Synthesis of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives as potential allosteric modulators of AMPA/kainate receptors

Article abstract of DOI:10.1021/jm025510d

A series of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives were synthesized and evaluated for their activity as allosteric modulators of kainate-activated currents in primary cultures of cerebellar granule neurons. Substitution of different groups at the 3-position of the benzothiadiazine ring distinguished between positive and negative allosteric modulatory properties.

Full text of DOI:10.1021/jm025510d

J . Med. Chem. 2002, 45, 2355-2357
2355
putative AMPA receptor modulatory activity for the
treatment of cognitive disorders gains importance.
Syn th esis of 3,4-Dih yd r o-2H-1,2,4-ben zo-
th ia d ia zin e 1,1-Dioxid e Der iva tives a s
P oten tia l Alloster ic Mod u la tor s of AMP A/
Ka in a te Recep tor s
As part of an ongoing project in this area, 3,4-dihydro-
2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives (3-
12) were synthesized and studied for their activity as
allosteric modulators of kainate-activated currents in
primary cultures of cerebellar granule neurons, since
kainic acid (KA) elicits a nondesensitizing current
mediated mainly by AMPA receptor.13,14 Particular
attention was paid in analyzing the influence on the
modulatory activity played by the nature and size of the
side chains in the 3-position of the benzothiadiazine
ring.
Daniela Braghiroli,*,† Giulia Puia,‡
Giuseppe Cannazza,‡ Annalisa Tait,‡ Carlo Parenti,‡
Gabriele Losi,‡ and Mario Baraldi‡
Dipartimento di Scienze del Farmaco, Universita` degli
Studi “G. D’Annunzio”sChieti, Via dei Vestini, 66013
Chieti, Italy, and Dipartimento di Scienze Farmaceutiche,
Universita` degli Studi di Modena e Reggio Emilia,
Via Campi 183, 41100 Modena, Italy
Ch em istr y. The 3,4-dihydro-2H-1,2,4-benzothiadi-
azine derivatives 3-12 were prepared from the corre-
sponding 2-aminobenzensulfonamides 13 and 14 by
condensation with a suitable aldehyde or the corre-
sponding ethyl hemiacetal in acidic medium, as outlined
in Scheme 1.
Received February 10, 2002
Abstr a ct: A series of 3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxide derivatives were synthesized and evaluated for
their activity as allosteric modulators of kainate-activated
currents in primary cultures of cerebellar granule neurons.
Substitution of different groups at the 3-position of the
benzothiadiazine ring distinguished between positive and
negative allosteric modulatory properties.
Synthesized compounds (3-12) possess a stereogenic
carbon atom in the 3-position of benzothiadiazine ring,
and as stereisomers often shown different pharmaco-
logical activities, it seemed advisable to resolve the
racemic mixture to investigate the biological properties
of each enantiomer. In a previous study15 an enanti-
oseparation method employing tris(3,5-dimethylphenyl-
carbamate) as a chiral stationary phase (Chiralcel OD
and Chiralcel OD-R) was developed for the separation
of the enantiomers of 2, and subsequent enantiomer-
ization studies16,17 revealed the rapid interconversion
of enantiomers in aqueous solvents. The HPLC method
has been applied to the structurally related chiral
compounds synthesized (3-12), and the enantiomeric
nature of eluates was confirmed by circular dichroism
spectra (Table 1). However, like for compound 2, up
evaporation of mobile phases of collected peak fractions
corresponding to the single enantiomers, racemization
occurred. Furthermore, the individual enantiomers of
the studied compounds were isolated by preparative
chromatography using poly(N-acryloyl-S-phenylalanine
ethyl ester) as chiral stationary phase, employing condi-
tions where no enantiomerization took place, followed
by dissolution at a concentration of 1 mM in the
extracellular medium used in the electrophysiological
tests. Rate constants of enantiomerization of individual
enantiomers were determined at room temperature,
revealing an enantiomerization half-life that is intrac-
tably short, averting efforts to measure stereospecific
or stereoselective biological activities of synthesized
compounds. Hence, racemates were used in pharmaco-
logical tests.
In tr od u ction . Recent studies indicate that com-
pounds that reduce the AMPA (R-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid) receptor desensitiza-
tion could improve impaired synaptic functions associated
with learning and cognition pathology and can be useful
in the treatment of attention disorders in children as
well as in senile dementias, including early stages of
Alzheimer disease.1,2 Among these compounds are two
benzothiadiazine derivatives: cyclothiazide (1) and
IDRA 21 (2).
Cyclothiazide (1) has been demonstrated to be one of
the most potent compounds in vitro that by removal of
AMPA receptors desensitization enhances synaptic
transmission.3-6 Since cyclothiazide does not cross the
blood-brain barrier, it is important to develop ana-
logues of this compound that manage to reach the
central nervous system and do not have peripheral
effects. IDRA 21 (2)7 fulfills these requirements, and it
has gained strong interest because of its potency in
abating pharmacologically induced cognitive impair-
ments in patas monkey,8,9 in improving cognition in
rats,10 and in promoting the induction of long-term
potentiation.11 Recent reports showed that 2 worsens
neuronal ischemic injury12 probably because of excessive
AMPA receptor activation, although the concentrations
used were higher than those needed for the nootropic
effect. Thus, the development of analogues of 1 or 2 with
P h a r m a cology. The pharmacological tests were
performed using the patch-clamp technique on primary
cultures of cerebellar granule neurons.18-20
The synthesized compounds were dissolved in DMSO
and diluted at the final concentration in extracellular
solution (DMSO at the final concentration was less than
1%) and were applied directly by gravity through a
Y-tube perfusion system.21 The low solubility of com-
pound 8 in DMSO and in other tested solvents precluded
* To whom correspondence should be addressed. E-mail: braghiroli@
unich.it. Fax: 39 871 3555267. Phone: 39 871 3555383.
† Universita` degli Studi “G. D’Annunzio”sChieti.
‡ Universita` degli Studi di Modena e Reggio Emilia.
10.1021/jm025510d CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/09/2002

2356 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Letters
Ta ble 2. Variation of KA-Evoked Currents Induced by
Benzothiadiazine Derivatives 2-7 and 9-12a
Sch em e 1
variation of
KA current, %
variation of
KA current, %
compd
compd
1
2
3
4
5
6
111 ( 21
115 ( 24
-22 ( 10
-26 ( 13
70 ( 9
7
9
10
11
12
-34 ( 9
-60 ( 8
-51 ( 3
-1 ( 3
-1 ( 3
72 ( 19
a
KA and 1 were tested at 100 µM, compounds 2-12 were tested
at 1 mM. Each value is the mean ( SEM of at least six cells.
Ta ble 1. Chromatographic Enantioseparation of Racemic
Benzothiadiazine Derivatives 2-12
CHIRALCEL ODa
CHIRALCEL OD-Rb
compd k′(-)
k′(+)
R
RS
k′(-) k′(+) RS
R
3
4
3.47
2.40
5.00 1.44 3.73 1.29 1.44 1.12 1.11
3.09 1.29 2.13 2.54 2.88 1.13 1.13
5
6
7
8
3.88 11.88 3.06 9.10 1.18 2.44 2.07 3.00
F igu r e 1. (A) Representative recording of inward currents
evoked by the application of KA and modulation by compounds
2, 9, and 10. The cells were voltage-clamped at -60 mV. Bars
at the top of the current traces show the duration of the drug
application. (B) Dose-dependent effect of compounds 9 and 10
on KA (100 µM)-evoked currents. Each bar is the mean ( SEM
of the percent reduction of KA-evoked currents measured on
at least five cells.
3.60
1.74
1.32
4.35
3.79
5.09 1.41 2.94
3.05 1.75 1.37
1.70 1.29 1.2
5.42 1.25 2.31 4.47 5.88 1.31 1.94
4.72 1.24 2.53
c
c
c
c
c
c
c
c
c
c
c
c
9
10
11
12
c
c
c
c
c
c
c
c
8.44 11.47 1.36 3.70
1.74
2.94 1.69 1.38 1.41 1.78 1.26 1.83
a
Column, Chiralcel OD; mobile phase, hexane/2-propanol 60:
40 (v/v); temperature, 25 °C; flow, 0.5 mL/min. b Column, Chiralcel
OD-R; mobile phase, water/acetonitrile 60:40 (v/v); temperature,
25 °C; flow, 1 mL/min. c No enantiomeric separation.
Substitution in position 3 of the methyl group of 2
with a bulkier substituent (compounds 3, 4, 7, 9, and
10) changes the pharmacological activity of the mol-
ecule, which becomes a negative modulator of KA-
activated currents (see Table 2 and Figure 1A). The
current evoked by application of 100 µM KA was
reduced in a dose-dependent fashion by increasing the
concentrations (see the histograms of Figure 1B for the
compounds 9 and 10). A further increase in the steric
hindrance of substituents as in 11 and 12 abolished the
pharmacological activity.
performing the pharmacological test. KA was dissolved
in the extracellular solution.
Resu lts a n d Discu ssion . We tested the activity of
2 and of synthesized derivatives (3-12) as allosteric
modulators of KA-activated currents in primary cultures
of cerebellar granule neurons. Application of KA (100
µM) evoked a nondesensitizing current that is mediated
by both AMPA and KA receptors activation. Compounds
1 (100 µM) and 2 (1 mM) potentiate the amplitude of
the KA current by 111 ( 21% (n ) 9) and 115 ( 24% (n
) 17), respectively.
In the series of synthesized 3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxide derivatives, 5 and 6 are the
only compounds that, similar to 2, act as positive
modulators of KA-evoked currents, even though to a
lesser extent. These compounds indeed have in position
3 a trifluoromethyl group that has a size similar to the
size of the methyl group present in the parent com-
pound. The lower modulatory activity could be ascribed
to the electron-withdrawing effect of fluorine atoms.
The presence of the chlorine atom in position 7 does
not seem to be an essential requisite for the studied
activity.
All the compounds were tested for their capability of
eliciting a response by itself. None of them were active
when applied alone.
We can surmise that these changes in the pharma-
cological activity of compounds 3, 4, 7, 9, and 10 could
be attributed to the addition of a substituent able to
interact with another recognition site within the recep-
tor structure. This interaction could lead to some

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2357
(8) Impagnatiello, F.; Oberto, A.; Longone, P.; Costa, E.; Guidotti,
A. 7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-
dioxide: a partila modulator of AMPA receptor desensitization
devoid of neurotoxicity. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
7053-7058.
(9) Thompson, D. M.; Guidotti, A.; Di Bella, M.; Costa, E. IDRA 21,
a congener of aniracetam, potently abates pharmacologically
induced cognitive impairments in patas monkey. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 7667-7671.
(10) Zivkovic, I.; Thomson, D. M.; Bertolino, M.; Uzunov, D.; Di Bella,
M.; Costa, E.; Guidotti, A. 7-Chloro-3-methyl-3,4-dihydro-2H-
1,2,4-benzothiadiazine S,S-dioxide (IDRA 21): a benzothiadiaz-
ine derivative that enhances cognition by attenuating D,L-alpha-
amino-2,3-dihydro-5-methyl-3-oxo-4-isoazolepropanoic acid (AM-
PA) receptor desensitization. J . Pharmacol. Exp. Ther. 1994, 272,
300-309.
(11) 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.
(12) Yamada, K. A. Modulating excitatory synaptic neurotransmis-
sion: potential treatment for neurological disease? Neurobiol.
Disease 1998, 5, 67-80.
(13) Lerma, J .; Patternain, A. V.; Salvador, N.; SomoHano, F.;
Morales, M. Excitatory aminoacid-activated channels. In Ion
Channel Pharmacology; Soria, B., Cena, V., Eds.; Oxford Uni-
versity Press: Oxford, 1998; Chapter 18; p 399.
(14) Dingledine, R.; Borges, K.; Bowie, D.; Traynelis S. F. The
Glutamate Receptor Ion Channels. Pharmacol. Rev. 1999, 51,
7-61.
conformational changes of the receptor protein that
ultimately resolve in a negative modulatory activity of
the compounds. When a dimethylamino group (com-
pounds 11 and 12) substitutes a phenolic hydroxyl group
(compounds 9 and 10), we could not measure any type
of modulatory activity either positive or negative. The
increase in the steric hindrance, the different spatial
arrangement, and the ability of this functional group
to act mainly as hydrogen bond acceptor are likely
responsible for the different activity of compounds 11
and 12. We are currently investigating this hypothesis.
These compounds represent novel tools for a better
understanding of the structural requirements for posi-
tive and negative modulation of the AMPA receptor.
Furthermore, they could be prototypes for a novel series
of compounds that may be useful in the treatment of
learning and cognition pathology.
Ack n ow led gm en t. This research was supported by
grants from the Universita` “G. D’Annunzio”sChieti,
Universita` di Modena e Reggio Emilia, and MURST.
(15) Cannazza, G.; Braghiroli, D.; Baraldi, M.; Parenti, C. Chiral
resolution of enantiomers of 7-chloro-3-methyl-3,4-diydro-2H-
1,2,4-benzothiadiazine 1,1-dioxide using high-performance liquid
chromatography on cellulose-based chiral stationary phases. J .
Pharm. Biomed. Anal. 2000, 23, 117-125.
(16) Cannazza, G.; Braghiroli, D.; Tait, A.; Baraldi, M.; Parenti, C.;
Lindner, W. Studies of enantiomerization of chiral 3,4-dihydro-
1,2,4-benzothiadiazine 1,1-dioxide type compounds. Chirality
2001, 13, 94-101.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails. This material is available free of charge via the Internet
at http://pubs.acs.org.
Refer en ces
(1) Bliss, T. V. P.; Collingridge, G. L. Synaptic model of memory:
long-term potentiation in the hippocampus. Nature 1993, 361,
31-39.
(17) Cannazza, G.; Puia, G.; Baraldi, M.; Braghiroli, D.; Tait, A.;
Mosciatti, B.; Rosa, C.; Parenti, C. Stereospecificy in (()IDRA
21 pharmacological action. J . Pharm. Biomed. Anal., in press
(18) Gallo, V.; Ciotti, M. T.; Coletti, A.; Aloisi, F.; Levi, G. Selective
release of glutamate from cerebellar granule cells differentiating
in culture. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7919-7923.
(19) Puia, G.; Losi, G.; Razzini, G.; Braghiroli, D.; Di Bella, M.;
Baraldi, M. Modulation of kainate-activated currents by diaz-
oxide and cyclothiazide analogues (IDRA) in cerebellar granule
neurons. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2000,
24, 1007-1015.
(20) Hamill, O. P.; Marty, A.; Neher, E.; Sackmann, B.; Sigworth, F.
J . Improved patch-clamp techniques for high-resolution current
recording from cell and cell-free membrane patches. Pfluegers
Arch. 1981, 391, 85-100.
(21) Murase, K.; Ryu, P. D.; Randic, M. Excitatory and inhibiting
aminoacids and peptide-induced responses in acutely isolated
rat spinal horn neurons. Neurosci. Lett. 1989, 103, 56-63.
(2) Nagarajan, N.; Quast, C.; Boxall, A. R.; Shahid, M.; Rosenmund,
C. Mechanism and impact of allosteric AMPA receptor modula-
tion by the Ampakine CX546. Neuropharmacology 2001, 41,
650-663.
(3) Vyklicky, L.; Patneau, D. K.; Mayer, M. L. Modulation of
excitatory synaptic transmission by drugs that reduce desensi-
tization at AMPA/kainate receptors. Neuron 1991, 7, 971-984.
(4) Patneau, D. K.; Vyklicky, L.; Mayer, M. L. Hipocampal neurons
exhibit cyclothiazide-sensitive rapidly desensitizing responses
to kainate. J . Neurosci. 1993, 13, 3496-3509.
(5) Partin, K. M.; Fleck, M. W.; Mayer, M. L. AMPA receptor flip/
flop mutants affecting deactivation, desensitization, and modu-
lation by cyclothiazide, aniracetam, and thiocyanate. J . Neurosi.
1996, 16, 6634-6647.
(6) Partin, K. M. Domain interactions regulating AMPA receptor
desensitization. J . Neurosci. 2001, 21, 1939-1948.
(7) Bertolino, M.; Baraldi, M.; Parenti, C.; Braghiroli, D.; Di Bella,
M.; Vicini, S.; Costa, E. Modulation of AMPA/kainate receptors
by analogues of diazoxide and cyclothiazide in thin slices of rat
hippocampus. Recept. Channels 1993, 1, 267-278.
J M025510D