5-arylbenzothiadiazine type compounds as positive allosteric modulators of AMPA/kainate receptors

Article abstract of DOI:10.1021/ml200184w

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPAr) has led to the search for new AMPAr positive modulators. On the basis of crystallographic data of the benzothiadiazines binding mode in the S1S2 GluA2 dimer interface, a set of 5-aryl-2,3-dihydrobenzothiadiazine type compounds has been synthesized and tested. Electrophysiological results suggested that 5-heteroaryl substituents on the benzothiadiazine core like 3-furanyl and 3-thiophenyl dramatically enhance the activity as positive modulators of AMPAr with respect to IDRA21 and cyclothiazide. Mouse brain microdialysis studies have suggested that 7-chloro-5-(3-furyl)-3-methyl-3,4- dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide crosses the blood-brain barrier after intraperitoneal injection. Biological results have been rationalized by a computational docking simulation that it has currently employed to design new AMPAr-positive modulator candidates.

Full text of DOI:10.1021/ml200184w

Letter
pubs.acs.org/acsmedchemlett
5-Arylbenzothiadiazine Type Compounds as Positive Allosteric
Modulators of AMPA/Kainate Receptors
Umberto M. Battisti,†,⊥ Krzysztof Jozwiak,‡,⊥ Giuseppe Cannazza,*,† Giulia Puia,§ Gabriella Stocca,§
Daniela Braghiroli,† Carlo Parenti,† Livio Brasili,† Marina M. Carrozzo,∥ Cinzia Citti,∥
and Luigino Troisi∥,⊥
†Dipartimento di Scienze Farmaceutiche, Universita
̀
degli Studi di Modena e Reggio Emilia, Via Campi 183, 41125 Modena, Italy
‡Laboratory of Medicinal Chemistry and Neuroengineering, Department of Chemistry, Medical University of Lublin, ul. W. Chodzki
4a, 20-093 Lublin, Poland
§Dipartimento di Scienze Biomediche, Universita
̀
degli Studi di Modena e Reggio Emilia, Via Campi 287, 41125 Modena, Italy
∥Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita
Italy
̀
del Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce,
S
* Supporting Information
ABSTRACT: The potential therapeutic benefit of compounds able to activate
AMPA receptors (AMPAr) has led to the search for new AMPAr positive
modulators. On the basis of crystallographic data of the benzothiadiazines
binding mode in the S1S2 GluA2 dimer interface, a set of 5-aryl-2,3-
dihydrobenzothiadiazine type compounds has been synthesized and tested.
Electrophysiological results suggested that 5-heteroaryl substituents on the
benzothiadiazine core like 3-furanyl and 3-thiophenyl dramatically enhance the
activity as positive modulators of AMPAr with respect to IDRA21 and
cyclothiazide. Mouse brain microdialysis studies have suggested that 7-chloro-
5-(3-furyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide
crosses the blood−brain barrier after intraperitoneal injection. Biological
results have been rationalized by a computational docking simulation that it has currently employed to design new AMPAr-
positive modulator candidates.
KEYWORDS: AMPA modulators, IDRA21, benzothiadiazines, cognitive enhancers
he principal excitatory neurotransmitter in the mammalian
T
central nervous system (CNS) is L-glutamate, and its
signal transduction is mediated by ionotropic and metabotropic
receptors.1 Different studies suggest that ionotropic α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAr)
is involved in learning processes and in memory establishment.2
The therapeutic potential of compounds able to activate
AMPAr has led to the search for new AMPAr-positive
modulators.3,4 Among these, one of the most investigated
chemical classes of compounds are benzothiadiazine derivatives
such as cyclothiadiazide (CTZ) (1), ( )7-chloro-3-methyl-3,4-
dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21) (2),
and ( ) 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-
de]-1,2,4-benzothiadiazine 1,1-dioxide (3), which are able to
inhibit desensitization of AMPAr-potentiating ionotropic
glutamatergic neurotransmission (Figure 1).5−7
Figure 1. Cyclothiazide (1), IDRA21(2), and 8-chloro-2,3,5,6-
tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-
dioxide (3).
inhibition of the receptor desensitization by stabilization of the
ligand-binding domain within a dimer interface.10,11 Notwith-
standing structure similarities of IDRA21 and cyclothiazide,
crystal structures of their complexes with S1S2 GluA2 domain
reveal different binding modes.10,11 In particular, IDRA21 and
cyclothiazide bind to the dimer interface S1S2 GluA2 with a
different orientation probably due to lower steric hindrance of
C3 methyl group of IDRA21 with respect to the C3 norbornyl
substituent of cyclothiazide (Figure 2).10,11
IDRA21, the first benzothiadiazine effective in increasing
learning and memory performance in behavioral tests,
represents an important lead compound since it is able to
cross the blood−brain barrier (BBB).8,9 Recent crystallographic
studies regarding the binding mode of benzothiadiazine
derivatives on S1S2 GluA2 subunits of AMPAr suggest that
these compounds bind to the dimer interface, leading to
Received: August 1, 2011
Accepted: November 14, 2011
Published: November 14, 2011
© 2011 American Chemical Society
25
dx.doi.org/10.1021/ml200184w | ACS Med. Chem. Lett. 2012, 3, 25−29

ACS Medicinal Chemistry Letters
Letter
Table 1. Potentiation of Kainate (100 μM)-Evoked Current
Induced by the Synthesized Compound at 10 μM
Figure 2. Comparison of binding modes of IDRA21 (A) (PDB code:
3IL1) and cyclothiazide (B) (PDB code: 1LBC) in the ligand binding
site of S1S2 GluA2 dimer interface. Overlap view of IDRA21 and
cyclothiazide (C).
The crystal structure of cyclothiazide in complex with S1S2
GluA2 subunits (PDB code: 1LBC) reveals that the benzene
ring and C7 sulfonamidic group of cyclothiazide fit into a
hydrophilic pocket defined by Lys763, Tyr424, Ser497, Phe495
and Ser729 residues. In contrast, the same hydrophilic pocket is
unfilled in the crystal structure of IDRA21 in complex with
S1S2 GluA2 subunits.
Detailed analysis of the latter crystal structure reveals that the
pocket contains four water molecules ordered to form a
network of hydrogen bonds with neighboring residues. One of
these water molecules is positioned on the plane of IDRA21
aromatic ring at a distance of 3.2 Å from the C5 atom.
Therefore, we hypothesized that the affinity of IDRA21
derivatives toward AMPAr could be increased by a substituent
at C5 position of IDRA21 core that could accommodate the
empty hydrophilic pocket and form additional hydrogen bonds
with the receptor and water molecules present in this cavity.
Molecular modeling studies indicate that heteroaromatic
substituents, like thiophenyl or furanyl, and aromatic
substituents like anilinic or benzoyl on C5 position of
IDRA21 can fit into the unfilled hydrophilic pocket, miming
the binding mode of benzene ring and C7 sulfonamidic group
of cyclothiazide (Figure 3).12
a
Each value is the mean SEM of 5−8 experiments.
a
Scheme 1.
Figure 3. Overlay of IDRA21 (red) and cyclothiazide (blue) led to the
design of new IDRA21 analogues.
Hence, several 5-aryl-7-chloro-3-methyl-3,4-dihydro-2H-
1,2,4-benzothiadiazine 1,1-dioxide derivatives were prepared
by a synthetic route based on the Pd-catalyzed Suzuki−Miyaura
coupling reaction (Table 1). The general synthetic pathway
employed is shown in Scheme 1.
Halogenation of 2-amino-5-chlorobenzensulfonamide (4a) in
acidic conditions gave 4b, which subsequently afforded
intermediate 4c by Pd-catalyzed cross-coupling reaction with
a suitable boronic acid. The 5-aryl-7-chloro-3-methyl-3,4-
dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives
were obtained by ring closure with a selected aldehyde under
catalytic acidic conditions. Compounds 11 and 12 were
obtained by an alternative synthetic pathway: The coupling
reaction with 3-aminophenylboronic acid was conducted on 7-
chloro-5-iodo-3-alkyl-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxide, since aldehyde reacted with the nitrogen atom of
a
(i) ICl, acetic acid, DMF. (ii) Na2CO3, RB(OH)2 tetrakis palladium,
H2O/dioxane. (iii) R1CHO, 2-propanol, HCl.
the anilinic group of the aromatic substituents at C5 during
benzothiadiazine ring closure (Scheme 2).
The prepared compounds described in Table 1, as well as
their intermediates, were fully characterized by IR, ESI, and
NMR spectroscopic analysis. Subsequently, the activities of
synthesized compounds were evaluated by patch-clamp
technique in primary cultures of cortical neurons as allosteric
modulators of AMPA/kainate receptors.13,14 The capability of
potentiating kainate-evoked current was tested for all of the
newly synthesized compounds. Kainate-evoked current was
mainly mediated by AMPA receptor activation because
26
dx.doi.org/10.1021/ml200184w | ACS Med. Chem. Lett. 2012, 3, 25−29

ACS Medicinal Chemistry Letters
Letter
a
has a critical influence . The preferred substituent appears to be
CH3 (5) since H or CH2CH3 led to compounds (6 and 7) with
lower activity as KA-current potentiators. Because of its activity,
9 represents a promising lead compound for the development
of new potent AMPAr-positive allosteric modulators.
Scheme 2.
Molegro Virtual Docker (MVD) software has been employed
to dock 5, 9, and 10 into the ligand binding site of S1S2 GluA2
dimer (PDB code: 3IL1) to understand their binding mode.12
The software MVD was evaluated on cyclothiazide, triflume-
thiazide, diazoxide, althiazide, hydrochlorothiazide, chlorothia-
zide, trichlormethiazide, and IDRA21. The average root-mean-
square distance (rmsd) of the best ranking pose of tested
compounds as compared to their binding pose in the respective
crystal structures was found to be 0.81 Å, proving that MVD is
able to accurately dock this type of compounds (see the
Supporting Information). Compounds 5, 9, and 10 compounds
were built by the software Spartan'08 and energy minimized
using Hartree−Fock ab initio method employing at the SCF
level with the 6-31G* basis set.
The docking output clearly indicates that 5, 9, and 10 bind
similarly to IDRA21 at the dimer interface of S1S2 GluA2
subunits with the C3 methyl substituent placed into the
hydrophobic cleft defined by Val750, Leu751, and Leu259
residues, the hydrogen atom on N4 of benzothiadiazine gave an
hydrogen bond with Ser754 (H-bond length 3.39 Å) residue,
and the sulfone oxygen atom of benzothiadiazine gave a polar
interaction with Gly731 residue (Figure 5) since it is located
a
R1CHO, 2-propanol, HCl. (ii) Na2CO3, RB(OH)2 tetrakis palladium,
H2O/dioxane.
application of GYKI 53655 (100 μM), an AMPA receptors
antagonist, almost completely abolished the current (data not
shown). Because 3 is one of the most active benzothiadiazine
derivatives reported in literature as a AMPAr-positive allosteric
modulator, it was selected as reference compound.4 Data
obtained indicate that 5, 9, and 10, at 10 μM, potentiate by 80
16, 358
80, and 214
41%, respectively, the kainate-
evoked currents at 10 μM (Tab.1). The dose−response curve
of the most active compound 9 reveals an EC50 of 1.3 μM,
suggesting that this benzothiadiazine derivative is one of the
most potent within this class of AMPAr-positive allosteric
modulators (Figure 4).
Figure 5. Binding of 9 and IDRA21 to the S1S2 GluA2 dimer
interface; the secondary structures of the two S1S2 monomers are
colored in yellow and magenta; green sticks illustrate plausible
hydrogen bond interactions between 9 and the receptor residues, both
colored in element color code; the IDRA21 molecule present in the
crystal model is colored in orange.
Figure 4. Modulation of KA-evoked current by compound 9. (a)
Representative whole cell recording showing KA (100 μM)-evoked
current recorded from a cortical neuron in culture in control
conditions (left traces), after application of 9 (300 nM) (middle
trace) and after washing (right trace). (b) Dose−response curve of the
effect of 9 on KA-evoked current. Each data point is the mean SEM
of at least five cells.
within 2.78 Å from the backbone of this residue. These
interactions are observed also in the crystal structure of
IDRA21 in complex with S1S2 GluA2 subunits.
The increased activity of 5, 9, and 10 with respect to IDRA21
can be explained by additional interactions with the ligand
binding site due to heteroaryl substituent in the C5 position of
the benzothiadiazine core that can be accommodated in the
unfilled hydrophilic pocket previously recognized in the crystal
structure of S1S2 GluA2 domain in complex with IDRA21.
Compounds 9 and 10 have shown higher activity with
respect to 5 probably because of a H-bond between the
heteroatom of 3-furanyl or 3-thiophenyl substituent with the
Ser497 residue of S1S2 GluA2 domain. In particular, the
heteroatom of the C5 substituent of 9 and 10 is within
hydrogen bond distance (2.71 Å) to the hydroxyl group of
Ser497. Moreover, 9 is more active than 10 probably due to the
major electronegativity of oxygen atom of 3-furanyl that allows
The analysis of these preliminary biological results suggests a
net of different molecular effects of these ligands, which affects
the potentiation of KA-current activity. In particular, the activity
significantly increases with a five-membered aromatic ring as
the C5 substituent, whereas compounds with a six-membered
aromatic ring show a very low activity. This effect is probably
due to a sterical hindrance that prevent them from fitting into
the hydrophilic pocket at S1S2 GluA2 dimer interface.
Moreover, the maximum of activity is achieved when the
heteroatom is at 3-position of the heteroaryl substituent. The
activities of compounds 5−7 indicate that the C3 substituent
27
dx.doi.org/10.1021/ml200184w | ACS Med. Chem. Lett. 2012, 3, 25−29

ACS Medicinal Chemistry Letters
Letter
a stronger H-bond with respect to the sulfur atom of the 3-
thiophenyl substituent.
route has been developed to prepare the designed 5-aryl-3,4-
dihydrobenzothiadiazine compounds. Electrophysiological re-
sults suggested that 5-heteroaryl substituents like 3-furanyl and
3-thiophenyl dramatically enhance the activity as positive
modulators of AMPAr with respect to IDRA21 and cyclo-
thiazide. Biological results have been rationalized by a
computational docking simulations that are employed to design
new AMPAr-positive modulator candidates. Moreover, mouse
cerebral microdialysis studies suggested that 9 crosses the BBB
after intraperitoneal injection since it has been detected at a
micromolar concentration in mouse nucleus accumbens
dialysates. Low levels of the corresponding unsatured 17
derivative have been detected in mouse nucleus accumbens
dialysates, suggesting a moderate action of cytocrome P450 on
9. Efforts aimed at a further optimization, as well as in-depth
biological studies, of the novel compounds are ongoing, and the
results will be reported in due course.
The good activity of 9 as positive allosteric modulator of
AMPAr in vitro suggests that it could exert cognition enhancing
properties when administered in vivo. With the aim to evaluate
if 9 can elicit AMPAr activity on the CNS, its capability to cross
the BBB was investigated. Recently, it has been reported that
3,4-dihydro-2H-1,2,4-benzothiadiazine derivatives in vivo can
hydrolyze in acidic conditions to corresponding 2-amino-
benzensulfonamide.15 Moreover, Pirrotte et al. have recently
reported that 4-alkyl-3,4-dihydro-2H-1,2,4-benzothiadiazine
derivatives undergo the action of hepatic cytochrome P450,
giving the corresponding 4-alkyl-2H-1,2,4-benzothiadiazine
derivative.16 As a consequence, it is possible that 9 in vivo
can be hydrolyzed to 5-chloro-3-(3-furyl)-2-aminobenzensulfo-
namide (16) and metabolized to give 7-chloro-5-(3-furyl)-3-
methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (17).
In the attempt to evaluate if 9 crosses the BBB and if it
produces 16 and 17 in vivo, cerebral microdialysis experiments
have been performed in mice to measure cerebral levels of 9,
16, and 17 after intraperitoneal administration of 9. A liquid
chromatography tandem mass spectrometry (LC/MS/MS)
method has been developed to evaluate concentrations of
parent ompound 9 and of their possible metabolites 16 and 17
in cerebral mouse dialysates. The identity of the compounds 9,
16, and 17 was confirmed by comparing their retention times
and their MS/MS profiles after injection of the corresponding
synthetic authentic compounds in the HPLC system. Figure 6
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, compound characterizations, compu-
tational methods, and pharmacology. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +39 059 2055136. Fax: +39 059 2055750. E-mail:
giuseppe.cannazza@unimore.it.
Author Contributions
⊥These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. M. Zoli and Dr. S. Guiducci for the help in
microdialysis experiments.
REFERENCES
■
(1) Dingledine, R.; Borges, K.; Bowie, D.; Traynelis, S. The glutamate
receptor ion channels. Pharmacol. Rev. 1999, 51, 7−61.
Figure 6. Time course of 9 and 17 outputs from mouse nucleus
accumbens after a single administration of 9 (20 mg/kg ip). Values are
expressed as μM of mean values SEMs of dialysates concentrations.
(2) Kew, J. N. C.; Kemp, J. A. Psychopharmacology 2005, 179, 4.
(3) Black, M. D. Therapeutic potential of positive AMPA modulators
and their relationship to AMPA receptor subunits. A review of
preclinical data. Psychopharmacology 2005, 179, 154−163.
(4) Swanson, G. T. Targeting AMPA and kainate receptors in
neurological disease: Therapies on the horizon?
Neuropsychopharmacology 2009, 34, 249−250.
(5) Vyklicky, L.; Patneau, D. K.; Mayer, M. L. Modulation of
excitatorysynaptic transmission by drugs that reduce desensitization at
AMPA/kainate receptors. Neuron 1991, 7, 971−984.
(6) Bertolino, M.; Baraldi, M.; Parenti, C.; Braghiroli, D.; DiBella, M.;
Vicini, S.; Costa, E. Recept. Channels 1993, 1, 267.
(7) Philips, D.; Sonnenberg, J.; Arai, A. C.; Vaswani, R.; Krutzik, P.
O.; Kleisli, T.; Kessler, M.; Granger, R.; Lynch, G; Chamberline, A. R.
5′-Alkyl-benzothiadiazides: A New Subgroup of AMPA Receptor
Modulators with Improved Affinity. Bioorg. Med. Chem. 2002, 10,
1229−1248.
(8) Arai, A.; Guidotti, A.; Costa, E.; Lynch, G. Effect of the AMPA
receptor modulator IDRA 21 on LTP in hippocampal slices.
NeuroReport 1996, 7, 2211−2215.
(9) Thompson, D. M.; Guidotti, A.; DiBella, M.; Costa, E. 7-Chloro-
3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide (IDRA
21), a congener of aniracetam, potently abates pharmacologically
shows the extracellular levels of 9 and 17 in mouse nucleus
accubens after intraperitoneal administration of 9 (20 mg/kg).
Dialysate concentrations of 16 were under the limit of
detection of the analytical method and are not reported in the
graph. The concentrations of 9 increased in the first 40 min
after administration to reach the concentration of 37 μM and
then gradually decreased and became undetectable after 2 h.
Levels of 17 increased in the first 1 h to reach the concentration
of 8 μM, then decreased slowly for the next 2 h to be
undetectable at 140 min. The microdialysis results indicate that
9 reaches the CNS after intraperitoneal injection. Moreover, 9
is subject to a moderate action of cytocrome P450 to give 17,
while it does not undergo hydrolysis after intraperitoneal
administration. In summary, a novel class of positive allosteric
modulators of AMPAr has been designed based on crystal
structures of IDRA21 and cyclothiazide in complex with the
S1S2 GluA2 dimer interface. An efficient and versatile synthetic
28
dx.doi.org/10.1021/ml200184w | ACS Med. Chem. Lett. 2012, 3, 25−29

ACS Medicinal Chemistry Letters
Letter
induced cognitive impairments in patas monkeys. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 7667−7671.
(10) Sun, Y.; Olson, R.; Horning, M.; Armstrong, N.; Mayer, M.;
Gouaux, E. Mechanism of glutamate receptor desensitization. Nature
2002, 417, 245−253.
(11) Ptak, C. P.; Ahmed, A. H.; Oswald, R. E. Probing the Allosteric
Binding Site of Glur2 with Thiazide derivatives. Biochemistry 2009, 48,
8594−8602.
(12) Thomsen, R.; Christensen, M. H. MolDock: A New Technique
for High-Accuracy Molecular Docking. J. Med. Chem. 2006, 49, 3315−
3321.
(13) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J.
Improved patch-clamp techniques for high-resolution currents
recording from cells and cell-free membrane patches. Pflugers Arch.
1981, 391, 85−100.
(14) Luo, J. H.; Fu, Z. Y.; Losi, G; Kim, B. G.; Prybylowski, K; Vissel,
B; Vicini, S. Functional expression of distinct NMDA channel subunits
tagged with green fluorescent protein in hippocampal neurons in
culture. Neuropharmacology 2002, 42, 306−318.
(15) Cannazza, G.; Jozwiak, K.; Parenti, C.; Braghiroli, D.; Carrozzo,
M. M.; Puia, G.; Losi, G.; Baraldi, M.; Lindner, W.; Wainer, I. W. A
Novel class of allosteric modulators of AMPA/Kainate receptors.
Bioorg. Med. Chem. Lett. 2009, 19, 1254−1257.
(16) 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.; de Tullio, P. 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. 2010, 53, 1700−1711.
29
dx.doi.org/10.1021/ml200184w | ACS Med. Chem. Lett. 2012, 3, 25−29