine atom,
allowing surface deformation leading to a convex region
(Figure 4). In addition, the red spot located on the HS around
the fluorine atom of 12b confirms a very short contact. For
both modulators, the π-stacking interaction with the hydrogen
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Figure 3. Hirshfeld surface of R-12b mapped over dnorm and viewed along two different axes (A and B), allowing the best observation of the
contacts. The water molecules and the residues surrounding 12b are shown with the most interacting residues being labeled.
atom of the Pro515 backbone was confirmed and appears as a
red colored convex domain on the HS mapped over the shape
index. This contact as well as those with the cyclopropyl group
of 12b are highlighted and surrounded by black circles (Figure
4). All of these similarities between the enantiomers of the
thiochroman 1,1-dioxide 12b and the benzothiadiazine 1,1-
dioxide 4 are probably at the origin of a similar
pharmacological profile.
The volume of the HS of each monomer was calculated and
compared to that generated for the dimer. The results give
almost identical values. The total surface volume obtained for
4 is slightly lower compared to that of R- and S-forms of 12b
with a value of 528.5 (4), 560.8 (R-12b), and 559.8 (S-12b)
Å3. In addition, the total surface area follows the same
tendency with a value of 467.3 (4), 483.4 (R-12b), and 479.9
(S-12b) Å2, respectively.
Spackman and co-workers showed that coupling the shape
of the molecule given by HS with surface properties like
electrostatic potential (ESP) mapped on the HS is a good way
to fully describe the molecular environment.25 Since the
electrostatic forces are long-range interactions, the ESP plays
an important role in molecular recognition by the receptor.26
The ESP surface mapped on the HS for 12b was calculated
and compared to that of 4. The electron rich groups with the
acceptor property (red color) are mainly located around the
sulfone oxygen atoms and the fluorine atoms (Figure 5). On
the contrary, the donor group’s domains (blue color) are not
well-defined on the surface of the two enantiomers of 12b.
Nevertheless, a low donor property was observed near
hydrogen atoms around both the cyclopropyl moiety and the
hydrogen atoms at the 4 and 6 positions on 12b. The large
white surface reveals no particular electrostatic property of
other regions of the modulator. Unlike 12b, the existence of
donor groups is well-defined as blue spots on 4 mapped with
ESP. In addition to the cyclopropyl group, the donor property
of the N−H hydrogen in 4 is clearly visible on the surface
(blue spot on the a axis), revealing a strong interaction (Figure
5). This observation confirms the presence of a hydrogen bond
with the carbonyl oxygen atom of Pro515.
2D histogram, named a fingerprint plot of the molecule. The
fingerprint plot for a selected molecule is unique due to the
fact that it is highly sensitive to the immediate environ-
ment.27,28 For the HS constructed for 12b embedded in the
GluA2-LBD crystal structure, the 2D fingerprint histogram
characterizes very well the interactions of 12b with the binding
site residues.
Since two molecules of the positive allosteric modulators are
present at the dimer interface of GluA2-LBD, the fingerprint of
each molecule was first constructed and compared. Although
the binding mode of the two enantiomers of 12b or those of 4
in the GluA2 pocket seems to be similar, the fingerprint
analysis reveals slight differences (Figure 6). However, these
differences remain trivial and are the consequence of the
immediate environment dissimilarity. Therefore, the finger-
print of the dimer was obtained as a global interaction contact
of the modulator (Figure 6, bottom). Analysis of these 2D
histograms reveals close resemblance of the two enantiomers
of 12b, but a notable dissimilarity with that of 4 is observed.
This result suggests that the modulator efficiency depends on
the global intermolecular contacts of the modulators and the
GluA2-LBD.
As the fingerprint plot indicates the contributions of
interatomic contacts, the decomposition of the fingerprint
pairwise component is a good way to identify the contacts
which have the most weight and to determine those which
could be at the source of the difference and therefore at the
origin of activity variation. The fingerprint obtained for 12b is
considered as a reference and its similarity with that of 4
bound to GluA2 could give an idea of the contacts, which are
at the origin of the activity.
For both 12b enantiomers and for 4, the fingerprint is
dominated by four contacts that contribute to 95.2%, 95.1%,
and 92.5% of the total fingerprint of R-12b, S-12b, and 4,
respectively (Figure 7). These dominant interactions corre-
spond to intermolecular H···H (Figure 7, first line), O···H/H···
O (Figure 7, second line), H···C/C···H (Figure 7, third line),
and F···H contacts (Figure 7, fourth line) that show the same
range values. The histogram of these contacts (Figure 7)
presents almost the same shape for the 12b enantiomers. On
the contrary, the decomposed fingerprint corresponding to
Distances from the HS to the nearest nucleus inside the
surface (di) and outside the surface (de) are used to create a
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Figure 4. Hirshfeld surface mapped over di (in the range −0.807 to 2.586 Å), de (in the range −0.821 to 2.526 Å), dnorm (in the range −0.300 to
1.000), and shape index (in the range −1.000 to 1.000). The surfaces correspond to compounds R-12b (A), S-12b (B), and 4 (C) in GluA2-LBD.
H···H and F···H contacts of 4 is different. The F···H contact
plot dissimilarity is in accordance with the contact depicted
earlier, demonstrating that in the case of 12b the fluorine atom
is closer to Ser518 but interacts also with a water molecule.
Contrary to 12b, the presence of the N−H bond in the
thiadiazine ring of 4 increases the N···H/H···N contacts that
contribute to 4% of the total fingerprint (Figures S7−S9).
Finally, both HSs and fingerprint plots seem a good way to
analyze and compare the intermolecular interactions of 12b
with that of 4 in the GluA2 macromolecular crystal structure.
These analyses suggest that the enhancement of ligand volume
occupancy in the LBD, the close contact of the ligands with
water molecules, and the absence of a N−H group forming
hydrogen bond with the carbonyl oxygen atom of Pro515
could be at the origin of the lower activity of thiochroman
derivatives.
CONCLUSIONS
■
The present study aims to assess the impact on biological
activity (AMPAR potentiation) of the replacement of the two
nitrogen atoms of the heterocyclic ring of benzothiadiazine 1,1-
dioxides by carbon atoms, leading to the corresponding 2H-
thiochromene 1,1-dioxide, and thiochroman 1,1-dioxide
analogues.
It was observed that some thiochroman 1,1-dioxides of
general formula 12 expressed in vitro a strong activity as
AMPAR PAMs, although usually lower than the corresponding
3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. It must be
noted, however, that the thiochroman 1,1-dioxides 12 are
racemates supporting the view that one of the two enantiomers
is expected to express a higher activity. As a perspective,
resolution of the racemic mixture is planned in the future in
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using the CE-B3LYP/6-31G(d,p) level of theory and mapped
on the Hirshfeld surfaces for 12b and compared to that of 4.
The electron rich groups are mainly located around the sulfone
oxygen atoms and the fluorine atom, and a low donor property
was observed near hydrogen atoms both around the cyclo-
propyl moiety and the hydrogen atoms at the 4 and 6 positions
on 12b.
Finally, the analysis of the fingerprint of the modulators
shows that both 12b and 4 interactions are dominated by H···
H, O···H/H···O, H···C/C···H, and F···H contacts that
contribute to 95.2%, 95.1%, and 92.5% of the total fingerprint
of R-12b, S-12b, and 4, respectively.
On the basis of this modeling approach, it is concluded that
enhancing ligand volume occupancy in the LBD, close contact
of the ligands with water molecules, and the absence of an N−
H group forming a hydrogen bond with the carbonyl oxygen
atom of Pro515 could be at the origin of the lower activity of
thiochroman 1,1-dioxides compared to their corresponding
3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides.
EXPERIMENTAL SECTION
■
Synthesis. General Procedures. Melting points were determined
1
on a Bu
̈
chi Tottoli capillary apparatus and are uncorrected. The H
and 13C NMR spectra were recorded on a Bruker Advance (500
MHz) instrument using deuterated dimethyl sulfoxide (d6-DMSO) as
the solvent with tetramethylsilane (TMS) as an internal standard;
chemical shifts are reported in δ values (ppm) relative to that of
internal TMS. The abbreviations s = singlet, d = doublet, t = triplet, q
= quadruplet, m = multiplet, and bs = broad singlet are used
throughout. Elemental analyses (C, H, N, S) were realized on a
Thermo Scientific Flash EA 1112 elemental analyzer and were within
0.4% of the theoretical values for carbon, hydrogen, and nitrogen; a
higher tolerance ( 0.6%) was admitted for sulfur, considering its
corresponding peak shape. This analytical method certified a purity of
≥95% for each tested compound. All reactions were routinely checked
by TLC on silica gel Merck 60 F254.
Figure 5. Hirshfeld surface mapped over ESP in the range −0.807 to
0.800 atomic units of R-12b, S-12b, and 4 in GluA2-LBD viewed
along the three crystallographic axes.
4-Cyclopropyl-4-hydroxythiochroman (9a). One milliliter of
cyclopropyl bromide (2.94 g, 24.3 mmol) in dry ether (10 mL) was
added dropwise to a suspension of magnesium chips (590 mg, 24.3
mmol) in dry ether (25 mL). The mixture was heated under stirring
until Grignard formation began. The remaining solution of cyclo-
propyl bromide was then added. After 10 min of stirring, the mixture
was cooled on an ice bath, and a solution of thiochroman-4-one (8a, 2
g, 12.2 mmol) was added dropwise. The reaction was stirred at 0 °C
for 20 min and then quenched using an aqueous solution of
ammonium chloride (5 mL, 10% w/v). Ether (20 mL) and water (20
mL) were added. The organic fraction was dried over magnesium
sulfate and concentrated under a vacuum. The residue was purified
using flash chromatography (dichloromethane/hexane 1:1) to afford a
clear oil and used in the next step without further characterization.
4-Cyclopropyl-7-fluoro-4-hydroxythiochroman (9b). The title
compound was obtained as described for 9a starting from 7-
fluorothiochroman-4-one (8b) and used in the next step without
further characterization.
4-Cyclopropyl-7-chloro-4-hydroxythiochroman (9c). The title
compound was obtained as described for 9a starting from 7-
chlorothiochroman-4-one (8c) and used in the next step without
further characterization.
4-Cyclopropyl-7-methoxy-4-hydroxythiochroman (9d). The title
compound was obtained as described for 9a starting from 7-
methoxythiochroman-4-one (8d) and used in the next step without
further characterization.
order to investigate the in vitro activity of the separated
enantiomers.
The in vivo transport across the blood−brain barrier of a
selection of compounds, i.e., 12a, 12b, and 12e was explored.
The plasma concentrations obtained 20 min after the oral
administration demonstrated that the three compounds
crossed the blood−brain barrier (conc. brain/conc. plasma
ratio near 1) and that compounds 12b and 12e were absorbed
more effectively from the gastrointestinal tract or less
metabolized than 12a.
The AMPAR potentiator 12b, which is one of the most
potent thiochroman-type AMPAR PAMs and which is the
analogue of the BTD reference compound BPAM344 (4), was
selected for further structural characterization and was
subjected to structural analysis by X-ray crystallography.
Interestingly, the electron density analysis for 12b in complex
with GluA2 (GluA2-LBD (L504Y/N775S)) clearly suggested
that both enantiomers of 12b can bind to the receptor.
However, R-12b seemed to be the preferred enantiomer of the
receptor.
The interactions of 12b with GluA2 were further analyzed
by mapping Hirshfeld surfaces and the 2D fingerprint of the
modulator at the dimer interface of the protein crystal
structure and compared to those of 4 in complex with
GluA2. The Hirshfeld surface mapped over dnorm highlights
that the residues Pro515, Phe516, and Ser518 form weak
hydrogen bonds with 12b. The ESP surface was calculated
4-Cyclopropyl-8-fluoro-4-hydroxythiochroman (9g). The title
compound was obtained as described for 9a starting from 8-
fluorothiochroman-4-one (8g) and used in the next step without
further characterization..
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Figure 6. Comparative Hirshfeld fingerprint plot of R-12b, S-12b, and 4 in GluA2-LBD (chain B and chain C dimer). The isolated fingerprint of
each monomer is presented in front of the molecule, and the total fingerprint is shown under the considered dimers.
4-Cyclopropyl-2H-thiochromene 1,1-Dioxide (11a). To sulfuric
acid 2.5 M (25 mL) was added 4-cyclopropyl-4-hydroxythiochroman
(9a, 1.3 g, 6 mmol). The mixture was heated at 60 °C for 90 min.
After reaching room temperature, the mixture was extracted with
dichloromethane (3 × 20 mL). The combined organic layers were
dried over magnesium sulfate, treated using activated charcoal, and
then concentrated. To the resulting oil in dichloromethane (25 mL)
was added metachloroperbenzoic acid (2 g, 11.5 mmol). The mixture
was stirred at 0 °C for 20 min. Water (50 mL) was added to the
medium, and the aqueous phase was basified up to a pH of 10 using
aqueous NaOH solution (10% w/v). The organic layer was collected,
and the aqueous layer was extracted with dichloromethane (2 × 20
mL). The combined organic layers were dried, treated using activated
charcoal, and then concentrated under a vacuum. The residue was
dispersed in hexane, heated for 5 min, and cooled. The resulting white
solid was collected by filtration. Yield = 35% over two steps. mp:
1
109−111 °C. H NMR (DMSO-d6): δ 0.57 (m, 2H, CH(CH2)2),
0.90 (m, 2H, CH(CH2)2), 1.84 (m, 1H, CH(CH2)2), 4.14 (dd, J = 5.2
Hz/1.6 Hz, 2H, 2−CH2), 6.02 (td, J = 5.2 Hz/1.4 Hz, 1H, 3-H), 7.61
(td, J = 7.6 Hz/1.0 Hz, 1H, 6-H), 7.79 (td, J = 7.7 Hz/1.3 Hz, 1H, 7-
H), 7.89 (dd, J = 7.7 Hz/1.2 Hz, 1H, 5-H), 8.00 (d, J = 7.6 Hz, 1H, 8-
H). 13C NMR (DMSO-d6): δ 5.7 (CH(CH2)2), 13.9 (CH(CH2)2),
48.4 (C-2), 117.4 (C-3), 122.3 (C-8), 126.4 (C-5), 128.9 (C-7),
133.3 (C-6), 135.3 (C-4a), 136.8 (C-8a). Anal. (C12H12O2S)
theoretical: C, 65.43; H, 5.49; S, 14.55. Found: C, 65.09; H, 5.46;
S, 14.61.
4-Cyclopropyl-7-fluoro-2H-thiochromene 1,1-Dioxide (11b). The
title compound was obtained as described for 11a starting from 4-
cyclopropyl-7-fluoro-4-hydroxythiochroman (9b). Yield = 72%. mp:
1
138−140 °C. H NMR (DMSO-d6): δ 0.56 (m, 2H, CH(CH2)2),
Figure 7. Comparative decomposed Hirshfeld fingerprint plot of
dominant interactions of R-12b, S-12b, and 4 in GluA2-LBD.
0.89 (m, 2H, CH(CH2)2), 1.83 (m, 1H, CH(CH2)2), 4.2 (d, J = 5.1
Hz, 2H, 2-CH2), 6.00 (t, J = 5.0 Hz, 1H, 3-H), 7.66 (td, J = 8.7 Hz/
2.2 Hz, 1H, 6-H), 7.71 (dd, J = 7.7 Hz/2.2 Hz, 1H, 8-H), 8.06 (dd, J
= 8.7 Hz/5.1 Hz, 1H, 5-H). 13C NMR (DMSO-d6): δ 5.8
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(CH(CH2)2), 14.0 (CH(CH2)2), 48.3 (C-2), 109.7 (d, J = 25 Hz, C-
8), 116.8 (C-3), 120.3 (d, J = 21 Hz, C-6), 129.4 (d, J = 8 Hz, C-5),
132.1 (d, J = 4 Hz, C-4a), 136.3 (C-4), 137.2 (d, J = 7 Hz, C-8a),
160.4−161.9 (d, J = 251 Hz, C-7). Anal. (C12H11FO2S) theoretical:
C, 60.49; H, 4.65; S, 13.46. Found: C, 60.34; H, 4.81; S, 12.91.
7-Chloro-4-cyclopropyl-2H-thiochromene 1,1-Dioxide (11c). The
title compound was obtained as described for 11a starting from 7-
chloro-4-cyclopropyl-4-hydroxythiochroman (9c). Yield = 68%. mp:
(dd, J = 8.8 Hz/5.3 Hz, 1H, 5-H). 13C NMR (DMSO-d6): δ 3.0
(CH(CH2)2), 6.3 (CH(CH2)2), 16.8 (CH(CH2)2), 27.0 (C-3), 40.5
(C-4), 48.0 (C-2), 109.4 (d, J = 24 Hz, C-8), 119.8 (d, J = 21 Hz, C-
6), 131.7 (d, J = 7 Hz, C-5), 136.6 (d, J = 3 Hz, C-4a), 139.6 (d, J = 6
Hz, C-8a), 160.0−161.4 (d, J = 248 Hz, C-7). Anal. (C12H13FO2S)
theoretical: C, 59.98; H, 5.45; S, 13.34. Found: C, 59.86; H, 5.52; S,
13.44.
4-Cyclopropyl-7-methoxythiochroman 1,1-Dioxide (12d). The
title compound was obtained as described for 12a starting from 4-
cyclopropyl-7-methoxy-2H-thiochromene 1,1-dioxide (9d). Yield =
1
121−123 °C. H NMR (DMSO-d6): δ 0.56 (m, 2H, CH(CH2)2),
0.89 (m, 2H, CH(CH2)2), 1.83 (m, 1H, CH(CH2)2), 4.22 (dd, J = 5.2
Hz/1.3 Hz, 2H, 2-CH2), 6.11 (td, J = 5.2 Hz/1.2 Hz, 1H, 3-H), 7.87
(m, 2H, 6-H/8-H), 8.01 (d, J = 8.3 Hz, 1H, 5-H). 13C NMR (DMSO-
d6): δ 5.8 (CH(CH2)2), 13.8 (CH(CH2)2), 48.4 (C-2), 118.0 (C-3),
122.1 (C-8), 128.7 (C-5), 133.2 (C-6), 134.3 (C-4/C-4a), 136.3 (C-
7), 136.9 (C-8a). Anal. (C12H11ClO2S) theoretical: C, 56.58; H, 4.35;
S, 12.59. Found: C, 56.53; H, 4.48; S, 12.85.
1
21%. mp: 134.5−136.5 °C. H NMR (DMSO-d6): δ 0.26 (m, 1H,
CH(CH2)2), 0.49 (m, 1H, CH(CH2)2), 0.55 (m, 1H, CH(CH2)2),
0.68 (m, 1H, CH(CH2)2), 0.97 (m, 1H, CH(CH2)2), 2.23 (m, 2H, 4-
H/3-Ha), 2.45 (m, 1H, 3-Hb), 3.47 (m, 1H, 2-Ha), 3.62 (m, 1H, 2-
Hb), 3.81 (s, 3H, OCH3), 7.19 (m, 2H, 6-H/8-H), 7.64 (d, J = 8.1 Hz,
1H, 5-H). 13C NMR (DMSO-d6): δ 2.8 (CH(CH2)2), 6.2
(CH(CH2)2), 16.9 (CH(CH2)2), 27.2 (C-3), 40.3 (C-4), 48.1 (C-
2), 55.6 (OCH3), 106.2 (C-8), 119.3 (C-6), 130.5 (C-5), 132.1 (C-
4a), 138.8 (C-8a), 158.2 (C-7). Anal. (C13H16O3S) theoretical: C,
61.88; H, 6.39; S, 12.71. Found: C, 61.51; H, 6.39; S, 12.58.
4-Cyclopropyl-7-methoxy-2H-thiochromene 1,1-Dioxide (11d).
The title compound was obtained as described for 11a starting from
4-cyclopropyl-7-methoxy-4-hydroxythiochroman (9d). Yield = 71%.
mp: 140−142 °C. 1H NMR (DMSO-d6): δ 0.54 (m, 2H, CH(CH2)2),
0.87 (m, 2H, CH(CH2)2), 1.80 (m, 1H, CH(CH2)2), 3.88 (s, 3H,
OCH3), 4.10 (dd, J = 5.2 Hz/1.5 Hz, 2H, 2-CH2), 5.87 (td, J = 5.1
Hz/1.3 Hz, 1H, 3-H), 7.33 (m, 2H, 6-H/8-H), 7.93 (d, J = 8.5 Hz,
1H, 5-H). 13C NMR (DMSO-d6): δ 5.7 (CH(CH2)2), 13.9
(CH(CH2)2), 48.5 (C-2), 55.9 (OCH3), 106.8 (C-8), 114.6 (C-3),
119.0 (C-6), 127.9 (C-4a), 128.4 (C-5), 136.6 (C-4/C-8a), 159.1 (C-
7). Anal. (C13H14O3S) theoretical: C, 62.38; H, 5.64; S, 12.81. Found:
C, 62.10; H, 5.65; S, 12.57.
4-Cyclopropyl-7-hydroxythiochroman 1,1-Dioxide (12e). To a
solution of 4-cyclopropyl-7-methoxythiochroman 1,1-dioxide (12d,
600 mg, 2.4 mmol) in dichloromethane (60 mL) at −30 °C was
added boron tribromide (2.2 mL), and the reaction was warmed to
room temperature. After 2 h of stirring at room temperature, the
mixture was cooled on an ice bath, and water (25 mL) was added to
the medium. After evaporation of dichloromethane under a vacuum,
extraction was performed using ethyl acetate. The combined organic
layers were dried under magnesium sulfate and evaporated to dryness.
The residue was dissolved in a minimum of methanol, and water was
added at 0 °C, resulting in a solid that was collected by filtration. Yield
4-Cyclopropyl-8-fluoro-2H-thiochromene 1,1-Dioxide (11g). The
title compound was obtained as described for 11a starting from 4-
cyclopropyl-8-fluoro-4-hydroxythiochroman (9g). Yield = 19%. mp:
1
111−113 °C. H NMR (DMSO-d6): δ 0.56 (m, 2H, CH(CH2)2),
1
= 89%. mp: 182−184 °C. H NMR (DMSO-d6): δ 0.23 (m, 1H,
0.89 (m, 2H, CH(CH2)2), 1.79 (m, 1H, CH(CH2)2), 4.25 (dd, J = 5.2
Hz/1.4 Hz, 2H, 2-CH2), 6.05 (td, J = 5.2 Hz/1.2 Hz, 1H, 3-H), 7.45
(t, J = 9 Hz, 1H, 6-H), 7.80 (m, 1H, 7-H), 7.84 (dd, J = 7.9 Hz/1.0
Hz, 1H, 5-H). 13C NMR (DMSO-d6): δ 5.9 (CH(CH2)2), 14.5
(CH(CH2)2), 50.1 (C-2), 117.0 (d, J = 21 Hz, C-7), 118.2 (C-3),
122.8 (d, J = 3 Hz, C-5), 123.1 (d, J = 12 Hz, C-8a), 134.9 (d, J = 9
Hz, C-6), 136.5 (d, J = 3 Hz, C-4a), 137.7 (C-4), 157.2−158.7 (d, J =
255 Hz, C-8). Anal. (C12H11FO2S) theoretical: C, 60.49; H, 4.65; S,
13.46. Found: C, 60.36; H, 4.77; S, 12.88.
CH(CH2)2), 0.47 (m, 1H, CH(CH2)2), 0.51 (m, 1H, CH(CH2)2),
0.68 (m, 1H, CH(CH2)2), 0.94 (m, 1H, CH(CH2)2), 2.14 (m, 1H, 4-
H), 2.22 (m, 1H, 3-Ha), 2.42 (m, 1H, 3-Hb), 3.42 (m, 1H, 2-Ha),
3.58 (m, 1H, 2-Hb), 6.98 (dd, J = 8.6 Hz/2.6 Hz, 1H, 6-H), 7.09 (d, J
= 2.6 Hz, 1H, 8-H), 7.53 (d, J = 8.6 Hz, 1H, 5-H), 10.05 (bs, 1H,
OH). 13C NMR (DMSO-d6): δ 2.7 (CH(CH2)2), 6.2 (CH(CH2)2),
16.9 (CH(CH2)2), 27.3 (C-3), 40.3 (C-4), 48.2 (C-2), 108.1 (C-8),
120.1 (C-6), 130.2 (C-4a), 130.4 (C-5), 138.6 (C-8a), 156.5 (C-7).
Anal. (C12H14O3S) theoretical: C, 60.48; H, 5.92; S, 13.46. Found: C,
60.49; H, 5.92; S, 13.32.
4-Cyclopropylthiochroman 1,1-Dioxide (12a). To a solution of 4-
cyclopropyl-2H-thiochromene 1,1-dioxide (11a, 400 mg, 1.8 mmol)
in ethanol (30 mL) was added 10% palladium on carbon (100 mg).
The mixture was placed in a hydrogenator under H2 (10 atm) for 1 h
at room temperature. The insoluble material was removed by
filtration, and the filtrate was concentrated to dryness under a
vacuum. The residue was taken up in hot dichloromethane (3 mL).
The addition of hexane gave a solid which was collected by hot
filtration and washed with hexane. The solid was purified by flash
chromatography using hexane/ethyl acetate 17:3. Yield = 28%. mp:
4-Cyclopropyl-7-(4-fluorophenoxy)thiochroman 1,1-Dioxide
(12f). To a solution of 4-cyclopropyl-7-hydroxythiochroman 1,1-
dioxide (12e, 400 mg, 1.7 mmol) in dichloromethane (60 mL) was
added 10 drops of pyridine, molecular sieves of 4 Å (5 g), 4-
fluorophenylboronic acid (345 mg, 2.5 mmol), and copper acetate
(450 mg, 2.5 mmol). The mixture was stirred for 5 h at 40 °C. Then,
dichloromethane was added (40 mL), and the solid was removed by
filtration. After evaporation of the solution to dryness under a
vacuum, acidic water (50 mL) was added, and the solution was
extracted by means of ethyl acetate (3 × 50 mL). The combined
organic layers were dried under magnesium sulfate and evaporated to
dryness under a vacuum. The resulting residue was then purified using
flash chromatography (dichloromethane/petroleum ether 5:15) to
afford a white solid. Yield = 23%. mp: 98−100 °C. 1H NMR (DMSO-
d6): δ 0.28 (m, 1H, CH(CH2)2), 0.51 (m, 1H, CH(CH2)2), 0.57 (m,
1H, CH(CH2)2), 0.70 (m, 1H, CH(CH2)2), 1.01 (m, 1H,
CH(CH2)2), 2.26 (m, 1H, 4-H/3−CH2), 2.46 (m, 1H, 3-CH2),
3.49 (m, 1H, 2−CH2), 3.64 (m, 1H, 2−CH2), 7.14 (d, J = 2.7 Hz, 1H,
8-H), 7.17 (m, 2H, 2′-H/6′-H), 7.27 (dd, J = 8.7 Hz/2.7 Hz, 1H, 6-
H), 7.30 (m, 2H, 3′-H/5′-H), 7.74 (d, J = 8.7 Hz, 1H, 5-H). 13C
NMR (DMSO-d6): δ 3.0 (CH(CH2)2), 6.3 (CH(CH2)2), 16.8
(CH(CH2)2), 27.1 (C-3), 40.5 (C-4), 48.0 (C-2), 110.2 (C-8), 117.0
(d, J = 23 Hz, C-3′/C-5′), 121.7 (d, J = 9 Hz, C-2′/C-6′), 122.0 (C-
6), 131.2 (C-5), 134.7 (C-4a), 139.2 (C-8a), 151.4 (C-1′), 156.6 (C-
7), 158.1−159.5 (d, J = 241 Hz, C-4′). Anal. (C18H17FO3S)
theoretical: C, 65.04; H, 5.16; S, 9.65. Found: C, 65.11; H, 5.19; S,
9.30.
1
149−150 °C. H NMR (DMSO-d6): δ 0.29 (m, 1H, CH(CH2)2),
0.52 (m, 1H, CH(CH2)2), 0.58 (m, 1H, CH(CH2)2), 0.70 (m, 1H,
CH(CH2)2), 1.02 (m, 1H, CH(CH2)2), 2.27 (m, 2H, 4-H/3-Ha),
2.49 (m, 1H, 3-Hb), 3.49 (m, 1H, 2-Ha), 3.64 (m, 1H, 2-Hb), 7.50
(td, J = 7.6 Hz/0.8 Hz, 1H, 7-H), 7.60 (dt, J = 7.7 Hz/1.4 Hz, 1H, 6-
H), 7.72 (d, J = 7.9 Hz, 1H, 5-H), 7.78 (dd, J = 7.9 Hz/1.2 Hz, 1H, 8-
H). 13C NMR (DMSO-d6): δ 3.0 (CH(CH2)2), 6.3 (CH(CH2)2),
16.8 (CH(CH2)2), 27.1 (C-3), 41.0 (C-4), 48.2 (C-2), 114.2 (C-8),
116.7 (C-5), 122.9 (C-8), 127.7 (C-7), 128.9 (C-5), 132.3 (C-6),
138.2 (C-8a), 140.2 (C-4a). Anal. (C12H14O2S) theoretical: C, 64.83;
H, 6.35; S, 14.42. Found: C, 64.44; H, 6.29; S, 13.93.
4-Cyclopropyl-7-fluorothiochroman 1,1-Dioxide (12b). The title
compound was obtained as described for 12a starting from 4-
cyclopropyl-7-fluoro-2H-thiochromene 1,1-dioxide (9b). Yield = 24%.
mp: 159−161 °C. 1H NMR (DMSO-d6): δ 0.29 (m, 1H, CH(CH2)2),
0.52 (m, 1H, CH(CH2)2), 0.59 (m, 1H, CH(CH2)2), 0.69 (m, 1H,
CH(CH2)2), 1.02 (m, 1H, CH(CH2)2), 2.27 (m, 2H, 3−CH2), 2.49
(m, 1H, 4-H), 3.53 (m, 1H, 2-Ha), 3.68 (m, 1H, 2-Hb), 7.49 (td, J =
8.6 Hz/2.8 Hz, 1H, 6-H), 7.59 (dd, J = 8.2 Hz/2.8 Hz, 1H, 8-H), 7.79
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4-Cyclopropyl-8-fluorothiochroman 1,1-Dioxide (12g). The title
compound was obtained as described for 12a starting from 4-
cyclopropyl-8-fluoro-2H-thiochromene 1,1-dioxide (9g). Yield = 48%.
mp: 182−183.5 °C. 1H NMR (DMSO-d6): δ 0.29 (m, 1H,
CH(CH2)2), 0.52 (m, 1H, CH(CH2)2), 0.58 (m, 1H, CH(CH2)2),
0.70 (m, 1H, CH(CH2)2), 1.03 (m, 1H, CH(CH2)2), 2.26 (m, 2H, 4-
H/3-Ha), 2.42 (m, 1H, 3-Hb), 3.55 (m, 1H, 2-Ha), 3.73 (m, 1H, 2-
Hb), 7.34 (t, J = 9.5 Hz, 1H, 6-H), 7.55 (d, J = 7.9 Hz, 1H, 5-H), 7.62
(m, 1H, 7-H). 13C NMR (DMSO-d6): δ 3.1 (CH(CH2)2), 6.4
(CH(CH2)2), 16.6 (CH(CH2)2), 26.8 (C-3), 41.2 (C-4), 50.4 (C-2),
115.2 (d, J = 20 Hz, C-7), 125.1 (d, J = 3 Hz, C-5), 126.6 (d, J = 13
Hz, C-8a), 133.6 (d, J = 9 Hz, C-6), 142.9 (C-4a), 158.0−159.4 (d, J
= 253 Hz, C-8). Anal. (C12H13FO2S) theoretical: C, 59.98; H, 5.45; S,
13.34. Found: C, 59.94; H, 5.53; S, 13.32.
4-Cyclopropyl-7-fluoro-4-hydroxythiochroman 1,1-Dioxide
(13b). The title compound was obtained as described for 11a in
the step using metachloroperbenzoic acid starting from 4-cyclopropyl-
7-fluoro-4-hydroxythiochroman (9b). Yield = 38%. mp: 142−145 °C.
1H NMR (DMSO-d6): δ 0.34 (m, 1H, CH(CH2)2), 0.40 (m, 1H,
CH(CH2)2), 0.51 (m, 1H, CH(CH2)2), 0.73 (m, 1H, CH(CH2)2),
1.19 (m, 1H, CH(CH2)2), 2.45 (m, 2H, 3-CH2), 3.71 (m, 2H, 2-
CH2), 5.44 (s, 1H, OH), 7.53 (td, J = 8.6 Hz/2.8 Hz, 1H, 6-H), 7.56
(dd, J = 8.2 Hz/2.7 Hz, 1H, 8-H), 7.84 (dd, J = 8.9 Hz/5.4 Hz, 1H, 5-
H). 13C NMR (DMSO-d6): δ 2.3 (CH(CH2)2), 21.4 (CH(CH2)2),
35.2 (C-3), 47.3 (C-2), 67.0 (C-4), 109.0 (d, J = 24 Hz, C-8), 120.1
(d, J = 21 Hz, C-6), 131.4 (d, J = 8 Hz, C-5), 139.0 (d, J = 6 Hz, C-
8a), 139.2 (d, J = 3 Hz, C-4a), 160.7−162.1 (d, J = 249 Hz, C-7).
Anal. (C12H13FO3S) theoretical: C, 56.24; H, 5.11; S, 12.51. Found:
C, 55.86; H, 5.21; S, 11.91.
7-Chloro-4-cyclopropyl-4-hydroxythiochroman 1,1-Dioxide
(13c). The title compound was obtained as described for 11a in the
step using metachloroperbenzoic acid starting from 7-chloro-4-
cyclopropyl-4-hydroxythiochroman (9c). Yield = 67%. mp: 147−
149 °C. 1H NMR (DMSO-d6): δ 0.37 (m, 2H, CH(CH2)2), 0.50 (m,
1H, CH(CH2)2), 0.73 (m, 1H, CH(CH2)2), 1.20 (m, 1H,
CH(CH2)2), 2.45 (m, 2H, 3-CH2), 3.72 (m, 2H, 2-CH2), 5.50 (s,
1H, OH), 7.73 (m, 2H, 6-H /8-H), 7.80 (s, 1H, 5-H). 13C NMR
(DMSO-d6): δ 1.8 (CH(CH2)2), 21.1 (CH(CH2)2), 34.9 (C-3), 47.2
(C-2), 66.9 (C-4), 121.8 (C-8), 130.6 (C-5), 132.5 (C-6), 133.2 (C-
7), 138.9 (C-4a), 141.6 (C-8a). Anal. (C12H13ClO3S) theoretical: C,
52.84; H, 4.80; S, 11.77. Found: C, 52.68; H, 4.83; S, 11.85.
AMPA-Evoked Membrane Depolarization. This assay con-
sisted of investigating AMPA-evoked membrane depolarization,
measured by fluorescent membrane potential dyes and an imaging-
based plate reader (FDSS, Hamamatsu, JP), on rat primary brain
cultures.10 Dissociated rat primary brain cells were prepared from
embryonic rats (E16) and were added to poly-D-lysine-coated 96-well
culture plates for 18 days at 37 °C and 5% CO2 (density 20 000 cells/
well). On the day of the experiment, ground medium was removed
from the cells and was replaced by 20 μL/well of membrane potential
dye loading solution (Molecular Devices), reconstituted according to
the manufacturer’s instructions. The plates were incubated for 1 h at
room temperature and then directly transferred to the fluorescence
imaging-based plate reader. Baseline fluorescence was monitored for
10 s followed by the addition of AMPA (300 μM).
Assessment of the Blood−Brain Barrier Permeability in
NRMI Mice. Male NMRI mice (18−20 g) were purchased from
Janvier (France). All animals were maintained in barrier facilities,
housed in a room kept at 21 1 °C with a 12 h light/dark cycle, and
allowed free access to tap water and normal diet food. Animal
protocols were approved by local ethical committees following the
principles of laboratory animal care with the European Communities
Council Directive 2010/63/UE. All efforts were made to minimize
animal suffering and to reduce the number of animals used. Thirty
and 60 min after oral compound administration at the dose of 3 mg/
kg, male NMRI mice of 18−20 g were euthanized by decapitation.
Compounds were suspended in distilled water with Tween80 2%.
Three animals were used for each time point. Total blood was
collected into lithium heparinized tubes. Plasma was removed
following 5 min of centrifugation at 3600 rpm and 4 °C, frozen in
dry ice and stored at −80 °C until bioanalysis. The brain was
collected, the cerebellum discarded, and the rest of the brain quickly
plunged into liquid nitrogen for 30 to 60 s and stored in a Precellys
tube at −80 °C until bioanalysis. Brain and plasma compound
concentrations were measured using solid-phase extraction (OSTRO)
and liquid chromatography using tandem mass spectrometry
detection (XEVO TQS).
X-ray Structure Determination. The GluA2-LBD (L504Y/
N775S) flip-like isoform from rat was prepared as previously
described.16 The purified protein (final concentration 4 mg/mL)
was mixed with L-glutamate (final concentration 5 mM) and 12b
(added in excess as soluble compound to ensure a saturated
concentration) in 10 mM Hepes at pH 7, 20 mM NaCl, and 1 mM
EDTA. For crystallization, the hanging-drop vapor diffusion method
was used, and crystallization plates were set up at 6 °C. The reservoir
volume was 500 μL and the drop sizes were 2 μL (1 μL protein/
ligand solution and 1 μL reservoir solution). The reservoir solution
consisted of 20% PEG 4000, 0.1 M zinc acetate, and 0.1 M cacodylate
at pH 6.5. Before cryo-protection in liquid nitrogen, the crystals were
briefly immersed in reservoir solution containing 20% glycerol. X-ray
diffraction data were collected at beamline ID29, ESRF, Grenoble,
France. The data were processed using XDS29 and CCP4i.30 The
structure of GluA2-LBD (L504Y/N775S) with glutamate and 12b
was determined using molecular replacement in PHASER31
implemented in CCP4i and the structure of GluA2-LBD (L504Y/
N775S) with glutamate and 1 (PDB code 3TKD, chain A) as a search
model. Initially, automated model building and refinement were
performed using AutoBuild in PHENIX.32 Atomic coordinates for
12b were created in Maestro33 and geometry optimized.34 A restraint
file for 12b was generated using eLBOW,35 keeping the geometry
from Maestro. The structure was refined in PHENIX36 using NCS,
TLS, isotropic B factors, and riding hydrogen atoms. Manual model
building was performed in COOT.37 The final structure of GluA2-
LBD (L504Y/N775S) with glutamate and 12b was analyzed in
COOT, PyMOL,38 and DynDom.39
Hirshfeld Surface Analysis. Molecular Hirshfeld surfa-
ces25,27,40−43 in the crystal structure were built based on the electron
distribution calculated as the sum of spherical atom electron
densities.44,45 The Hirshfeld surfaces mapped with different properties
like di, de, dnorm, shape-index, and 2D fingerprint plots were generated
using CrystalExplorer 17.5.46
The distance from the surface to the nearest nucleus external to the
surface (de), the distance to the nearest nucleus internal to the surface
(di) as well as the normalized contact distance (dnorm) were calculated
to identify the regions of intermolecular interactions on the Hirshfeld
surfaces (HS).25
Hydrogen atoms were added to the molecules as well as to oxygen
atoms of water molecules using the “Add Hydrogen” utility tool in
PyMOL. The two enantiomers were separated to obtain two clusters
for each form using PyMOL. The presence of water molecules is
maintained to generate the different shape-contact surfaces.22 The
PyMOL software was used for the molecular cluster preparation.38
The combination of de and di in the form of a 2D histogram, the so-
called fingerprint plot,47,48 provides decomposition of Hirshfeld
surfaces into a contribution of different intermolecular interactions
present in the crystal structure.25
The colored property called the shape index is sensitive to very
subtle changes in surface shape, is based on the local curvature of the
surface, and can also be mapped on the HS.49 Shape index allows
highlighting of the π−π stacking interaction as adjacent blue and red
colored triangles. On the HS mapped with shape index, convex blue
regions represent donor groups and concave red regions represent
acceptor groups.
Electrostatic potentials mapped on HS provide direct insight into
intermolecular interactions in crystals. The molecular electrostatic
potential (MEP) map analysis allows a quantitative evaluation of the
electron-rich and electron-deficient sites in the molecule and can be
used as a good indicator for HB donors as well as HB acceptors. The
map is obtained at the B3LYP/6-31G(d,p) level of theory. The blue
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and red colors on the surface indicate the positive and negative
potentials, respectively.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00255.
■
ACKNOWLEDGMENTS
■
sı
The technical assistance of Stéphane Counerotte is gratefully
acknowledged. We would like to thank technician Heidi
Peterson for help with production of GluA2-LBD and the
beamline scientist at ESRF, Grenoble, France for help during
X-ray data collection.
Analysis of the eletron density for 12b (X-ray structure
determination) and Hirshfeld surface analysis figures
(PDF)
ABBREVIATIONS
■
AUTHOR INFORMATION
Corresponding Authors
■
AMPARs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate
receptors; AMPAR PAMs, AMPAR positive allosteric modu-
lators; BDNF, brain-derived neurotrophic factor; BTDs, 1,2,4-
benzothiadiazine 1,1-dioxides; clogP, lipophilicity; ESP,
electrostatic potential; GluA2-LBD (L504Y/N775S), flip-like
isoform of the ligand-binding domain protein of GluA2; HS,
Hirshfeld surface; iGluRs, ionotropic glutamate receptors;
LBD, ligand-binding domain; MEP, molecular electrostatic
potential
Pierre Francotte − Center for Interdisciplinary Research on
Medicines (CIRM) − Laboratory of Medicinal Chemistry,
̀
̀
University of Liege, B-4000 Liege, Belgium; orcid.org/
0000-0001-9252-7798; Email: Pierre.Francotte@uliege.be
Jette Sandholm Kastrup − Research Cluster on Molecular
Neuroprotection and Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, DK-2100 Copenhagen, Denmark;
orcid.org/0000-0003-2654-1510; Email: jsk@
sund.ku.dk
REFERENCES
■
(1) Traynelis, S. F., Wollmuth, L. P., McBain, C. J., Menniti, F. S.,
Vance, K. M., Ogden, K. K., Hansen, K. B., Yuan, H., Myers, S. J., and
Dingledine, R. (2010) Glutamate receptor ion channels: structure,
regulation, and function. Pharmacol. Rev. 62, 405−496.
(2) Greger, I. H., Watson, J. F., and Cull-Candy, S. G. (2017)
Structural and functional architecture of AMPA-type glutamate
receptors and their auxiliary proteins. Neuron 94, 713−730.
(3) Sobolevsky, A. I., Rosconi, M. P., and Gouaux, E. (2009) X-ray
Structure, Symmetry and Mechanism of an AMPA-subtype Glutamate
Receptor. Nature 462, 745−756.
(4) Schwenk, J., Harmel, N., Brechet, A., Zolles, G., Berkefeld, H.,
Muller, C. S., Bildl, W., Baehrens, D., Huber, B., Kulik, A., Klöcker, N.,
̈ ̈
Schulte, U., and Fakler, B. (2012) High-resolution proteomics unravel
architecture and molecular diversity of native AMPA receptor
complexes. Neuron 74, 621−633.
(5) Jin, R., Clark, S., Weeks, A. M., Dudman, J. T., Gouaux, E., and
Partin, K. M. (2005) Mechanism of Positive Allosteric Modulators
Acting on AMPA Receptors. J. Neurosci. 25, 9027−9036.
(6) Francotte, P., de Tullio, P., Fraikin, P., Counerotte, S., Goffin, E.,
and Pirotte, B. (2006) In Search of Novel AMPA Potentiators. Recent
Pat. CNS Drug Discovery 1, 239−246.
(7) Murray, T. K., Whalley, K., Robinson, C. S., Ward, M. A., Hicks,
C. A., Lodge, D., Vandergriff, J. L., Baumbarger, P., Siuda, E., Gates,
M., Ogden, A. M., Skolnick, P., Zimmerman, D. M., Nisenbaum, E. S.,
Bleakman, D., and O’Neill, M. J. (2003) LY503430, a Novel α-
Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptor Po-
tentiator with Functional, Neuroprotective and Neurotrophic Effects
in Rodent Models of Parkinson’s Disease. J. Pharmacol. Exp. Ther.
306, 752−762.
(8) O’Neill, M. J., Bleakman, D., Zimmerman, D. M., and
Nisenbaum, E. S. (2004) AMPA receptor potentiators for the
treatment of CNS disorders. Curr. Drug Targets: CNS Neurol. Disord.
3, 181−194.
(9) Pirotte, B., Francotte, P., Goffin, E., and de Tullio, P. (2013)
AMPA receptor positive allosteric modulators: a patent review. Expert
Opin. Ther. Pat. 23, 615−628.
(10) Francotte, P., Goffin, E., Fraikin, P., Lestage, P., Van Heugen, J.-
C., Gillotin, F., Danober, L., Thomas, J.-Y., Chiap, P., Caignard, D.-H.,
Pirotte, B., and De Tullio, P. (2010) New Fluorinated 1,2,4-
Benzothiadiazine 1,1-Dioxides: Discovery of an Orally Active
Cognitive Enhancer Acting through Potentiation of the 2-Amino-3-
(3-hydroxy-5-methylisoxazol-4-yl)propionic Acid Receptors. J. Med.
Chem. 53, 1700−1711.
Authors
̀
Koffi Sénam Etse − Center for Interdisciplinary Research on
Medicines (CIRM) − Laboratory of Medicinal Chemistry,
̀
̀
University of Liege, B-4000 Liege, Belgium
Jerzy Dorosz − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, DK-2100 Copenhagen, Denmark
Katrine McLain Christensen − Department of Drug Design
and Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, DK-2100 Copenhagen, Denmark
Jean-Yves Thomas − Institut de Recherches Servier, F-78290
Croissy-sur-Seine, France
Iuliana Botez Pop − Institut de Recherches Servier, F-78290
Croissy-sur-Seine, France
Eric Goffin − Center for Interdisciplinary Research on
Medicines (CIRM) − Laboratory of Medicinal Chemistry,
̀
̀
University of Liege, B-4000 Liege, Belgium
Thomas Colson − Center for Interdisciplinary Research on
Medicines (CIRM) − Laboratory of Medicinal Chemistry,
̀
̀
University of Liege, B-4000 Liege, Belgium
Pierre Lestage − Institut de Recherches Servier, F-78290
Croissy-sur-Seine, France
Laurence Danober − Institut de Recherches Servier, F-78290
Croissy-sur-Seine, France
Bernard Pirotte − Center for Interdisciplinary Research on
Medicines (CIRM) − Laboratory of Medicinal Chemistry,
̀
̀
University of Liege, B-4000 Liege, Belgium
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.1c00255
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
We acknowledge funding from the Danish Council for
Independent ResearchMedical Sciences and Danscatt
́
(J.D., J.S.K.) and the Leon Fredericq Foundation (T.C.).
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(11) Francotte, P., Nørholm, A. B., Deva, T., Olsen, L., Frydenvang,
K., Goffin, E., Fraikin, P., de Tullio, P., Challal, S., Thomas, J. Y., Iop,
F., Louis, C., Botez-Pop, I., Lestage, P., Danober, L., Kastrup, J. S., and
Pirotte, B. (2014) Positive Allosteric Modulators of 2-Amino-3-(3-
hydroxy-5-methylisoxazol-4-yl)propionic Acid Receptors Belonging to
4-Cyclopropyl-3,4-dihydro-2H-1,2,4-pyridothiadiazine Dioxides and
Diversely Chloro-Substituted 4-Cyclopropyl-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-Dioxides. J. Med. Chem. 57, 9539−9553.
(12) Goffin, E., Drapier, T., Larsen, A. P., Geubelle, P., Ptak, C. P.,
Laulumaa, S., Rovinskaja, K., Gilissen, J., Tullio, P. d., Olsen, L.,
Frydenvang, K., Pirotte, B., Hanson, J., Oswald, R. E., Kastrup, J. S.,
and Francotte, P. (2018) 7-Phenoxy-Substituted 3,4-Dihydro-2H-
1,2,4-benzothiadiazine 1,1-Dioxides as Positive Allosteric Modulators
of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)
Receptors with Nanomolar Potency. J. Med. Chem. 61, 251−264.
(13) Drapier, T., Geubelle, P., Bouckaert, C., Nielsen, L., Laulumaa,
S., Goffin, E., Dilly, S., Francotte, P., Hanson, J., Pochet, L., Kastrup, J.
S., and Pirotte, B. (2018) Enhancing Action of Positive Allosteric
Modulators through the Design of Dimeric Compounds. J. Med.
Chem. 61, 5279−5291.
(14) Laulumaa, S., Hansen, K. V., Masternak, M., Drapier, T.,
Francotte, P., Pirotte, B., Frydenvang, K., and Kastrup, J. S. (2019)
Crystal Structures of Potent Dimeric Positive Allosteric Modulators at
the Ligand-Binding Domain of the GluA2 Receptor. ACS Med. Chem.
Lett. 10, 243−247.
(15) Lima, L. M., and Barreiro, E. J. (2005) Bioisosterism: a useful
strategy for molecular modification and drug design. Curr. Med. Chem.
12, 23−49.
(16) Ciapetti, P., and Giethlen, B. Molecular Variations Based on
Isosteric Replacements, in (2015) The Practice of Medicinal Chemistry,
4th ed., Elsevier.
(17) Nørholm, A. B., Francotte, P., Olsen, L., Krintel, C.,
Frydenvang, K., Goffin, E., Challal, S., Danober, L., Botez-Pop, I.,
Lestage, P., Pirotte, B., and Kastrup, J. S. (2013) Synthesis,
pharmacological and structural characterization, and thermodynamic
aspects of GluA2-positive allosteric modulators with a 3,4-dihydro-
2H-1,2,4-benzothiadiazine 1,1-dioxide scaffold. J. Med. Chem. 56,
8736−8745.
(18) Krintel, C., Francotte, P., Pickering, D. S., Juknaite, L.,
Pøhlsgaard, J., Olsen, L., Frydenvang, K., Goffin, E., Pirotte, B., and
Kastrup, J. S. (2016) Enthalpy-entropy compensation in the binding
of modulators at ionotropic glutamate receptor GluA2. Biophys. J. 110,
2397−2406.
(19) Pøhlsgaard, J., Frydenvang, K., Madsen, U., and Kastrup, J. S.
(2011) Lessons from more than 80 structures of the GluA2 ligand-
binding domain in complex with agonists, antagonists and allosteric
modulators. Neuropharmacology 60, 135−150.
(20) SwissADME. http://www.swissadme.ch/index.php.
(21) Armstrong, N., and Gouaux, E. (2000) Mechanisms for
activation and antagonism of an AMPA-sensitive glutamate receptor:
crystal structures of the GluR2 ligand binding core. Neuron 28, 165−
181.
(22) Grimme, S., Ehrlich, S., and Goerigk, L. (2011) Effect of the
damping function in dispersion corrected density functional theory. J.
Comput. Chem. 32, 1456−1465.
(23) Spackman, P. R., Yu, L. J., Morton, C. J., Parker, M. W., Bond,
C. S., Spackman, M. A., Jayatilaka, D., and Thomas, S. P. (2019)
Bridging Crystal Engineering and Drug Discovery by Utilizing
Intermolecular Interactions and Molecular Shapes in Crystals.
Angew. Chem., Int. Ed. 58, 16780−16784.
(24) McKinnon, J. J., Spackman, M. A., and Mitchell, A. S. Novel
tools for visualizing and exploring intermolecular interactions in
molecular crystals. (2004) Acta Crystallogr., Sect. B: Struct. Sci. B60,
627−668.
Hirshfeld Surface Analysis and DFT calculations of 1-phenyl-N-
(benzomethyl)-N-({1-[(2-benzo-4-methyl-4,5-dihydro-1,3-oxazol-4-
yl)methyl]-1H-1,2,3-triazol-4-yl}methyl)methanamine. J. Mater. Envi-
ron. Sci. 9, 2254−2262.
(27) Spackman, M. A., and Jayatilaka, D. (2009) Hirshfeld surface
analysis. CrystEngComm 11, 19−32.
(28) Munshi, P., Skelton, B. W., McKinnon, J. J., and Spackman, M.
A. (2008) Polymorphism in 3-methyl-4-methoxy-4′-nitrostilbene
(MMONS), a highly active NLO material. CrystEngComm 10, 197−
206.
(29) Kabsch, W. (2010) XDS. Acta Crystallogr., Sect. D: Biol.
Crystallogr. D66, 125−132.
(30) Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J.,
Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G.,
McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S.,
Potterton, E. A., Powell, H. R., Read, J., Vagin, A., and Wilson, K. S.
(2011) Overview of the CCP4 suite and current developments. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 67, 235−242.
(31) McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M.
D., Storoni, L. C., and Read, R. J. J. (2007) Phaser crystallographic
software. J. Appl. Crystallogr. 40, 658−674.
(32) Terwilliger, T. C., Grosse-Kunstleve, R. W., Afonine, P. V.,
Moriarty, N. W., Zwart, P. Z., Hung, L. W., Read, R. J., and Adams, P.
D. (2008) Iterative model building, structure refinement and density
modification with the PHENIX AutoBuild wizard. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 64, 61−69.
̈
(33) Maestro version 2.3; Schrodinger, LLC: New York, 2013.
̈
(34) MacroModel 10.1; Schrodinger, LLC: New York, 2013.
(35) Moriarty, N. W., Grosse-Kunstleve, R. W., and Adams, P. D.
(2009) electronic Ligand Builder and Optimization Workbench
(eLBOW): a tool for ligand coordinate and restraint generation. Acta
Crystallogr., Sect. D: Biol. Crystallogr. D65, 1074−1080.
(36) Liebschner, D., Afonine, P. V., Baker, M. L., Bunkoczi, G.,
Chen, V. B., Croll, T. I., Hintze, B., Hung, L.-W., Jain, S., McCoy, A.
J., Moriarty, N. W., Oeffner, R. D., Poon, B. K., Prisant, M. G., Read,
R. J., Richardson, J. S., Richardson, D. C., Sammito, M. D., Sobolev,
O. V., Stockwell, D. H., Terwilliger, T. C., Urzhumtsev, A. G., Videau,
L. L., Williams, C. J., and Adams, P. D. (2019) Macromolecular
structure determination using X-rays, neutrons and electrons: recent
developments in Phenix. Acta Crystallogr. D75, 861−877.
(37) Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010)
Features and development of Coot. Acta Crystallogr., Sect. D: Biol.
Crystallogr. D66, 486−501.
(38) The PyMOL Molecular Graphics System, version 2.0;
̈
Schrodinger, LLC.
(39) Hayward, S., and Berendsen, H. J. (1998) Systematic analysis of
domain motions in proteins from conformational change: New results
on citrate synthase and T4 lysozyme. Proteins: Struct., Funct., Genet.
30, 144−154.
(40) McKinnon, J. J., Jayatilaka, D., and Spackman, M. A. (2007)
Towards quantitative analysis of intermolecular interactions with
Hirshfeld surfaces. Chem. Commun. 37, 3814−3816.
(41) Spackman, M. A., McKinnon, J. J., and Jayatilaka, D. (2008)
Electrostatic potentials mapped on Hirshfeld surfaces provide direct
insight into intermolecular interactions in crystals. CrystEngComm 10,
377−388.
(42) Hirshfeld, F. L. (1977) Bonded-atom fragments for describing
molecular charge densities. Theoret. Chim. Acta 44, 129−138.
(43) Clausen, H. F., Chevallier, M. S., Spackman, M. A., and Iversen,
B. B. (2010) Three new co-crystals of hydroquinone: crystal
structures and Hirshfeld surface analysis of intermolecular inter-
actions. New J. Chem. 34, 193−199.
(44) Hunter, C. A., Singh, J., and Thornton, J. M. (1991) π-π
interactions: the geometry and energetics of phenylalanine-phenyl-
alanine interactions in proteins. J. Mol. Biol. 218, 837−846.
(45) Singh, J., and Thornton, J. M. (1990) SIRIUS. An automated
method for the analysis of the preferred packing arrangements
between protein groups. J. Mol. Biol. 211, 595−615.
(25) Spackman, M. A., and McKinnon, J. J. (2002) Fingerprinting
intermolecular interactions in molecular crystals. CrystEngComm 4,
378−392.
(26) Boukhssas, S., Aouine, Y., Faraj, H., Alami, A., Hallaoui, A.,
Tijani, N., Yamni, K., Zouihri, H., Mrani, D., and Lachkar, M. (2018)
(46) CrystalExplorer17; University of Western Australia, 2017.
2691
https://doi.org/10.1021/acschemneuro.1c00255
ACS Chem. Neurosci. 2021, 12, 2679−2692

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(47) Rohl, A. L., Moret, M., Kaminsky, W., Claborn, K., McKinnon,
J. J., and Kahr, B. (2008) Hirshfeld Surfaces Identify Inadequacies in
Computations of Intermolecular Interactions in Crystals: Pentam-
orphic 1,8-Dihydroxyanthraquinone. Cryst. Growth Des. 8, 4517−
4525.
(48) Parkin, A., Barr, G., Dong, W., Gilmore, C. J., Jayatilaka, D.,
McKinnon, J. J., Spackman, M. A., and Wilson, C. C. (2007)
Comparing entire crystal structures: structural genetic fingerprinting.
CrystEngComm 9, 648−652.
(49) Koenderink, J. J., and van Doorn, A. J. (1992) Surface shape
and curvature scales. Image Vision Comput. 10, 557−564.
2692
https://doi.org/10.1021/acschemneuro.1c00255
ACS Chem. Neurosci. 2021, 12, 2679−2692