Synthesis, pharmacological and structural characterization, and thermodynamic aspects of GluA2-positive allosteric modulators with a 3,4-dihydro-2 h -1,2,4-benzothiadiazine 1,1-dioxide scaffold

Article abstract of DOI:10.1021/jm4012092

Positive allosteric modulators of ionotropic glutamate receptors are potential compounds for treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind within the dimer interface of the ligand-binding domain (LBD) and stabilize the agonist-bound conformation, thereby slowing receptor desensitization and/or deactivation. Here we describe the synthesis and pharmacological testing at GluA2 of a new generation of 3,4-dihydro-2H-1,2,4- benzothiadiazine 1,1-dioxides. The most potent modulator 3 in complex with GluA2-LBD-L483Y-N754S was subjected to structural analysis by X-ray crystallography, and the thermodynamics of binding was studied by isothermal titration calorimetry. Compound 3 binds to GluA2-LBD-L483Y-N754S with a K _d of 0.35 μM (ΔH = -7.5 kcal/mol and -TΔS = -1.3 kcal/mol). This is the first time that submicromolar binding affinity has been achieved for this type of positive allosteric modulator. The major structural factor increasing the binding affinity of 3 seems to be interactions between the cyclopropyl group of 3 and the backbone of Phe495 and Met496.

Full text of DOI:10.1021/jm4012092

Article
pubs.acs.org/jmc
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
Ann-Beth Nørholm,†,∥ Pierre Francotte,‡,∥ Lars Olsen,† Christian Krintel,† Karla Frydenvang,†
Eric Goffin,‡ Sylvie Challal,§ Laurence Danober,§ Iuliana Botez-Pop,§ Pierre Lestage,§ Bernard Pirotte,‡,⊥
and Jette S. Kastrup*,†,⊥
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark
‡Department of Medicinal Chemistry, Drug Research Centre (CIRM), University of Liege, Avenue de l’Ho
̂ ̀
pital 1, B36, 4000 Liege,
Belgium
§Institut de Recherches Servier, Chemin de Ronde 125, F-78290 Croissy-sur-Seine, France
ABSTRACT: Positive allosteric modulators of ionotropic gluta-
mate receptors are potential compounds for treatment of
cognitive disorders, e.g., Alzheimer’s disease. The modulators
bind within the dimer interface of the ligand-binding domain
(LBD) and stabilize the agonist-bound conformation, thereby
slowing receptor desensitization and/or deactivation. Here we
describe the synthesis and pharmacological testing at GluA2 of a
new generation of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxides. The most potent modulator 3 in complex with
GluA2-LBD-L483Y-N754S was subjected to structural analysis
by X-ray crystallography, and the thermodynamics of binding was studied by isothermal titration calorimetry. Compound 3 binds
to GluA2-LBD-L483Y-N754S with a Kd of 0.35 μM (ΔH = −7.5 kcal/mol and −TΔS = −1.3 kcal/mol). This is the first time
that submicromolar binding affinity has been achieved for this type of positive allosteric modulator. The major structural factor
increasing the binding affinity of 3 seems to be interactions between the cyclopropyl group of 3 and the backbone of Phe495 and
Met496.
finally an intracellular C-terminal domain.1 The structure of the
full-length homotetrameric AMPA receptor GluA2 showed that
the LBDs are arranged as a dimer of dimers.5 While the
endogenous neurotransmitter (S)-glutamate binds at the
agonist-binding site in the LBD,6 positive allosteric modulators
bind at the LBD dimer interface.7,8 Positive allosteric
modulators can potentiate GluA2 currents in two ways: (i)
by stabilizing the (S)-glutamate-bound conformation and
thereby slowing deactivation or (ii) by stabilizing the interface
between two subunits and slowing the entrance into the
desensitized state, which involves a rearrangement of the
interface and closure of the ion channel with (S)-glutamate still
bound to the receptor.
INTRODUCTION
■
Ionotropic glutamate receptors (iGluRs) mediate the majority
of fast excitatory neurotransmission in the mammalian central
nervous system (CNS). iGluRs are divided into three subtypes:
α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA) receptors, kainic acid receptors, and N-methyl-D-
aspartic acid (NMDA) receptors. All three subtypes have been
shown to be involved in long-term potentiation (LTP) and
long-term depression (LTD), including learning and memory
formation (see ref 1 and references therein). iGluR agonists
function by direct activation of the receptors and, thus, may
cause severe side effects, e.g., neurotoxicity. On the contrary,
positive allosteric modulators of iGluRs only potentiate the
effects of (S)-glutamate and therefore lead to a fine-tuning of
receptor signaling. Several studies support positive allosteric
modulation of AMPA receptors as a promising therapeutic
strategy in the treatment of dysfunctions in the CNS, such as
attention deficit hyperactivity disorder (ADHD),2 Alzheimer’s
disease,3 and schizophrenia.4
In 1998, Pirotte et al.9 reported a series of 4H-1,2,4-
pyridothiadiazine 1,1-dioxides and 2,3-dihydro-4H-1,2,4-pyri-
dothiadiazine 1,1-dioxides with various alkyl and aryl
substituents in the 2-, 3-, and 4-positions as positive allosteric
modulators of AMPA receptors. This study was later followed
up by synthesis and pharmacological testing of new derivatives
in an attempt to enhance modulatory activity, e.g., by
iGluRs form homo- or heterotetrameric ligand-gated ion
channels, where each of the four subunits is composed of an
extracellular part with an N-terminal domain and a ligand-
binding domain (LBD), a membrane-spanning region, and
Received: August 6, 2013
© XXXX American Chemical Society
A
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a
addressing lipophilicity to obtain in vivo active compounds as
well as the steric and electronic impact of different
substituents.10−12 In a recent study, we employed isothermal
titration calorimetry (ITC) to determine the binding affinity of
compound 1 (Figure 1) at the GluA2 LBD,13 as 1 is the most
Scheme 1
Figure 1. Chemical structure of the parent compound 1 and a general
formula of the newly synthesized 4-cyclopropyl-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxides 2−9.
potent compound within the series of 1,2,4-benzothiadiazine
1,1-dioxides.10−12 It was noticed that the ethyl substituent of 1
forms van der Waals contacts to GluA2 residues, primarily to
backbone atoms of Met496.
Here, we report the synthesis and pharmacological character-
ization of eight compounds in which the 4-ethyl substituent has
been substituted with a 4-cyclopropyl group and with various
substituents introduced in the 7-position (Figure 1). We show
that the introduction of a cyclopropyl group leads to an
approximately 10-fold improvement of potency at GluA2
compared to that of ethyl, using an in vitro test on rat primary
brain cells measuring the effect of the new compounds on
AMPA-evoked membrane depolarization (fluorescence assay).
The most potent compound 3, containing a fluorine atom in
the 7-position, was selected for studies of thermodynamics of
binding at GluA2 LBD using ITC. Together with an X-ray
structural analysis, these studies provide a molecular explan-
ation for the improved binding affinity of the new generation of
3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides.
a
Reagents and conditions: (i) (1) HNO2, −5 °C; (2) SO2, CuCl2, 15
min; (3) NH4OH (50−55%); (ii) cyclopropylamine, 100 °C, 24 h
(90%); (iii) HC(OEt)3, 130 °C, 3 h (85%); (iv) H2, Pd/C (10%), rt,
30 min (80%); (v) (1) HNO2, 0 °C; (2) Cu2Cl2, rt, 30 min (75%);
(vi) NaBH4, 2-propanol, 55 °C, 10 min (60%).
The use of the same reagent on intermediate 13 gave rise to the
corresponding 7-nitro-substituted target compound 8.
The other final compounds (2, 3, 5, 6, 7, and 9) were
obtained by means of an original pathway starting from
commercially available 2-fluorobenzenesulfonamides 16
(Scheme 2). Independent of the nature of the substituent at
the 5-position of 2-fluoro-substituted benzenesulfonamides,
nucleophilic substitution of the fluorine atom by cyclopropyl-
amine occurred, providing the corresponding 2-(cyclopropyla-
RESULTS AND DISCUSSION
■
Synthesis. Access to the diversely 7-substituted 4-cyclo-
propyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides 2−9
is described in Schemes 1 and 2.
a
Scheme 2
The synthesis of the 7-chloro- and 7-nitro-substituted
compounds 4 and 8 was initially performed according to the
pathway reported in Scheme 1. The commercially available 2-
chloro-5-nitroaniline (10) was converted into the correspond-
ing benzenesulfonamide 11 according to the Meerwein
variation of the Sandmeyer reaction.14 Nucleophilic substitu-
tion of the chlorine atom by cyclopropylamine, providing
intermediate 12, was facilitated by the fact that the halogen
atom was located at the ortho and para positions of two
electron-withdrawing groups. Ring closure of 12 into the
corresponding 4-cyclopropyl-4H-1,2,4-benzothiadiazine 1,1-
dioxide 13 was achieved by heating 12 in triethyl orthoformate.
Reduction of the nitro group of 13 into the corresponding 7-
amino-substituted 4-cyclopropyl-4H-1,2,4-benzothiadiazine 1,1-
dioxide (14) by catalytic hydrogenation, followed by
diazotization of 14 in the presence of cuprous chloride
provided 7-chloro-4-cyclopropyl-4H-1,2,4-benzothiadiazine
1,1-dioxide (15). The latter intermediate was converted into
the 7-chloro-substituted target compound 4 after saturation of
the N2−C3 double bond by means of sodium borohydride.
a
Reagents and conditions: (i) cyclopropylamine, dioxane, 100−110
°C, 24−96 h (85−95%); (ii) HC(OEt)3, 130−150 °C, 1−24 h (70−
75%); (iii) NaBH4, 2-propanol, 50−55 °C, 5−10 min (80%).
B
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mino)-substituted benzenesulfonamides 17. Such a reaction
may be due to the greater lability to nucleophilic substitution of
the aromatic fluorine atom compared to other halogen atoms,
provided that only the electron-withdrawing sulfonamide group
was present at the ortho position. Ring closure of 17 by means
of triethyl orthoformate provided intermediates 18, which were
converted into the target compounds (2, 3, 5, 6, 7, and 9) after
saturation of the N2−C3 double bond of 18 by reaction with
sodium borohydride.
Pharmacological Testing. The newly synthesized 4-
cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides
2−9 were evaluated in vitro as AMPA 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 EC2× 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). Moreover, the maximum effect of each modulator
an EC2× value of 0.75 μM in the fluorescence assay (see Table
1).
In a previous work, we reported that compound 1, the 4-
ethyl analogue of compound 3, markedly potentiated the effect
of AMPA on its receptors expressed in Xenopus oocytes with an
EC2× value of 3.2 μM.10 The replacement of the ethyl chain at
the 4-position of compound 1 by a cyclopropyl moiety, leading
to compound 3, markedly increased the effect on AMPA
receptors. The EC2× value of 3, determined in the fluorescence
assay on rat neuronal cells, was found to be 0.27 μM,
corresponding to a 10-fold increase in potency.
Interestingly, the fluorine atom appeared to be the best
choice of halogen atom at the 7-position of 4-cyclopropyl-3,4-
dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. The rank order
of efficacy as AMPA potentiators was found to be F > Cl > Br
(see compounds 3, 4, and 5, Table 1). Moreover, the fluoro
compound 3 also expressed the highest maximal effect since
this compound was able to increase by 15-fold the AMPA-
evoked response (Table 1).
The presence of a hydrogen atom at the 7-position (see
compound 2) was found to produce the same effect as that of a
chlorine atom and clearly appeared to be more suitable than the
presence of a methyl group (see compound 6). Considering the
introduction of an electron-withdrawing group, such as a
trifluoromethyl, nitro, or cyano group, a great variability was
observed. The 7-cyano-substituted compound 9 was the most
potent on AMPA receptors with an EC2× value of 0.47 μM, not
far from that of the most potent 7-fluoro-substituted compound
3. However, introduction of a trifluoromethyl group led to a
complete loss of activity, which was the same trend as the one
already observed with 4-ethyl-substituted 3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxides.10
Thermodynamic Details of Binding. As compound 3 was
shown to be a highly effective positive allosteric modulator of
GluA2 with submicromolar potency, it was selected for studies
of the thermodynamics of binding to GluA2 using isothermal
titration calorimetry. We used the rat GluA2-LBD-L483Y-
N754S double mutant,13 which exists as a dimer in solution due
to the L483Y mutation (numbering without the signal peptide).
The preformed dimer enables measurement of modulator
binding affinity independently of the dimerization process. In
the original rat GluA2 structure reported by Armstrong and
Gouaux,6 an asparagine is present at position 754, correspond-
ing to the flop-splice isoform of GluA2. In the flip-splice
isoform, a serine is located at this position. Ser754 was
introduced in the protein construct as it has previously been
shown that a similar compound, cyclothiazide, displays
preference for the flip-splice isoform.15 This preference has
been attributed to sterical hindrance in the flop-splice isoform.7
The ITC experiments showed that the binding of 3 to
GluA2-LBD-L483Y-N754S is an exothermic process (Figure
2). The binding affinity (Kd) of 3 at GluA2-LBD-L483Y-N754S
is 0.35 μM with ΔH = −7.5 kcal/mol and −TΔS = −1.3 kcal/
mol (Table 2). This means that the complex formation is
primarily enthalpy-driven. Binding of compound 1 to GluA2-
LBD-L483Y-N754S was also found to be enthalpy-driven13
(Table 2). The increased binding affinity of 3 compared to 1 is
seen to be primarily due to a large enthalpy gain combined with
a small entropy loss.
Table 1. Effects of 7-Substituted 4-Cyclopropyl-3,4-dihydro-
2H-1,2,4-benzothiadiazine 1,1-Dioxides on the Fluorescence
Induced by 300 μM AMPA on Primary Cultures of Neurons
from Rat Embryonic Cortex
a
b
c
compd
X
EC2× (μM)
EC50 (μM)
Emax
2
3
4
5
6
7
8
9
H
0.73 0.15 (3)
0.27 0.03 (3)
0.75 0.21 (4)
1.27 0.37 (3)
5.13 1.67 (3)
>100 (3)
4.17 0.60 (3)
0.90 0.10 (3)
3.13 0.72 (4)
4.83 1.17 (3)
nd
× 8.5 0.8 (3)
× 15.0 2.6 (3)
× 10.9 2.7 (4)
× 9.3 0.7 (3)
× 8.7 1.9 (3)
nd
F
Cl
Br
CH3
CF3
NO2
CN
nd
d
d
d
14.0 3.6 (2)
22.5 (1/2)
× 7 (1/2)
× 9.2 0.4 (3)
0.47 0.03 (3)
2.50 0.50 (3)
a
EC2× = modulator concentration giving a 2-fold increase of the
b
fluorescence induced by AMPA (300 μM) (mean SEM (n)). EC50
= modulator concentration responsible for 50% of the maximal effect
c
(mean SEM (n)). Emax = maximal effect normalized to unity for
d
AMPA-evoked response (×1). FLIPR (fluorescence imaging plate
reader). The principle of this technique is identical to that of the
fluorescence assay (use of a fluorescent probe sensitive to variation of
the membrane potential), but it uses another plate reader (FLIPR,
Molecular Devices, US).
(Emax value normalized to unity for AMPA-evoked response
taken as ×1) and its EC50 value (concentration of modulator
responsible for 50% of the maximal effect) were also
determined (Table 1).
Compared to the voltage clamp assay on Xenopus laevis
oocytes used in our previous studies for the evaluation of
AMPA potentiators (measurement of AMPA-induced currents
on X. laevis oocytes injected with rat cortex poly(A+) mRNA),
the fluorescence assay on primary neuronal cultures isolated
from rat embryonic cortex was found to provide similar EC2×
values (personal observation). For comparison purposes,
compound 4 was found to express an EC2× value of 0.8 μM
in the voltage clamp assay (data not shown), while it expressed
Structure Determination. Cocrystallization of compound
3 with the GluA2 LBD and examination of the interactions at
the level of the modulator-binding site are expected to help the
understanding of the unique impact of the cyclopropyl group
C
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Table 3. X-ray Data Collection and Refinement Statistics
Data Collection
space group
P21212
a, b, c (Å)
113.9, 164.0, 47.3
29.13−1.87 (1.97−1.87)
74220
a
resolution (Å)
no. of unique reflns
redundancy
4.1 (4.1)
completeness (%)
99.9 (100)
5.7 (38.5)
b
Rmerge (%)
I/σI
10.9 (2.0)
Refinement
c
d
Rwork /Rfree (%)
17.5/21.1
no. of non-hydrogen atoms
no. of residues (molecules A/B/C)
7134
262/260/262
3/3/687/13/1/6/10
no. of (S)-glutamate/3/water/
acetate/cacodylate/glycerol/zinc
ions
RMS deviations
bond lengths (Å)
bond angles (deg)
0.007
1.1
no. of residues in allowed areas of
100
e
Ramachandran plot (%)
av B values (Å2)
molecules A/B/C
21.2/26.2/25.3
(S)-glutamate/3/water/
acetate/cacodylate/glycerol/
zinc ions
16.4/14.3/31.5/38.4/22.6/38.0/42.0
a
Values in parentheses correspond to the outermost resolution shell.
Rmerge = ∑hkl∑i|Ii(hkl) − I(hkl)|/∑hkl∑i|Ii(hkl)|, where Ii(hkl) is the
b
intensity of an individual measurement of the reflection with Miller
indices hkl and I(hkl) is the intensity from multiple observations.
Figure 2. Isothermal titration calorimetry study of binding of
compound 3 to GluA2-LBD-L483Y-N754S. The raw data (top
panel) and isotherm (bottom panel) are presented. The graphs
show that heat is developed after each injection (exothermic reaction)
and that the signal is diminished when the protein becomes saturated
with 3. The experiment shown is a representative of three experiments.
c
Rwork = ∑hkl|Fobsd − Fcalcd|/∑hkl|Fobsd|, where |Fobsd| and |Fcalcd| are the
d
observed and calculated structure factor amplitudes. Rfree is equivalent
to Rwork, but calculated with reflections omitted from the refinement
e
process (5% of reflections omitted). The Ramachandran plot was
calculated according to PROCHECK.30
Table 2. Isothermal Titration Calorimetry Using GluA2-
LBD-L483Y-N754S
LBD adopts a closed clamshell-like structure as a result of
movement of lobe D2 of ca. 20° compared to the unliganded
(apo) structure of GluA2 LBD.6 In the present structure, the
closure of lobe D2 toward D1 is 20.7°, 19.4°, and 20.2° for
molecules A, B, and C, respectively, compared to the apo
structure (PDB code 1FTO, molecule A). These values are
similar to those of agonist-bound structures without modu-
lators.16
The core of 3 binds with the same contacts to the protein as
observed for 1.13 The primary polar contacts of 3 to GluA2
residues are to Pro494 and Ser497 (Figure 3B). The N2 atom
of 3 forms a hydrogen bond to the carbonyl oxygen atom of
Pro494. No hydrogen bonds from 3 to water molecules are
seen.
As seen from Figure 3C, shape complementarities are present
between the GluA2 modulator-binding site and compound 3.
The N4 cyclopropyl moiety is at van der Waals distance from
the backbone atoms of Phe495 and Met496. In addition, there
is an extensive water molecule network (Figure 4A), also at van
der Waals distance from the cyclopropyl group. Analysis of the
binding-site water molecules shows 5−7 water molecules are
located within a 4 Å distance of compound 3. However, these
water molecules do not form hydrogen-bonding interactions
with 3, but are engaged in the formation of hydrogen bonds to
residues Phe495, Ser497, Ser729, Lys730, Ser754, and Asp760.
Compared to the GluA2-LBD-L483Y-N754S with 1 bound,
small differences in the pocket accommodating the cyclopropyl
a
3
1
b
Kd (μM)
0.35 0.02
−7.5 0.2
−1.3 0.2
5.6 0.9
−4.9 0.4
−2.3 0.4
2.3 0.1
ΔH (kcal/mol)
−TΔS (kcal/mol)
nH
2.7 0.1
a
b
The values are from Krintel et al.13 Standard deviation.
and fluorine atom on the improvement of the binding affinity
for AMPA receptors. To get such detailed information on the
binding mode of 3, we determined a high-resolution structure
of GluA2-LBD-L483Y-N754S in complex with (S)-glutamate
and 3. Data collection and refinement statistics are shown in
Table 3. The complex crystallizes as a dimer with (S)-glutamate
located at the agonist-binding site and two molecules of 3 at the
dimer interface (Figure 3A). We have previously shown that the
L483Y mutation does not alter the GluA2 dimer interface and
binding mode of cyclothiazide,13 and as Tyr483 is located
approximately 9 Å from compound 3, it most likely does not
affect the binding mode of the modulator. Two different dimers
are observed; one dimer is composed of molecule A and a
symmetry-related molecule A and the second dimer of
molecules B and C. The agonist-binding site is positioned in
a cavity between the so-called D1 and D2 lobes that are
connected via a hinge region. Upon (S)-glutamate binding, the
D
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Figure 3. X-ray structure of dimeric GluA2-LBD-L483Y-N754S in complex with compound 3. (A) Cartoon representation of the GluA2 dimer
(molecules B and C). Two molecules of 3 (in yellow stick representation) bind at the dimer interface, and (S)-glutamate (in black stick
representation) binds at the agonist-binding site. The protein molecules are shown in cyan (molecule B) and salmon (molecule C). (B) Zoom on
the modulator-binding site. A simulated annealing 2Fo − Fc OMIT electron-density map for 3 (in dark gray, contoured at the 1σ level and carved 2.0
Å around 3) is shown. Possible hydrogen-bonding interactions between GluA2 residues and 3 within 3.5 Å are shown as dashed lines. (C) Surface
representation of the modulator-binding site, showing shape complementarities between GluA2 and 3.
group of 3 or the ethyl group of 1 are seen. In particular, the
cyclopropyl ring seems to form stacking interactions with the
peptide backbone (Figure 4A), probably as a result of the fact
that the cyclopropyl ring has some π-character.17 All these
nonbonded interactions are likely to contribute to the better
affinity and enthalpy of binding of 3 compared to 1.
mediated contact to Ser729 through two water molecules at the
B−C dimer interface and in a water-mediated contact to Ser497
of a symmetry-related molecule A through one water molecule
in the A−A(sym) dimer. Thus, both conformations of the side
chain of Ser497 will contribute to stabilization of the dimer
interface.
The oxygen atoms of the sulfonamide are also engaged in van
der Waals interactions, one oxygen to the side chains of Lys493
and Pro494 and the other oxygen to the side chain of Lys730
(not shown). The fluorine atom in the 7-position of 3 is
directed toward the side chain of Ser497, which was located in
two conformations. In one conformation, a fluorine hydrogen
bond to the side-chain hydroxyl group of Ser497 (3.2−3.3 Å) is
seen, Figure 3B. The occupancy of this conformation has been
refined to 0.42, 0.33, and 0.46 for molecules A, B, and C,
respectively, whereas compound 3 is present with full
occupancy. As the fluorine hydrogen-bonding distance is
above 3.2 Å and as Ser497 is present in two conformations,
this hydrogen bond is not very strong. Analysis of the
hydrogen-bonding pattern of the other side-chain conformation
of Ser497 shows the hydroxyl group to be engaged in a water-
Among compounds 2−9, most of the substituents in the 7-
position are of electron-withdrawing character, and generally,
such substituents will enhance the binding due to more
favorable π-stacking interactions of 3 to the peptide backbone
of Lys730 and Gly731 and the other molecule of 3 in the
binding site.18 There is, however, not much space in this
position since the cyclopropyl group of the other molecule of 3
is within 4.5 Å of the substituent. To accommodate larger
substituents in the 7-position than, e.g., a cyano group, such
compounds will need to adopt another binding mode to avoid
steric clashes.
Compound 3 shows a binding mode similar to that of IDRA-
21, which belongs to the shifted thiazide class.19 Compound 3
is a much more potent and efficacious compound than IDRA-
21 (the concentration of IDRA-21 giving a 2-fold increase of
E
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Figure 4. Binding mode of 3 compared to that of 1 (A) and cyclothiazide (B) at GluA2-LBD-L483Y-N754S. The structures of GluA2-LBD-L483Y-
N754S in complex with 3 (modulator shown in yellow and protein molecules in cyan and salmon for molecules B and C, respectively), 1 (residues in
light gray and 1 in dark gray, PDB code 3TDJ, molecules A and B), and cyclothiazide (residues in light gray and cyclothiazide in dark gray, PDB code
3TKD, molecules A and B) have been superimposed on lobe D1 residues. Selected residues are shown. Water molecules are shown as spheres, in red
for the structure with 3, in dark gray for the structure with 1, and in dark gray for the structure with cyclothiazide.
compound. All reactions were routinely checked by TLC on silica gel
Merck 60 F254
the magnitude of the current induced by (S)-AMPA being 134
μM10). A comparison of the binding mode of compound 1 and
IDRA-21 has previously been reported,13 suggesting that the
major structural factors increasing the potency of 1 over IDRA-
21 are increased van der Waals contacts. Compound 3 shows a
different binding mode compared to cyclothiazide, which
belongs to the classical thiazide class (Figure 4B). However, in
both classes, two modulator molecules are bound at the dimer
interface.
.
General Synthetic Pathway to 7-Substituted 4-Cyclopropyl-
3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides 2−9.
Finely ground NaBH4 (1 g, 26.4 mmol) was added to a solution of
the appropriate 7-substituted 4-cyclopropyl-4H-1,2,4-benzothiadiazine
1,1-dioxide 13 or 18a−f (1.9 g, 6.3−8.6 mmol) or 15 (1.2 g, 4.68
mmol) in 2-propanol (40−50 mL) and then heated at 50−55 °C for
5−10 min. The solvent was removed by evaporation under reduced
pressure. The residue was taken up in water (50 mL) and brought to
acidic pH by adding 6 N HCl. The title compound was extracted with
dichloromethane (3 × 30 mL). The organic phase was dried over
MgSO4 and filtered. The filtrate was evaporated to dryness, and the
residue was recrystallized from methanol/water (1/1) (60 mL) (yield
80%) except for compound 4, which was purified by chromatography
on a silica column (mobile phase chloroform) and then recrystallized
from methanol (yield 60%).
CONCLUSION
■
We report the synthesis of eight new allosteric modulators
containing a 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide
scaffold. Compound 3 with a cyclopropyl group in the 4-
position and fluorine in the 7-position was found in a functional
study to potentiate AMPA-evoked currents by a factor of 15
with an EC50 of 0.90 μM. The binding affinity of 3 determined
by isothermal titration calorimetry was seen to be in the
submicromolar range, with a Kd of 0.35 μM. The detailed
molecular interactions of 3 at GluA2-LBD-L483Y-N754S were
studied by X-ray crystallography. Compared to compound 1,
the cyclopropyl group was found to possess shape comple-
mentarity to the GluA2 LBD and to form stacking interactions
with the peptide backbone. On the basis of these compounds,
new routes of synthesis of related compounds might lead to
positive allosteric modulators with an improved activity profile
in future pharmacological and structural studies.
Data for 4-Cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine
1
1,1-Dioxide (2). White solid. Mp: 165−166 °C. H NMR (DMSO-
d6): δ 0.65 (m, 2H, CH(CH2)2), 0.91 (m, 2H, CH(CH2)2), 2.48 (m,
1H, CH(CH2)2), 4.66 (s, 2H, 3-CH2), 6.86 (t, J = 7.5 Hz, 1H, 7-H),
7.27 (d, J = 8.5 Hz, 1H, 5-H), 7.45 (t, J = 7.9 Hz, 1H, 6-H), 7.53 (dd, J
= 7.9 Hz, 0.9 Hz, 1H, 8-H), 7.85 (s, 1H, NH). 13C NMR (DMSO-d6):
δ 8.4 (CH(CH2)2), 29.6 (CH(CH2)2), 61.0 (C-3), 114.5 (C-5), 117.3
(C-7), 123.2 (C-8a), 124.2 (C-8), 132.9 (C-6), 144.1 (C-4a). Anal.
Calcd for C10H12N2O2S: C, 53.55; H, 5.39; N, 12.49; S, 14.29. Found:
C, 53.49; H, 5.30; N, 12.63; S, 14.04.
Data for 4-Cyclopropyl-7-fluoro-3,4-dihydro-2H-1,2,4-benzothia-
1
diazine 1,1-Dioxide (3). White solid. Mp: 163−164 °C. H NMR
(DMSO-d6): δ 0.64 (m, 2H, CH(CH2)2), 0.90 (m, 2H, CH(CH2)2),
2.49 (m, 1H, CH(CH2)2), 4.65 (s, 2H, 3-CH2), 7.31 (dd, J = 9.3 Hz,
4.5 Hz, 1H, 5-H), 7.38 (m, 1H, 6-H), 7.41 (m, 1H, 8-H), 7.99 (br s,
1H, NH). 13C NMR (DMSO-d6): δ 8.4 (CH(CH2)2), 29.8
(CH(CH2)2), 61.0 (C-3), 110.3 (d, J = 24 Hz, C-8), 116.6 (d, J = 7
Hz, C-5), 120.5 (d, J = 23 Hz, C-6), 123.1 (d, J = 6 Hz, C-8a), 141.1
(C-4a), 152.9−154.8 (d, J = 238 Hz, C-7). Anal. Calcd for
C10H11FN2O2S: C, 49.57; H, 4.58; N, 11.56; S, 13.23. Found: C,
49.34; H, 4.66; N, 11.70; S, 13.12.
EXPERIMENTAL SECTION
General Procedures. Melting points were determined on a Buchi
■
̈
Tottoli capillary apparatus and are uncorrected. The 1H and 13C NMR
spectra were recorded on a Bruker Avance (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 br = broad 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.
This analytical method certified a purity of ≥95% for each tested
Data for 7-Chloro-4-cyclopropyl-3,4-dihydro-2H-1,2,4-benzo-
thiadiazine 1,1-Dioxide (4). White solid. Mp: 159−161 °C. 1H
NMR (DMSO-d6): δ 0.66 (m, 2H, CH(CH2)2), 0.92 (m, 2H,
CH(CH2)2), 2.53 (m, 1H, CH(CH2)2), 4.68 (s, 2H, 3-CH2), 7.29 (d, J
= 9.1 Hz, 1H, 5-H), 7.50 (dd, J = 9.1 Hz, 2.6 Hz, 1H, 6-H), 7.53 (d, J =
2.5 Hz, 1H, 8-H), 8.01 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 8.4
F
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(CH(CH2)2), 29.7 (CH(CH2)2), 60.9 (C-3), 116.7 (C-5), 120.9 (C-
7), 123.3 (C-8), 123.9 (C-8a), 132.8 (C-6), 143.0 (C-4a). Anal. Calcd
for C10H11ClN2O2S: C, 46.42; H, 4.29; N, 10.83; S, 12.39. Found: C,
46.44; H, 4.22; N, 11.01; S, 12.82.
Data for 7-Bromo-4-cyclopropyl-3,4-dihydro-2H-1,2,4-benzo-
thiadiazine 1,1-dioxide (5). White solid. Mp: 178−179 °C. 1H
NMR (DMSO-d6): δ 0.66 (m, 2H, CH(CH2)2), 0.91 (m, 2H,
CH(CH2)2), 2.52 (m, 1H, CH(CH2)2), 4.68 (s, 2H, 3-CH2), 7.24 (d, J
= 9 Hz, 1H, 5-H), 7.60 (dd, J = 9 Hz, 2.4 Hz, 1H, 6-H), 7.63 (d, J =
2.2 Hz, 1H, 8-H), 8.02 (s, 1H, NH). 13C NMR (DMSO-d6): δ 8.4
(CH(CH2)2), 29.6 (CH(CH2)2), 60.9 (C-3), 108.0 (C-7), 117.1 (C-
5), 124.4 (C-8a), 126.0 (C-8), 135.5 (C-6), 143.3 (C-4a). Anal. Calcd
for C10H11BrN2O2S: C, 39.62; H, 3.66; N, 9.24; S, 10.57. Found: C,
39.69; H, 3.67; N, 9.38; S, 10.58.
Data for 4-Cyclopropyl-7-methyl-3,4-dihydro-2H-1,2,4-benzo-
thiadiazine 1,1-Dioxide (6). White solid. Mp: 144−145 °C. 1H
NMR (DMSO-d6): δ 0.63 (m, 2H, CH(CH2)2), 0.88 (m, 2H,
CH(CH2)2), 2.24 (s, 3H, CH3), 2.44 (m, 1H, CH(CH2)2), 4.61 (s,
2H, 3-CH2), 7.18 (d, J = 8.6 Hz, 1H, 5-H), 7.27 (dd, J = 8.7 Hz, 1.9
Hz, 1H, 6-H), 7.34 (d, J = 2 Hz, 1H, 8-H), 7.82 (s, 1H, NH). 13C
NMR (DMSO-d6): δ 8.3 (CH(CH2)2), 19.6 (CH3), 29.6 (CH-
(CH2)2), 61.1 (C-3), 114.7 (C-5), 123.2 (C-8a), 123.9 (C-8), 126.5
(C-7), 133.7 (C-6), 142.0 (C-4a). Anal. Calcd for C11H14N2O2S: C,
55.44; H, 5.92; N, 11.76; S, 13.45. Found: C, 55.41; H, 5.94; N, 11.92;
S, 13.05.
part of the ammonia were removed by evaporation under reduced
pressure. The aqueous solution/suspension obtained was adjusted to
neutral pH by adding 6 N HCl. The precipitate formed was collected
by filtration and washed with water. The crude product was suspended
in water (200 mL), and 10% NaOH was added until the pH was
clearly alkaline. The suspension was gently heated to facilitate
dissolution of the title product. The residual insoluble material was
removed by filtration, and the cooled filtrate was adjusted to neutral or
slightly acidic pH by adding 6 N HCl. The resulting precipitate was
collected by filtration, washed with water, and dried. Yield: 50−55%.
Mp: 180−183 °C. 1H NMR (DMSO-d6): δ 7.97 (d, J = 8.7 Hz, 1H, 3-
H), 8.00 (s, 2H, SO2NH2), 8.43 (dd, J = 8.7 Hz, 2.7 Hz, 1H, 4-H),
8.67 (d, J = 2.7 Hz, 1H, 6-H). 13C NMR (DMSO-d6): δ 123.6 (C-6),
127.8 (C-4), 133.3 (C-3), 137.1 (C-2), 142.1 (C-1), 145.9 (C-5).
2-(Cyclopropylamino)-5-nitrobenzenesulfonamide (12). 11
(5 g, 21 mmol) was introduced into a hermetically closed vessel
containing a mixture of dioxane (70 mL) and cyclopropylamine (3.5
mL, 50 mmol). The hermetically closed vessel was placed in an oven at
100 °C for 24 h. After this time period, the solvent and the reagent
were removed by distillation under reduced pressure. The residue was
taken up in methanol (20 mL), and the insoluble material, which
contains the title product, was collected by filtration, washed with
methanol, and dried (yield: 90%). This compound was used in the
1
next step without further purification. H NMR (DMSO-d6): δ 0.63
(m, 2H, CH(CH2)2), 0.91 (m, 2H, CH(CH2)2), 2.67 (m, 1H,
CH(CH2)2), 6.96 (br s, 1H, NH), 7.28 (d, J = 9.3 Hz, 1H, 3-H), 7.72
(br s, 2H, SO2NH2), 8.28 (dd, J = 9.3 Hz, 2.7 Hz, 1H, 4-H), 8.48 (d, J
= 2.7 Hz, 1H, 6-H). 13C NMR (DMSO-d6): δ 7.4 (CH(CH2)2), 25.0
(CH(CH2)2), 113.1 (C-3), 124.5 (C-1), 125.0 (C-6), 128.7 (C-4),
135.5 (C-5), 150.1 (C-2).
4-Cyclopropyl-7-nitro-4H-1,2,4-benzothiadiazine 1,1-Diox-
ide (13). In a round-bottom flask, a mixture of 12 (5 g, 19.4
mmol) and ethyl orthoformate (50 mL) was heated in the open state
at 130 °C for 3 h. The resulting suspension was cooled on an ice bath,
and the insoluble material was collected by filtration, washed with
ether, and dried. Yield: 85%. Mp: 233−235 °C. 1H NMR (DMSO-d6):
δ 1.08 (m, 2H, CH(CH2)2), 1.20 (m, 2H, CH(CH2)2), 3.46 (m, 1H,
CH(CH2)2), 8.07 (d, J = 9.3 Hz, 1H, 5-H), 8.29 (s, 1H, 3-H), 8.56 (d,
J = 2.6 Hz, 1H, 8-H), 8.60 (dd, J = 9.3 Hz, 2.6 Hz, 1H, 6-H). 13C
NMR (DMSO-d6): δ 7.5 (CH(CH2)2), 32.8 (CH(CH2)2), 118.9 (C-
5), 120.2 (C-8), 122.0 (C-8a), 127.8 (C-6), 141.7 (C-7), 144.6 (C-4a),
152.5 (C-3).
7-Amino-4-cyclopropyl-4H-1,2,4-benzothiadiazine 1,1-Diox-
ide (14). To a solution of 13 (5 g, 18.7 mmol) in ethanol (180 mL)
was added 10% palladium on carbon (500 mg). The suspension was
placed in a hydrogenator under H2 (10 atm) for 30 min at room
temperature. The suspension was concentrated to dryness under
reduced pressure. The residue was taken up in boiling acetone (300
mL). The insoluble material was removed by filtration in the hot state
and was washed with boiling acetone. The filtrate was concentrated to
dryness, and the residue was recrystallized from methanol. Yield: 80%.
Mp: 283−285 °C. 1H NMR (DMSO-d6): δ 0.95 (m, 1H, CH(CH2)2),
1.11 (m, 1H, CH(CH2)2), 3.28 (m, 1H, CH(CH2)2), 5.72 (s, 2H,
NH2), 6.95 (d, J = 2.5 Hz, 1H, 8-H), 6.98 (dd, J = 9 Hz, 2.6 Hz, 1H, 6-
H), 7.54 (d, J = 9 Hz, 1H, 5-H), 7.92 (s, 1H, 3-H). 13C NMR (DMSO-
d6): δ 7.0 (CH(CH2)2), 31.9 (CH(CH2)2), 105.3 (C-8), 117.9 (C-5),
119.1 (C-6), 123.6 (C-8a), 125.8 (C-4a), 147.9 (C-7), 149.4 (C-3).
7-Chloro-4-cyclopropyl-4H-1,2,4-benzothiadiazine 1,1-Diox-
ide (15). A solution of CuSO4·5H2O (84 g, 336 mmol) and NaCl
(22.5 g, 385 mmol) in water (200 mL) was cooled on an ice bath, and
an aqueous solution of Na2S2O5 (22.5 g in 100 mL, 118 mmol) was
added dropwise. After the resulting solution was stirring for 15 min,
the precipitate of Cu2Cl2 was collected by filtration and washed with
water. A solution of 14 (3.48 g, 14.7 mmol) in 6 N HCl (40 mL) was
cooled on an ice bath, and then an aqueous solution of NaNO2 (2 g in
15 mL, 29 mmol) was added dropwise. The resulting solution was
added gradually to a solution of Cu2Cl2 (3 g, 15.2 mmol) in
concentrated HCl (30 mL). After the resulting solution was stirred for
30 min at room temperature, water (150 mL) was added to the
reaction mixture, and the resulting precipitate was collected by
Data for 4-Cyclopropyl-7-trifluoromethyl-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-Dioxide (7). White solid. Mp: 155−157 °C. 1H
NMR (DMSO-d6): δ 0.71 (m, 2H, CH(CH2)2), 0.96 (m, 2H,
CH(CH2)2), 2.64 (m, 1H, CH(CH2)2), 4.78 (s, 2H, 3-CH2), 7.42 (d, J
= 9.5 Hz, 1H, 5-H), 7.77 (m, 1H, 6-H/8-H), 8.10 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 8.4 (CH(CH2)2), 29.9 (CH(CH2)2), 60.9 (C-3),
115.3 (C-5), 116.7 (d, J = 33 Hz, C-7), 121.3 (d, J = 4 Hz, C-8), 122.4
(C-8a), 123.1−125.2 (d, J = 271 Hz, CF3), 129.4 (d, J = 3 Hz, C-6),
146.7 (C-4a). Anal. Calcd for C11H11F3N2O2S: C, 45.20; H, 3.79; N,
9.58; S, 10.97. Found: C, 45.41; H, 4.02; N, 9.67; S, 11.02.
Data for 4-Cyclopropyl-3,4-dihydro-7-nitro-2H-1,2,4-benzothia-
1
diazine 1,1-Dioxide (8). White solid. Mp: 198−201 °C. H NMR
(DMSO-d6): δ 0.77 (m, 2H, CH(CH2)2), 1.01 (m, 2H, CH(CH2)2),
2.76 (m, 1H, CH(CH2)2), 4.86 (s, 2H, 3-CH2), 7.41 (d, J = 9.2 Hz,
1H, 5-H), 8.29 (m, 3H, 6-H/8-H/NH). 13C NMR (DMSO-d6): δ 8.4
(CH(CH2)2), 30.5 (CH(CH2)2), 61.1 (C-3), 115.1 (C-5), 120.4 (C-
8), 121.6 (C-8a), 128.0 (C-6), 136.3 (C-7), 148.7 (C-4a). Anal. Calcd
for C10H11N3O4S: C, 44.60; H, 4.12; N, 15.60; S, 11.91. Found: C,
44.75; H, 4.51; N, 16.00; S, 12.23.
Data for 7-Cyano-4-cyclopropyl-3,4-dihydro-2H-1,2,4-benzothia-
1
diazine 1,1-Dioxide (9). White solid. Mp: 259−261 °C. H NMR
(DMSO-d6): δ 0.71 (m, 2H, CH(CH2)2), 0.97 (m, 2H, CH(CH2)2),
2.65 (m, 1H, CH(CH2)2), 4.79 (s, 2H, 3-CH2), 7.35 (d, J = 9 Hz, 1H,
5-H), 7.82 (dd, J = 9 Hz, 2 Hz, 1H, 6-H), 8.02 (d, J = 2 Hz, 1H, 8-H),
8.13 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 8.4 (CH(CH2)2), 30.0
(CH(CH2)2), 60.9 (C-3), 98.1 (C-7), 115.4 (C-5), 118.6 (CN), 123.0
(C-8a), 128.8 (C-8), 135.9 (C-6), 146.9 (C-4a). Anal. Calcd for
C11H11N3O2S: C, 53.00; H, 4.45; N, 16.86; S, 12.86. Found: C, 53.01;
H, 4.35; N, 16.68; S, 13.12.
2-Chloro-5-nitrobenzenesulfonamide (11). A portion of glacial
acetic acid (160 mL) was saturated for 30 min with gaseous sulfur
dioxide. To the resulting solution, cooled on an ice bath, was added
under stirring an aqueous solution of CuCl2 (7 g in 20 mL)
(suspension A). 2-Chloro-5-nitroaniline (10) (15 g, 87 mmol) was
dissolved in a mixture of glacial acetic acid (160 mL) and 16 N HCl
(40 mL). To the resulting solution, cooled in an ice−salt bath (−5
°C), was added dropwise under stirring an aqueous solution of NaNO2
(8 g in 20 mL, 116 mmol). At the end of the addition, the resulting
solution was slowly mixed with suspension A. After being stirred for 15
min, the suspension was poured onto ice (400 g). The resulting
precipitate was collected by filtration, washed with water, and
immediately redissolved in dioxane (150 mL). The solution obtained
was added gradually, under stirring, to a concentrated ammonium
hydroxide solution (300 mL) previously cooled on an ice bath. After
the resulting solution was stirred for 30 min, the organic solvent and
G
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filtration, washed with water, and dried. Yield: 75%. Mp: 229−231 °C.
1H NMR (DMSO-d6): δ 1.02 (m, 2H, CH(CH2)2), 1.16 (m, 2H,
flask, a mixture of the appropriate 5-substituted 2-(cyclopropylamino)-
benzenesulfonamide 17 (2.5 g, 8.6−11.8 mmol) and ethyl
orthoformate (25 mL) was heated in the open state at 130−150 °C
for 1−24 h. The resulting suspension was cooled on an ice bath, and
the insoluble material was collected by filtration, washed with ether,
and dried. The solid was redissolved in a hot mixture of acetone and
methanol, and the hot solution was treated with charcoal and filtered.
The filtrate was concentrated to dryness, and the residue was
recrystallized from methanol. Yield: 70−75%. The following
compounds were obtained.
CH(CH2)2), 3.38 (m, 1H, CH(CH2)2), 7.87−7.90 (m, 2H, 5-H/6-H),
7.94 (d, J = 7.8 Hz, 1H, 8-H), 8.17 (s, 1H, 3-H). 13C NMR (DMSO-
d6): δ 7.3 (CH(CH2)2), 32.3 (CH(CH2)2), 119.4 (C-5), 123.3 (C-8),
123.4 (C-8a), 130.6 (C-7), 133.3 (C-6), 135.4 (C-4a), 151.6 (C-3).
General Synthetic Pathway to 5-Substituted 2-
(Cyclopropylamino)benzenesulfonamides 17. The solution of
the appropriate 5-substituted 2-fluorobenzenesulfonamide 16 (3 g,
12−17 mmol) in dioxane (30 mL) supplemented with cyclopropyl-
amine (3 mL, 43.1 mmol) was heated at 100−110 °C in a hermetically
closed vessel for 24−96 h. The solvent and the excess amine were
removed by distillation under reduced pressure, and the residue was
dissolved in methanol (20 mL). The methanolic solution was cooled
on an ice bath, and water (60 mL) was added. The resulting precipitate
of the title compound was collected by filtration, washed with water,
and dried. It was used in the next step without further purification.
Yield: 85−95%. The following compounds were obtained.
Data for 4-Cyclopropyl-4H-1,2,4-benzothiadiazine 1,1-Dioxide
(18a). White solid. Mp: 191−193.5 °C. 1H NMR (DMSO-d6): δ 1.01
(m, 2H, CH(CH2)2), 1.17 (m, 2H, CH(CH2)2), 3.38 (m, 1H,
CH(CH2)2), 7.57 (m, 1H, 7-H), 7.81−7.85 (m, 2H, 5-H/6-H), 7.88
(d, J = 7.8 Hz, 1H, 8-H), 8.15 (s, 1H, 3-H). 13C NMR (DMSO-d6): δ
7.3 (CH(CH2)2), 32.1 (CH(CH2)2), 116.8 (C-5), 122.3 (C-8a), 124.1
(C-7), 126.8 (C-8), 133.2 (C-6), 136.5 (C-4a), 151.5 (C-3).
Data for 4-Cyclopropyl-7-fluoro-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (18b). White solid. Mp: 170−172 °C. 1H NMR (DMSO-d6):
δ 1.02 (m, 2H, CH(CH2)2), 1.16 (m, 2H, CH(CH2)2), 3.39 (m, 1H,
CH(CH2)2), 7.73 (m, 1H, 6-H), 7.79 (dd, J = 7.4 Hz, 2.9 Hz, 1H, 8-
H), 7.92 (dd, J = 9.3 Hz, 4.4 Hz, 1H, 5-H), 8.14 (s, 1H, 3-H). 13C
NMR (DMSO-d6): δ 7.3 (CH(CH2)2), 32.3 (CH(CH2)2), 110.2 (d, J
= 25 Hz, C-8), 120.0 (C-5), 121.1 (d, J = 23 Hz, C-6), 123.3 (C-8a),
133.3 (C-4a), 151.3 (C-3), 158.4−160.4 (d, J = 248 Hz, C-7).
Data for 7-Bromo-4-cyclopropyl-4H-1,2,4-benzothiadiazine 1,1-
Data for 2-(Cyclopropylamino)benzenesulfonamide (17a). Heat-
1
ing time: 96 h. H NMR (DMSO-d6): δ 0.51 (m, 2H, CH(CH2)2),
0.79 (m, 2H, CH(CH2)2), 2.46 (m, 1H, CH(CH2)2), 6.11 (s, 1H,
NH), 6.72 (t, J = 7.5 Hz, 1H, 5-H), 7.14 (d, J = 8.3 Hz, 1H, 3-H), 7.30
(s, 2H, SO2NH2), 7.40 (t, 1H, 4-H), 7.60 (dd, J = 7.8 Hz, 0.8 Hz, 1H,
6-H). 13C NMR (DMSO-d6): δ 7.25 (CH(CH2)2), 24.5 (CH(CH2)2),
112.9 (C-3), 115.4 (C-5), 125.0 (C-1), 128.0 (C-6), 133.2 (C-4),
145.4 (C-2).
1
Dioxide (18c). White solid. Mp: 242−244 °C. H NMR (DMSO-d6):
Data for 2-(Cyclopropylamino)-5-fluorobenzenesulfonamide
1
δ 1.02 (m, 2H, CH(CH2)2), 1.16 (m, 2H, CH(CH2)2), 3.37 (m, 1H,
CH(CH2)2), 7.80 (d, J = 8.8 Hz, 1H, 5-H), 8.00−8.02 (m, 2H, 6-H/8-
H), 8.18 (s, 1H, 3-H). 13C NMR (DMSO-d6): δ 7.25 (CH(CH2)2),
32.23 (CH(CH2)2), 118.2 (C-7), 119.6 (C-5), 123.6 (C-8a), 126.1 (C-
8), 135.8 (C-4a), 136.1 (C-6), 151.6 (C-3).
(17b). Heating time: 96 h. H NMR (DMSO-d6): δ 0.50 (m, 2H,
CH(CH2)2), 0.78 (m, 2H, CH(CH2)2), 2.45 (m, 1H, CH(CH2)2),
5.98 (s, 1H, NH), 7.14 (dd, J = 9.1 Hz, 4.6 Hz, 1H, 3-H), 7.33 (td, J =
8.6 Hz, 3.1 Hz, 1H, 4-H), 7.38 (dd, J = 8.9 Hz, 3.1 Hz, 1H, 6-H), 7.46
(br s, 2H, SO2NH2). 13C NMR (DMSO-d6): δ 7.3 (CH(CH2)2), 24.8
(CH(CH2)2), 114.1 (d, J = 25 Hz, C-6), 114.3 (d, J = 7 Hz, C-3),
120.2 (d, J = 22 Hz, C-4), 125.3 (d, J = 6 Hz, C-1), 142.3 (C-2), 152.7
(d, J = 234 Hz, C-5).
Data for 4-Cyclopropyl-7-methyl-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (18d). White solid. Mp: 192−195 °C. 1H NMR (DMSO-d6):
δ 0.99 (m, 2H, CH(CH2)2), 1.15 (m, 2H, CH(CH2)2), 2.42 (s, 3H,
CH3), 3.35 (m, 1H, CH(CH2)2), 7.63 (dd, J = 8.7 Hz, 1.7 Hz, 1H, 6-
H), 7.69 (d, J = 2 Hz, 1H, 8-H), 7.73 (d, J = 8.6 Hz, 1H, 5-H), 8.10 (s,
1H, 3-H). 13C NMR (DMSO-d6): δ 7.2 (CH(CH2)2), 20.2 (CH3),
32.0 (CH(CH2)2), 116.7 (C-5), 122.2 (C-8a), 123.4 (C-8), 134.0 (C-
6), 134.2 (C-7), 136.8 (C-4a), 151.1 (C-3).
Data for 5-Bromo-2-(cyclopropylamino)benzenesulfonamide
1
(17c). Heating time: 24 h. H NMR (DMSO-d6) d 0.51 (m, 2H,
CH(CH2)2), 0.80 (m, 2H, CH(CH2)2), 2.47 (m, 1H, CH(CH2)2),
6.16 (s, 1H, NH), 7.11 (d, J = 8.9 Hz, 1H, 3-H), 7.50 (s, 2H,
SO2NH2), 7.56 (dd, J = 8.9 Hz, 2.4 Hz, 1H, 4-H), 7.69 (d, J = 2.4 Hz,
1H, 6-H). 13C NMR (DMSO-d6): δ 7.26 (CH(CH2)2), 24.6
(CH(CH2)2), 105.8 (C-5), 115.3 (C-3), 126.5 (C-1), 129.9 (C-6),
135.6 (C-4), 144.6 (C-2).
Data for 4-Cyclopropyl-7-(trifluoromethyl)-4H-1,2,4-benzothia-
1
diazine 1,1-Dioxide (18e). White solid. Mp: 160−162 °C. H NMR
(DMSO-d6): δ 1.06 (m, 2H, CH(CH2)2), 1.18 (m, 2H, CH(CH2)2),
3.43 (m, 1H, CH(CH2)2), 8.04 (d, J = 9.4 Hz, 1H, 5-H), 8.19 (m, 2H,
6-H/8-H), 8.25 (s, 1H, 3-H). 13C NMR (DMSO-d6): δ 7.4
(CH(CH2)2), 32.5 (CH(CH2)2), 118.5 (C-5), 121.7 (d, J = 4 Hz,
C-8), 122.1−124.3 (d, J = 272 Hz, CF3), 122.4 (C-8a), 126.7 (d, J =
33 Hz, C-7), 129.8 (d, J = 3 Hz, C-6), 139.6 (C-4a), 152.3 (C-3).
Data for 7-Cyano-4-cyclopropyl-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (18f). White solid. Mp: 268−270 °C. 1H NMR (DMSO-d6): δ
1.04 (m, 2H, CH(CH2)2), 1.18 (m, 2H, CH(CH2)2), 3.40 (m, 1H,
CH(CH2)2), 7.98 (d, J = 8.9 Hz, 1H, 5-H), 8.23 (s, 1H, 3-H), 8.24
(dd, J = 8.9 Hz, 1.8 Hz, 1H, 6-H), 8.50 (d, J = 1.9 Hz, 1H, 8-H). 13C
NMR (DMSO-d6): δ 7.4 (CH(CH2)2), 32.5 (CH(CH2)2), 109.1 (C-
7), 117.2 (CN), 118.3 (C-5), 122.7 (C-8a), 129.4 (C-8), 136.3 (C-6),
139.8 (C-4a), 152.3 (C-3).
Data for 2-(Cyclopropylamino)-5-methylbenzenesulfonamide
1
(17d). Heating time: 72 h. H NMR (DMSO-d6): δ 0.48 (m, 2H,
CH(CH2)2), 0.77 (m, 2H, CH(CH2)2), 2.43 (m, 1H, CH(CH2)2),
5.97 (s, 1H, NH), 7.05 (d, J = 8.4 Hz, 1H, 3-H), 7.23 (dd, J = 8.4 Hz,
1.8 Hz, 1H, 4-H), 7.43 (d, J = 2 Hz, 1H, 6-H), 7.54 (br s, 2H,
SO2NH2). 13C NMR (DMSO-d6): δ 7.2 (CH(CH2)2), 19.8 (CH3),
24.6 (CH(CH2)2), 113.1 (C-3), 124.1 (C-1), 124.9 (C-5), 128.0 (C-
6), 133.8 (C-4), 143.3 (C-2).
Data for 2-(Cyclopropylamino)-5-(trifluoromethyl)-
1
benzenesulfonamide (17e). Heating time: 24 h. H NMR (DMSO-
d6): δ 0.58 (m, 2H, CH(CH2)2), 0.86 (m, 2H, CH(CH2)2), 2.58 (m,
1H, CH(CH2)2), 6.53 (s, 1H, NH), 7.29 (d, J = 8.8 Hz, 1H, 3-H), 7.58
(s, 2H, SO2NH2), 7.75 (dd, J = 8.8 Hz, 1.9 Hz, 1H, 4-H), 7.88 (d, J =
1.7 Hz, 1H, 6-H). 13C NMR (DMSO-d6): δ 7.3 (CH(CH2)2), 24.6
(CH(CH2)2), 113.4 (C-3), 115.3 (C-5), 123.4−125.6 (d, J = 270 Hz,
CF3), 124.7 (C-1), 125.4 (d, J = 4 Hz, C-6), 129.9 (d, J = 3 Hz, C-4),
148.0 (C-2).
Data for 5-Cyano-2-(cyclopropylamino)benzenesulfonamide
(17f). Heating time 24 h. 1H NMR (DMSO-d6): δ 0.57 (m, 2H,
CH(CH2)2), 0.86 (m, 2H, CH(CH2)2), 2.58 (m, 1H, CH(CH2)2),
6.68 (s, 1H, NH), 7.24 (d, J = 8.8 Hz, 1H, 3-H), 7.59 (br s, 2H,
SO2NH2), 7.81 (dd, J = 8.8 Hz, 2 Hz, 1H, 4-H), 7.91 (d, J = 2 Hz, 1H,
6-H). 13C NMR (DMSO-d6): δ 7.3 (CH(CH2)2), 24.6 (CH(CH2)2),
96.5 (C-5), 113.7 (C-3), 119.1 (CN), 125.5 (C-1), 132.5 (C-6), 136.4
(C-4), 148.3 (C-2).
Effect on AMPA-Evoked Membrane Depolarization. This
assay consisted of investigating AMPA-evoked membrane depolariza-
tion, measured by fluorescent membrane potential dyes and an
imaging based plate reader (FDSS, Hamamatsu, JP), on rat primary
brain cultures.12 Dissociated rat primary brain cells were prepared
from embryonic rat (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)
General Synthetic Pathway to 7-Substituted 4-Cyclopropyl-
4H-1,2,4-Benzothiadiazine 1,1-Dioxides 18. In a round-bottom
H
dx.doi.org/10.1021/jm4012092 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
over 3 min and then the compound in the presence of AMPA over 3
min. Subsequent monitoring of fluorescence changes was performed
during these two periods of 3 min. Responses were averaged over the
last 15 s of each 3 min recording period. AMPA concentration−
response curves were calculated in the absence or presence of different
concentrations of the compound. The results are expressed as the area
under the curve of the AMPA-mediated concentration−response effect
in the absence or presence of the compound. EC2× corresponds to the
concentration of compound that evoked a 2-fold increase of all AMPA-
mediated responses. EC50 corresponds to the concentration of
compound that evoked 50% of the maximal effect of the compound.
Protein Expression and Purification. GluA2-LBD-L483Y-
N754S was expressed and purified as previously described.13 In
brief, Escherichia coli Origami B (DE3) cells were transformed with the
GluA2-LBD-L483Y-N754S pET-22b(+) plasmid. The cells were
grown to an OD600 of 0.9 in LB medium and cooled to 20 °C.
Protein expression was induced by addition of 0.5 mM isopropyl β-D-
thiogalactopyranoside. After 18 h, the cells were harvested and lysed,
and the protein was purified by Ni2+-affinity chromatography. The N-
terminal His tag was removed by trypsin digestion, and the cleaved
protein was purified by anion-exchange chromatography and size-
exclusion chromatography.
Crystallization and X-ray Structure Determination. The
protein solution consisted of 6 mg/mL GluA2-LBD-L483Y-N754S
in 10 mM HEPES, 20 mM NaCl, 1 mM EDTA, and 1 mM (S)-
glutamate, pH 7.0. Solid compound 3 (0.5−1 mg) was added to 100
μL of protein solution and incubated at 4 °C for at least 24 h prior to
the setup of the crystallization experiments.
The hanging drop vapor diffusion method was used with drops
consisting of 1 μL of protein solution and 1 μL of reservoir solution.
The crystallization conditions were 16.5% PEG4000, 0.15 M zinc
acetate, and 0.1 M cacodylate, pH 6.5. The crystals were briefly soaked
in cryoprotectant (reservoir solution with 20% glycerol) and flash-
cooled in liquid nitrogen.
AUTHOR INFORMATION
Corresponding Author
*Phone: +45 3533 6486. E-mail: jsk@sund.ku.dk.
■
Author Contributions
∥A.-B.N. and P.F. contributed equally to this work.
Author Contributions
⊥B.P. and J.S.K. equally supervised this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank MAX-lab (Lund, Sweden) for providing beam time
and The Danish Council for Independent Research (Mobility
Ph.D. grant) (A.-B.N., K.F., L.O., J.S.K.) and GluTarget and
Danscatt (A.-B.N., C.K., K.F., L.O., J.S.K.) for financial support.
ABBREVIATIONS USED
■
ADHD, attention deficit hyperactivity disorder; AMPA, α-
amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CNS,
central nervous system; iGluRs, ionotropic glutamate receptors;
ITC, isothermal titration calorimetry; LBD, ligand-binding
domain; LTD, long-term depression; LTP, long-term potentia-
tion; NMDA, N-methyl-D-aspartic acid
REFERENCES
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ASSOCIATED CONTENT
■
Accession Codes
The structure coordinates and corresponding structure factor
file of GluA2-LBD-L483Y-N754S with 3 have been deposited
in the Protein Data Bank under the accession code 4N07.
I
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Journal of Medicinal Chemistry
Article
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