Development of thiophenic analogues of benzothiadiazine dioxides as new powerful potentiators of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors

Article abstract of DOI:10.1021/jm400676g

On the basis of the results obtained in previous series of AMPA potentiators belonging to 3,4-dihydro-2H-benzo- and 3,4-dihydro-2H-pyrido-1,2,4- thiadiazine 1,1-dioxides, the present work focuses on the design of original isosteric 3,4-dihydro-2H-thieno-1,2,4-thiadiazine 1,1-dioxides. Owing to the sulfur position, three series of compounds were developed and their activity as AMPA potentiators was characterized. In each of the developed series, potent compounds were discovered. After screening the selected active compounds on a safety in vivo test, 6-chloro-4-ethyl-3,4-dihydro-2H-thieno[2,3-e]-1,2,4- thiadiazine 1,1-dioxide (24) appeared as the most promising compound and was further evaluated. Its effects on long-term potentiation in vivo and on AMPA-mediated noradrenaline release were measured to predict its potential cognitive enhancing properties. Finally, an object recognition test performed in mice revealed that 24 was able to significantly enhance cognition, after oral administration, at doses as low as 0.3 mg/kg. This study validates the interest of the isosteric replacement of the benzene or pyridine nuclei by the thiophene nucleus in the ring-fused thiadiazine dioxides class of AMPA potentiators.

Full text of DOI:10.1021/jm400676g

Article
pubs.acs.org/jmc
Development of Thiophenic Analogues of Benzothiadiazine Dioxides
as New Powerful Potentiators of 2‑Amino-3-(3-hydroxy-5-
methylisoxazol-4-yl)propionic Acid (AMPA) Receptors
,
†
†
‡
†
†
‡
‡
Pierre Francotte,* Eric Goffin, Pierre Fraikin, E. Graindorge, Pierre Lestage, Laurence Danober,
‡
‡
‡
†,§
Sylvie Challal, Nathalie Rogez, Olivier Nosjean, Daniel-Henri Caignard, Bernard Pirotte,
and Pascal de Tullio†,§
†
Centre Interfacultaire de Recherche du Med
pital 1, B36, 4000 Liege, Belgium
Institut de Recherches Servier, 125 Chemin de Ronde, F-78290 Croissy-sur-Seine, France
́
icament (CIRM)Laboratoire de Chimie Pharmaceutique, University of Liege, Avenue
de l′Ho
̂
̀
‡
*
S Supporting Information
ABSTRACT: On the basis of the results obtained in previous series of
AMPA potentiators belonging to 3,4-dihydro-2H-benzo- and 3,4-dihydro-
2
H-pyrido-1,2,4-thiadiazine 1,1-dioxides, the present work focuses on the
design of original isosteric 3,4-dihydro-2H-thieno-1,2,4-thiadiazine 1,1-
dioxides. Owing to the sulfur position, three series of compounds were
developed and their activity as AMPA potentiators was characterized. In
each of the developed series, potent compounds were discovered. After
screening the selected active compounds on a safety in vivo test, 6-chloro-
4
-ethyl-3,4-dihydro-2H-thieno[2,3-e]-1,2,4-thiadiazine 1,1-dioxide (24)
appeared as the most promising compound and was further evaluated. Its effects on long-term potentiation in vivo and on
AMPA-mediated noradrenaline release were measured to predict its potential cognitive enhancing properties. Finally, an object
recognition test performed in mice revealed that 24 was able to significantly enhance cognition, after oral administration, at doses
as low as 0.3 mg/kg. This study validates the interest of the isosteric replacement of the benzene or pyridine nuclei by the
thiophene nucleus in the ring-fused thiadiazine dioxides class of AMPA potentiators.
INTRODUCTION
receptor in its channel-open state following the binding of the
released transmitter (L-glutamate).
On the basis of these findings and considering the potential
■
L-Glutamic acid is the major excitatory neurotransmitter in our
central nervous system, activating metabotropic (coupled to G-
proteins) and ionotropic receptors (coupled to an ionic
channel). Among the latter, AMPA-type receptors mediate
fast excitatory postsynaptic currents at the great majority of the
synapses and are involved in the expression of long-term
potentiation, a phenomenon closely linked to learning and
memory processes. On this basis, the AMPA receptor
therapeutic interest of such compounds (e.g., Alzheimer’s
disease treatment, cognitive disorders, schizophrenia, or
9−11
depression),
several distinct chemical classes of compounds
were investigated, among which the 1,2,4-benzothiadiazine 1,1-
dioxide-type chemical class (i.e., compounds 3−6, 8, and 10) is
12−14
one of the most studied.
Structural modifications starting
from IDRA-21 (3) were achieved, leading to the preparation of
(
AMPAR) has been proposed as an interesting target to
pyridinic analogues (pyrido-1,2,4-thiadiazine 1,1-dioxides) such
15
develop cognitive enhancers. Given the fact that excessive
glutamatergic activity generates toxic effects, such as excitotox-
icity and seizure, the enhancement of glutamate signals through
AMPAR modulators has been anticipated to produce
unacceptable side effects. This hypothesis was however rejected
in the beginning of the 1990s when cyclothiazide (1), diazoxide
as 7. This first work was followed by a SAR study focused on
benzenic compounds 8, studying the impact of substitution at
16
the 7-position. The combination of SARs established from
pyridinic and benzenic series led to the design of a second
17
generation of pyrido-1,2,4-thiadiazine 1,1-dioxides such as 9.
Furthermore, 4-fluoroalkylated benzo-1,2,4-thiadiazine 1,1-
(
2), IDRA-21 (3), and S18986 (4) were characterized as
dioxides characterized by an enhanced metabolic stability
were prepared. Among the compounds designed in this study,
S70340 (10) was found to be active in vivo in the object
recognition test after oral administration in rats at the dose of
1
−4
18
AMPA positive allosteric modulators and when it was shown
that this new pharmacological class enhances memory without
causing severe side effects (Figure 1).
4
−7
These compounds,
0
.1 mg/kg. AMPA potentiation mediated by this compound has
called ampakines or “AMPApams”, are now well-known to act
through binding to allosteric sites located at the level of the
8
ligand binding domain of the AMPA receptor. They have thus
Received: May 7, 2013
no agonist or antagonist effects but instead stabilize the
Published: October 3, 2013
©
2013 American Chemical Society
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Figure 1. An overview of benzothiadiazines and related pyridinic isosteres developed as AMPApams.
been recently shown to activate genes and functional processes
bearing a chlorine atom at the 6-position (the closest to the 7-
position in the benzenic series). For the 6-thieno system, we
envisaged a series of thieno-1,2,4-thiadiazine 1,1-dioxides
bearing two chlorine atoms at the 5- and 7-positions.
1
9
involved in neuroprotective and cognitive effects. In
particular, it was confirmed that one of the most relevant
effects of 10, like other ampakines, was the increase of the
brain-derived neurotrophic factor (BDNF) expression, a major
feature which determines the therapeutic potentialities of
ampakines (neuroprotection, neural stem cells differentiation).
In the present paper, we disclose the results of our studies on
a new series of AMPApams. With the aim of obtaining
compounds with a better activity and taking into account that a
CHEMISTRY
■
Considering at first that 2-amino-5-chlorothiophene-3-sulfona-
mide (15) is the key intermediate in the access to the 6-chloro-
3
,4-dihydro-2H-thieno[2,3-e]-1,2,4-thiadiazine 1,1-dioxide ser-
21
20
ies, we first followed the approach reported by Nielsen to
prepare 6-chloro-3-imino-3,4-dihydro-2H-thieno[2,3-e]-1,2,4-
thiadiazine 1,1-dioxide 14, which had to be hydrolyzed to
give the expected key product 15. Despite numerous attempts,
this step was unsuccessful and we finally based our synthesis on
a strategy described by Topliss. The reaction between 1-(5-
chlorothiophen-2-yl)ethanone 16 and hydroxylamine provided
a mixture of two oximes in an equimolar proportion (Scheme
small change of a ligand can easily alter receptor functionality,
we have focused our efforts on the aromatic part of benzo-1,2,4-
thiadiazine 1,1-dioxides and prepared new compounds in which
the benzene ring of known benzothiadiazines was substituted
by thiophene providing thieno-1,2,4-thiadiazine 1,1-dioxide
isosteres. This pharmacomodulation has been used with success
in many therapeutic fields, such as the benzodiazepine class
22
(
leading to clotiazepam) or the oxicam class (leading to
1
). Then, these two isomers underwent a Beckmann rearrange-
tenoxicam).
ment, resulting in a mixture of the carboxamide 19 and the
acetamide 20, depending on the starting oxime. After a
chromatographic separation of the isomers, a chlorosulfonation
was achieved on the acetamide 20, directly followed by a
reaction with gaseous ammonia (interestingly, reaction with an
aqueous solution of ammonia led to a complete loss of the
material). Because the acidic hydrolysis of the amide 21 into
the expected 15 was characterized by a very low yield, the
amide was reduced using lithium aluminum hydride to obtain
The benzene/thiophene switch is expected to give rise to
three different thiophenic regioisomeric systems: 3,4-dihydro-
2H-thieno[2,3-e]-1,2,4-thiadiazine 1,1-dioxides (“5-thieno” ser-
ies, 11), 3,4-dihydro-2H-thieno[3,4-e]-1,2,4-thiadiazine 1,1-
dioxides (“6-thieno” series, 12), and 3,4-dihydro-2H-thieno-
[
3,2-e]-1,2,4-thiadiazine 1,1-dioxides (“7-thieno” series, 13)
(
Figure 2).
Keeping in mind that the best in vitro results were previously
obtained with a halogen atom, i.e., a fluorine or a chlorine atom,
at the 7-position of benzo-1,2,4-thiadiazine 1,1-dioxides, we
focused our effort toward 5-thieno and 7-thieno compounds
22. The usual ring formation with triethyl orthoformate and the
subsequent reduction using sodium borohydride gave rise to
the obtention of the final compound 24. While other 5-acyl-2-
chlorothiophenes could theoretically have given access to final
compounds bearing a bigger alkyl group on the nitrogen atom
at the 4-position, we limited our exploration in this series to 24,
considering the SAR trends observed in pyridinic and in
12−14
benzenic series.
While the strategy for the synthesis of the key intermediate 4-
amino-2,5-dichlorothiophene-3-sulfonamide (28) could follow
23
a previously reported scheme approach, we preferred starting
from the commercially available sulfonyl chloride derivative 25
(Scheme 2). Nitration was directly performed on the sulfonyl
Figure 2. The three thiophenic regioisomeric systems investigated in
this work.
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a
Scheme 1
a
(
i) NH OH; (ii) PCl ; (iii) (1) ClSO H, (2) NH gas; (iv) HCl; (v) LiAlH ; (vi) HC(OEt) , 170 °C, 30 min; (vii) NaBH ; (viii) HCl.
2 5 3 3 4 3 4
a
Scheme 2
a
(
i) HNO /H SO ; (ii) NH OH; (iii) Fe/NH Cl; (iv) HC(OEt) , 150 °C, 90 min; (v) NaBH , H O; (vi) CH I, K CO , C H NO /DMF; (vii)
3 2 4 4 4 3 4 2 3 2 3 2 5 2
NaBH , 2-propanol; (viii) HC(OEt) , 170 °C, 35 min; (ix) RCOCl, pyridine, dioxane; (x) LiAlH .
4
3
4
7
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Journal of Medicinal Chemistry
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a
Scheme 3
a
(
i) (CH ) CNH ; (ii) n-BuLi; (iii) p-toluenesulfonyl azide; (iv) hexadecyltributylphosphonium bromide, NaBH ; (v) HCl; (vi) HC(OEt) , 160 °C,
3 3 2 4 3
3
0 min; (vii) NaBH , water; (viii) R-X, K CO , CH CN/DMF; (ix) NaBH , 2-propanol; (x) CHF COCl, pyridine, dioxane; (xi) LiAlH , Et O; (xii)
4
2
3
3
4
2
4
2
HCOOH, 80 °C, 5 h.
chloride derivative to obtain the 4-nitro-substituted compound
6. Reaction of the latter with diluted ammonia followed by
reduction of the nitro group using powdered iron in acidic
medium led to 28. From this intermediate, ring closure was
performed with triethyl orthoformate, affording 29a. While the
methylation of the latter on the nitrogen atom at the 4-position
The reduction step using sodium borohydride was
successfully performed with 29a using water as the solvent,
while this reaction was carried out in 2-propanol with 29b−f.
Concerning the 6-chloro-3,4-dihydro-2H-thieno[3,2-e]-1,2,4-
thiadiazine 1,1-dioxide series, the reaction between 5-
chlorothiophene-2-sulfonyl chloride (33) and tert-butylamine
afforded N-(tert-butyl)-5-chlorothiophene-2-sulfonamide (34)
2
(
using methyl iodide and potassium carbonate in nitroethane
(
Scheme 3). The latter was then converted into the key
and dimethylformamide) required 2 h at 60 °C to give 29b,
ethylation in the same conditions did not occur at all, probably
due to the steric hindrance induced by the chlorine atom at the
24
intermediate 35 in four steps, as previously reported. Then,
the ring formation using triethyl orthoformate (leading to 36a)
followed by alkylation of the 4-position (leading to 36b−h) and
finally reduction of the CN double bond of the thiadiazine ring
5-position. On the basis of this hypothesis, N-methylation could
have occurred at the 2-position, leading to the obtention of an
isomer of 29b. Selectivity of the alkylation at the 4-position was
checked after the final reduction step: as expected, 30b was
found soluble in alkaline water as this compound has a weak
acidic function (NH at the 2-position included in a
sulfonamide).
by means of NaBH led to expected final compounds 37b−h.
4
Attempts to prepare the 4-fluoromethyl derivative by this way
were unsuccessful because 37f was found to result from the
substitution of the fluorine atom of intermediate 36f by 2-
propanol during the reduction step. The alternative pathway
described in the 6,7-dichloro-5-thieno series was used to obtain
the 4-(2,2-difluoroethyl)-substituted derivative 37i. In this case,
the ring formation was performed using formic acid instead of
triethyl-orthoformate.
Unexpectedly, 29c was obtained from a side reaction taking
place when prolonging the heating of 28 in triethyl
orthoformate above 150 °C. Considering the reactivity of 29a
18
toward alkylating agents, an alternative strategy was followed
to insert a n-propyl chain or fluoroalkyl chains at the 4-position:
selective acylation on the amino function of 28 with the
appropriate acyl chloride at low temperature afforded the
corresponding amides 31a−c. Then, owing to the nature of the
acyl chain, 31a−c were reduced either using lithium aluminum
hydride or borane in diethyl ether at low temperature, affording
RESULTS AND DISCUSSION
■
The thieno-1,2,4-thiadiazine 1,1-dioxides were evaluated as
AMPA potentiators using the voltage-clamp method and a
FlipR technique developed by the Servier Institute. For each
compound, among other parameters, the EC2x value was
determined, which corresponds to the concentration of drug
responsible for a 2-fold increase of the amplitude of the current
induced by AMPA (at 10 μM).
Activity data on AMPA receptors obtained by means of the
voltage-clamp protocol on Xenopus oocytes with compounds
bearing an ethyl chain at the 4-position and one chlorine atom
32a−c. Ring closure into the corresponding thieno-1,2,4-
thiadiazine 1,1-dioxides 29d−f was performed with triethyl
orthoformate. Because of instability issues of intermediates 31b
and 32b, the conversion of 28 into 29e was achieved through a
one-pot synthesis.
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Table 1. Effects of the Different Thiophenic Compounds on the Amplitude of the Current Induced by (S)-AMPA (10 μM) in
Xenopus laevis Oocytes Injected with Rat Cortex mRNA
a
b
c
d
compd
EC2X (μM)
EC5X (μM)
EC50 (μM)
max effect
e
e
e
e
8
9
2
3
b
5.6 ± 0.9
20 ± 3
4.4 ± 0.6
3.6 ± 0.1
14 ± 3
28 ± 2
nd
3682
e
e
e
e
b
44 ± 4
1093 ± 302
2830 ± 1086
2266 ± 88
4
24 ± 4
18.5 ± 2.3
11 ± 3
9.2 ± 0.7
7c
a
b
c
Concentration of drug giving a 2-fold increase of the magnitude of the current induced by (S)-AMPA (10 μM; mean ± SEM; n ≥ 3).
Concentration of drug giving a 5-fold increase of the magnitude of the current induced by (S)-AMPA (10 μM; mean ± SEM; n ≥ 3).
d
Concentration of drug responsible for 50% of the maximal effect (mean ± SEM; n ≥ 3). Maximum effect of the drug on the AMPA-evoked current
e
16,17
(
expressed in % of the current evoked by AMPA, taken as 100%). Published results.
nd: not determined.
on the aromatic ring appears to be within the same range (see
Table 1). Whereas no marked difference was observed
comparing 8b and 24, a slight improvement of in vitro potency
must be pointed out after switching from the “7-chloro-benzo”
to the “6-chloro-7-thieno” series (see 8b vs 37c). However, 37c
displayed a distinct pharmacological profile at high concen-
trations (from 30 to 300 μM) because it was able to act as an
agonist. These trends were not observed in one of our previous
the 4-position (37a) by replacing the hydrophobic interaction
normally occupied by the small alkyl group.
In contrast to previous observations that saturated
compounds were systematically found to be more active than
their corresponding unsaturated precursors (focused on
13,15−18
pyridinic, benzenic, and thiophenic series),
it was
interesting to note that the unsaturated derivatives in the 5,7-
dichloro-6-thieno series were found to be more active than their
saturated counterparts (see 29c and 29e vs 30c and 30e).
Considering the two thiophenic series developed here, the
insertion of fluorine atoms at the alkyl chain did not
significantly alter the EC2X parameter (compare 29c vs 29e,
30c vs 30e, 37c vs 37g or 37i, 37e vs 37h) while it was found
to double the maximal increase in some cases (see 37c/37g,
37e/37h). Taken together and contrary to observations made
in the benzenic series, these data suggest that insertion of one
or two fluorine atoms at such position of 7-thieno compounds
do not have a deleterious impact on the binding interactions.
It could be deduced from the EC2X value, the EC5X value, and
the maximal effect obtained with 24 and 37c (see Table 2) that
the two isomers displayed a similar pharmacological profile.
However, it is worth mentioning that 37c, but not 24, was able
to act as an AMPA agonist at high drug concentrations (30−
300 μM). This property precluded any further interest of 37c as
a potential cognitive enhancer.
1
7
studies focused on pyridinic series (compare 8b and 9b).
The two thiophenic series developed in this work clearly
displayed distinct trends.
Taking into account the in vitro results reported in Table 2,
SARs in the 6-chloro-7-thieno series (compounds 36 and 37)
were in accordance with the trends observed in our previous
published studies. First of all, the absence of an alkyl substituent
at the 4-position was clearly unfavorable for activity on AMPA
receptors (see 37a), as already reported for pyrido-1,2,4-
15
thiadiazine 1,1-dioxides. It was also noticed that the in vitro
activity improved with the size of the alkyl chain at the 4-
position, with the optionally mono or difluorinated ethyl chains
(37c, 37g, and 37i) being optimal for in vitro activity.
Interestingly, the insertion of a third carbon at the end of the
ethyl chain (n-propyl chain) was clearly deleterious for the
activity (see 37e), while the 4-isopropyl-substituted compound
37d displayed an activity close to that observed with the 4-
ethyl-substituted compound 37c. Finally, a dramatic increase of
the maximum effect was observed with the 4-monofluoroalkyl-
substituted compounds (see 37g and 37h). In particular, 37g
was able to potentiate more than 50 times the AMPA current in
the voltage clamp experiment (max increase: 5604%).
Comparing the data obtained using the voltage clamp and
the flipR methods, we may observe that the EC2X values
obtained by voltage clamp and FlipR approaches corroborate
each other and may be thus both considered as a predictive
value, allowing a preliminary screening. The FlipR method
confirms the in vitro potency of 24, 37g, and 37i on AMPA
receptors expressed on rat primary brain cells cultures.
Unexpectedly, the most active compound in the 5,7-dichloro-
6
-thieno series was 30b (with a methyl group at the 4-position).
The ethyl analogue 30c displayed a high EC2X value, identical
to that observed with 30a. The poor activity of 30c seemed
surprising, considering the usual observation made in previous
series and correlated in the 6-chloro-7-thieno series that an
ethyl chain is preferred to hydrogen or the methyl chain at the
According to their pronounced activity as AMPA potentia-
tors revealed by the in vitro screening, three compounds (24,
37g, and 37i) were selected for further evaluations, on rat
hippocampal slices, in an ex vivo electrophysiological test
1
6,18
already described in previous studies.
We studied the effects
4
-position (37c vs 37a/37b). This atypical activity trend could
of 24, 37g, and 37i at 10 μM on the excitatory postsynaptic
field potentials (EPSfPs) evoked in the CA1 region after
electrical stimulation of the Schaffer collateral. Because of the
presence of 1.2 mM Mg2+ in the perfusion medium, EPSfPs
recorded in this test are primarily mediated by AMPAR
activation. Data in Table 3 clearly show that the three
be explained by the chlorine atom via steric interactions
changing the binding orientation of the ethyl group into a less
favorable binding interaction. The “interference” exerted by this
chlorine atom could also be responsible for the surprising
activity displayed by the compound devoid of an alkyl chain at
7
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Table 2. Effects of the Different Thiophenic Compounds on the Amplitude of the Current Induced by (S)-AMPA (10 μM) in
Xenopus laevis Oocytes Injected with Rat Cortex mRNA and on the Fluorescence induced by 300 μM AMPA on Primary
Cultures of Neurons from Rat Embryonic Cortex
a
b
c
d
compd
R4
NS/S
EC2X (μM)
max increase (%)
FlipR (μM)
2
2
2
3
2
3
2
3
2
3
2
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
CH CH
NS
S
nd
nd
2830
nd
>100
10.4
nd
2
3
3
4
CH CH
4.4 ± 0.6
>100
2
9a
0a
9b
0b
9c
0c
9d
0d
9e
0e
9f
H
NS
S
H
72 ± 18
>973
>413
2916
>1003
>955
nd
>100
nd
e
CH3
CH3
NS
S
81
5.4 ± 2.8
42 ± 15
71 ± 14
nd
nd
CH CH
NS
S
nd
2
3
CH CH
>55
>100
2
3
CH CH CH
NS
S
2
2
3
3
e
e
CH CH CH
>300
>126
>2327
>485
nd
>100
2
2
e
e
CH CH F
NS
S
26.5
20.3
2
2
e
e
CH CH F
113
>100
2
2
e
CH CHF
NS
S
nd
>100
2
2
e
e
0f
CH CHF
235
>227
253
>100
2
2
e
e
7a
6b
7b
6c
7c
6d
7d
6e
7e
6f
H
S
216
>100
e
CH3
CH3
CH CH
NS
S
166; >300
>100
36.0 ± 4.4
172
>462
>175
2266
>277
217
nd
e
e
NS
S
>100
2
3
3
CH CH
3.6 ± 0.1
>100
2
e
CH(CH3)2
CH(CH3)2
CH CH CH
NS
S
>100
4.2 ± 0.7
nd
9.55
e
NS
S
2066
2092
nd
>100
2
2
3
3
e
CH CH CH
15
25.1
2
2
e
CH2F
NS
S
nd
nd
>100
7f
CH OCH(CH )
nd
nd
2
3
2
e
6g
7g
6h
7h
6i
CH CH F
NS
S
>300
204
>100
2
2
CH CH F
2.0 ± 0.6
5604
107
3.32
2
2
e
e
CH CH CH F
NS
S
>300
>100
2
2
2
e
CH CH CH F
12
>3781
nd
45.7 (2)
2
2
2
e
CH CHF
NS
S
nd
>100
2
2
e
7i
CH CHF
1.6
2558
2.9 ± 0.1
2
2
a
b
NS, not saturated; S, saturated. Drug concentration giving a 2-fold increase of the amplitude of the current induced by AMPA (10 μM) with the
c
voltage clamp method (n = 3, mean ± SEM) . Maximum effect of the drug on the AMPA-evoked current (expressed in % of the current evoked by
AMPA, taken as 100%). Drug concentration giving a 2-fold increase of the fluorescence induced by AMPA (300 M) with the flipR method (n = 3,
mean ± SEM) . n = 2, no SEM; nd, not determined.
d
e
compounds increased the signals at the concentration of 10
μM, suggesting that they were able to interact with postsynaptic
AMPAR located on hippocampal CA1 neurons, as it was
Before evaluating the in vivo efficacy of the three compounds
(24, 37g, and 37i), their safety was assessed with the Irwin’s
test, well-known in the pharmaceutical industry to estimate the
potential acute neurotoxicity of new compounds and performed
to select nontoxic doses and most promising candidates for in
vivo testing. After oral administration of a high dose of
compound (30 mg/kg), the changes in the behavioral and
physiological states of the mice were recorded. This screening
first highlighted 37g’s acute toxicity with five cases of
convulsion followed by apathy after administration in five
animals. Administration of 37i triggered hypothermia in 60% of
the tested animals and convulsion, leading to death in 2/5
animals. 24 was found to be atoxic, although a reversible 2 °C
decrease in body temperature was observed 30 min after its oral
administration (30 mg/kg). It should be noted here that the
observed hypothermia could not be related to activity on
18
demonstrated for our previous hit compound 10. However
epileptiform activity was observed after application of 37g,
probably reflecting additional effects on other targets as
25
26
demonstrated for cyclothiazide and CX546. We can also
not exclude that 37g, 37i, and 24 may represent different
subclasses of AMPApams, with different modulations of AMPA
receptors kinetics (e.g., deactivation or desensitization) or
different subunits preferences (GluR1−4, flip and flop splice
variants) that may also explain why 37g as other ampakines
2
5
26
(
cyclothiazide or CX546 ) was observed to cause epilepti-
form discharges in hippocampal slices. This activity did not
permit measurement of the half-width and constituted a first
warning for a potential toxicity.
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Table 3. Effects of Compounds 24, 37g, and 37i (10 μM) on
the AMPA-Mediated Postsynaptic Response Recorded in
CA1 Area of Rat Hippocampal Slices
recordings of the EPSfPs in the dentate gyrus assessed the
results obtained in the in vitro test (see Figure 3A). Then, 1 h
after ip administration, LTP was induced by a tetanic
stimulation and the EPSfPs continued to be measured during
3
h. As can be seen in Figure 3, 24 increased the amplitude of
the measured potentials and extended the maintenance of LTP
compared to vehicle rats. This activity on LTP is a key feature
for a potential new cognitive enhancer. Moreover, these first in
vivo results clearly highlighted the ability of 24 to cross the
blood−brain barrier and to reach the CNS.
Because the role of presynaptic AMPA receptors has been
27
a
a
a
established with noradrenaline release, several AMPApams
have been reported to potentiate noradrenaline release in rat
compd
area (%)
amplitude (%)
half-width (%)
2
3
3
4
18.3 ± 5.2
9.3 ± 5.5
22.7 ± 3.5
3.7 ± 2.0
33.7 ± 3.8
15.3 ± 4.3
11.7 ± 7.2
16,28,29
b
hippocampal slices.
This activity could explain the
7g
7i
nd
cognitive enhancing properties of AMPA potentiators. There-
fore, the neurochemical effects of 24 were investigated on rat
hippocampal slices. When tested alone at 300 μM, 24 was
14.3 ± 5.0
a
Area, amplitude, and half-width of the AMPA-mediated EPSfP
induced by the compounds were normalized as a percentage of that
evoked before application (basal value taken as 100%), n = 3. nd: not
3
b
found to slightly increase the [ H]-NA release compared to
determined.
basal DMSO control release levels (+34%). Moreover, 24
3
induced a 281% enhancement of the (S)-AMPA evoked [ H]-
NA release (100% representing the effects shown by (S)-AMPA
alone). This result clearly indicated that the selected compound
was able to act on presynaptic AMPA receptors.
AMPA receptors because 36e, inactive in vitro (FlipR data,
Table 2), was able to induce a 4.6 °C hypothermia after
intraperitoneal injection (30 mg/kg). This trend was
concordant with hypothermic effects observed with a previously
To assess its potential interest as a new cognitive enhancer,
effects of 24 were evaluated in vivo in an object recognition test
with CD1 mice. This three-session test, considered as a
paradigm for episodic memory in rodents, was inspired by the
18
published series of AMPApams. Further studies should be
performed to elucidate the mechanism underlying such effect.
As 24 was atoxic compared to 37g and 37i, further in vivo
evaluations were focused on 24. No in vitro metabolic stability
or in vivo evaluation of the blood−brain barrier crossing of the
compound could unfortunately be performed in order to
confirm the adequate brain levels of 24 due to difficulties in
detecting the compound using LC-MS-MS methods. Therefore
the first test that was performed in vivo was electrophysiological
recording on the hippocampus of anesthetized Wistar rats in
order to confirm the previous in vitro effects observed on
EPSfPs on rats hippocampal slices and also to measure its
effects on long-term potentiation. Within 1 h after intra-
peritoneal injection of 24 (30 mg/kg) in the animals,
30
model developed by Ennaceur and Delacour and was based
on the fact that animals remembering a familiar object spend
less time exploring it compared to exploring a new object. As
can be seen in Figure 4, oral administration of 24 1 h before the
three sessions significantly increased the performance of mice at
doses as low as 0.3 mg/kg. This activity became highly
significant at 1 mg/kg. However, also in the absence of
corroborative brain penetration studies, the effect of 24 both on
EPSFPs recorded in vivo on anesthetized rats and on object
recognition test in mice strongly suggest that 24 is absorbed in
mice after oral administration and is able to reach the CNS.
Figure 3. (A) Effects of 24 on the amplitude of the EPSfP recorded in the dentate gyrus of the hippocampus in vivo on anesthetized rats. (B) Effects
of 24 on LTP induced in the dentate gyrus of the hippocampus on anesthetised Wistar rat. The amplitude of the EPSfP was averaged over four
stimuli and was normalized as a percentage of the averaged amplitude of the response during the 1 h baseline period prior to ip injection (control
value taken as 100%).
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NMR spectra were taken on a Bruker Advance 500 (500 MHz)
instrument in DMSO-d with TMS as an internal standard. Chemical
6
shifts are reported in δ values (ppm) relative to internal TMS. The
abbreviations s = singlet, d = doublet, t = triplet, q = quadruplet, quint
=
quintuplet, m = multiplet, and b = broad are used throughout.
Elemental analysis results (C, H, N, S) were realized on a Carlo-Erba
EA 1108 elemental analyzer or on a FlashEA 1112 series (Thermo-
Interscience) and were within (0.4% of the theoretical values (see
Supporting Information). All compounds showed >95% purity,
according to the elemental analyses. All reactions were routinely
checked by TLC on silica gel Merck 60F254.
N-(5-Chloro-2-thienyl)acetamide (20). The title compound was
2
2
obtained according to the process described in the literature; mp
Figure 4. Effect of treatment with 24 per os on the object recognition
test in CD1 mice. The discrimination index (δnew − fam) was the
difference between the exploration times of the new and familiar
objects on the last session with intersessions interval of 24 h. Under
such conditions, control rats did no longer recognize the familiar
object and spent similar times in exploring the familiar and new
objects. Compound 24 significantly improved at 0.3 and 1 mg/kg per
os the recognition of the familiar object as evidenced by increased
exploration toward the new object (n = 13). *p ≤ 0.05, **p ≤ 0.01 vs
control, one way ANOVA.
31
1
1
77−178 °C (lit. 179 °C). H NMR (DMSO-d , 500 MHz) δ 2.06
3H, s, CH ), 6.43 (1H, d, 3-H), 6.85 (1H, d, 4-H), 11.36 (1H, s,
NH).
N-[3-(Aminosulfonyl)-5-chloro-2-thienyl]acetamide (21). To
PCl (3.5 g, 17 mmol) dissolved in chlorosulfonic acid (18 mL) was
added in the cold state (+5 °C) N-(5-chloro-2-thienyl)-acetamide
(20) (3.5 g, 20 mmol). The resulting mixture was heated at 70 °C for
45 min. The reaction medium was poured on ice and extracted with
diethylether. Gaseous ammonia was bubbled into the dried organic
phase during 15 min. After clarification of the ether suspension, the
solvents were removed under reduced pressure. The solid residue was
taken up in water (20 mL) and the insoluble material was collected by
filtration, washed with water, and dried to yield the expected
compound (1.20 g, 24%) used in the next step without further
6
(
3
5
CONCLUSION
■
The present work explored new 3,4-dihydro-2H-thieno-1,2,4-
thiadiazine 1,1-dioxides bearing at least one chlorine atom on
the aromatic ring and one alkyl/fluoroalkyl chain on the
nitrogen atom at the 4-position of the heterocycle. Three series
of compounds were examined by varying the position of the
sulfur atom in the thiophene ring.
1
purification. H NMR (DMSO-d , 500 MHz) δ 2.24 (3H, s, CH ),
6
3
7
.09 (1H, s, 4-H), 7.60 (2H, s, SO NH ), 10.19 (1H, s, NH).
2
2
5
-Chloro-2-(ethylamino)-thiophene-3-sulfonamide (22). N-
[3-(Aminosulfonyl)-5-chloro-2-thienyl]-acetamide (21) (500 mg, 1.96
mmol) was suspended in dry ether (20 mL) prior to the addition of
LiAlH (250 mg). After being stirred during 40 min at room
The 6-chloro-3,4-dihydro-2H-thieno[3,2-e]-1,2,4-thiadiazine
4
temperature, the medium was cooled in an ice bath and water was
slowly added. The mixture was then carefully adjusted to pH 4 by
adding concentrated HCl prior extraction with ethyl acetate (3 × 15
mL). The combined organic layers were dried over MgSO4 and
filtered, and the filtrate was concentrated under reduced pressure.
After treatment with charcoal in methanol, the expected compound
1
,1-dioxide series appeared to possess SAR close to that
observed with their benzo-1,2,4-thiadiazine 1,1-dioxides
counterparts. However, 5,7-dichloro-3,4-dihydro-2H-thieno-
[
3,4-e]-1,2,4-thiadiazine 1,1-dioxides clearly displayed an
atypical behavior, with some saturated final compounds being
found to be less active than their corresponding unsaturated
counterparts.
was crystallized from a mixture of methanol/water (1:2) (290 mg,
1
6
(
1%); mp 134−140 °C (dec.). H NMR (DMSO-d , 500 MHz) δ 1.19
6
Different attempts to prepare examples of 6-chloro-3,4-
dihydro-2H-thieno[2,3-e]-1,2,4-thiadiazine 1,1-dioxides resulted
in the synthesis of 24, bearing an ethyl chain on the nitrogen
atom at the 4-position. In vitro and ex vivo tests revealed its
strong biological efficiency on postsynaptic AMPA receptors
and demonstrated its safety in vivo up to a dose of 30 mg/kg
3H, t, NHCH CH ), 3.12 (2H, m, NHCH CH ), 6.49 (1H, t,
2 3 2 3
NHCH CH ), 6.83 (1H, s, 4-H), 7.18 (2H, s, SO NH ). Anal.
2
3
2
2
(C H ClN O S ) C, H, N, S.
6
9
2
2 2
6-Chloro-4-ethyl-4H-thieno[2,3-e]-1,2,4-thiadiazine 1,1-di-
oxide (23). 5-Chloro-2-(ethylamino)-thiophene-3-sulfonamide (22)
(380 mg, 1.58 mmol) was suspended in triethyl orthoformate (20
mL). The mixture was heated at 170 °C in an open vessel for 30 min.
After the mixture was cooled, the insoluble material that appeared was
(
no plasma/brain concentration data available). Therefore, 24
appeared to possess the most interesting pharmacological
profile among all the new synthesized thiophenic compounds.
Compound 24 was further explored in vivo and was found to
modulate LTP and to increase AMPA-mediated noradrenaline
release, two mechanisms underlying potential cognition-
enhancing properties. This assumption was confirmed thanks
to the object recognition test in mice, where 24 was found to
improve cognition after oral administration of doses of the drug
as low as 0.3 mg/kg.
collected by filtration, washed with diethyl ether, and dried (0.32 g,
1
8
1%); mp 226−228 °C. H NMR (DMSO-d , 500 MHz) δ 1.37 (3H,
6
t, CH CH ), 3.97 (2H, q, CH CH ), 7.61 (1H, s, 7-H), 8.11 (1H, s, 3-
2
3
2
3
H). Anal. (C H ClN O S ) C, H, N, S.
7
7
2
2 2
6-Chloro-4-ethyl-3,4-dihydro-2H-thieno[2,3-e]-1,2,4-thiadia-
zine 1,1-dioxide (24). A solution of 6-chloro-4-ethyl-4H-thieno[2,3-
e]-1,2,4-thiadiazine 1,1-dioxide (23) (180 mg, 0.72 mmol) in 2-
propanol (15 mL) was supplemented under stirring with sodium
borohydride (150 mg, 3.97 mmol). After the mixture was stirred for 5
min at room temperature, the solvent was removed by distillation
under reduced pressure and the residue was suspended in water (25
mL). The alkaline suspension was adjusted to pH 7 with 6 N HCl and
extracted 3-fold with chloroform (3 × 10 mL). The combined organic
layers were dried over MgSO4 and filtered. The filtrate was
concentrated to dryness under reduced pressure, and the residue of
Taken as a whole, 3,4-dihydro-2H-thieno-1,2,4-thiadiazine
,1-dioxides constitute a new class of powerful AMPA
1
potentiators. Further exploration of this series will be
performed with the objective of discovering new candidates
for the treatment of cognitive deficits.
the title compound was recrystallized in methanol/water, 1:2 and dried
EXPERIMENTAL SECTION
Synthesis. Melting points were determined on a Stuart smp3
capillary apparatus and are uncorrected. IR spectra were recorded as
KBr pellets on a Perkin-Elmer 1750 FT spectrophotometer. The H
1
(
0.09 g, 50%); mp 150−154 °C. H NMR (DMSO-d , 500 MHz) δ
■
6
1.16 (3H, t, CH
CH ), 3.30 (2H, q, CH CH ), 4.70 (2H, d, 3-H ),
2 3 2 3 2
7.13 (s, 1H, 7-H), 7.90 (t, 1H, NH). Anal. (C H ClN O S ) C, H, N,
7
9
2
2 2
1
S.
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Journal of Medicinal Chemistry
Article
2
,5-Dichloro-4-nitrothiophene-3-sulfonyl Chloride (26). 2,5-
then cooled on ice. The precipitate that formed was collected by
dichlorothiophene-3-sulfonyl chloride (25) (10 g, 40 mmol) was
slowly added to an ice-cold mixture of nitric acid (30 mL) and sulfuric
acid (30 mL). After 1 h of stirring on an ice bath, the mixture was
poured on ice. The resulting solid was collected by filtration, washed
with water, and dried. The title compound was used in the next step
without further purification. (11.3 g, 96%).
filtration, washed with n-hexane, and dried (1.55 g, 35%); mp 172−
−1 1
175 °C. IR (KBr): 1605, 1547, 1314, 1147 cm . H NMR (DMSO-d ,
6
500 MHz) δ 1.30 (t, 3H, NCH CH ), 4.20 (q, 2H, NCH CH ), 7.90
2
3
2
3
(s, 1H, 3-H). Anal. (C H Cl N O S ) C, H, N, S.
7
6
2
2
2 2
5,7-Dichloro-4-ethyl-3,4-dihydro-2H-thieno[3,4-e]-1,2,4-
thiadiazine 1,1-Dioxide (30c). The title compound was obtained as
described for 24 starting from 5,7-dichloro-4-ethyl-4H-thieno[3,4-e]-
1,2,4-thiadiazine 1,1-dioxide (29c) (0.5 g, 1.7 mmol) (380 mg, 75%);
mp 144−147 °C. IR (KBr): 3313, 3281, 2963, 1582, 1548, 13224,
2
,5-Dichloro-4-nitrothiophene-3-sulfonamide (27). A solu-
tion of 2,5-dichloro-4-nitrothiophene-3-sulfonyl chloride (26) (11 g,
7 mmol) in dioxane (150 mL) was added dropwise to a 10% w/v
3
−
1
1
ammonium hydroxide solution (300 mL). After 1 h, ammonia and
dioxane were removed by evaporation under reduced pressure. The
white precipitate that formed was collected by filtration, washed with
water, and dried (7.7 g, 75%); mp 125−126 °C. IR (KBr): 3390, 3291,
1172 cm . H NMR (DMSO-d
NCH CH ), 3.20 (q, 2H, NCH CH
1H, NH). Anal. (C Cl ) C, H, N, S.
6
, 500 MHz) δ 1.10 (t, 3H,
), 5.40 (s, 2H, 3-H ), 8.30 (s,
2
3
2
3
2
H
8
N O S
2 2 2 2
7
2,5-Dichloro-4-(propionamido)thiophene-3-sulfonamide
31a). To 4-amino-2,5-dichlorothiophene-3-sulfonamide (28) (500
−1
(
1
505, 1541, 1306, 1369, 1170 cm . Anal. (C H Cl N O S ) C, H, N,
4 2 2 2 4 2
mg, 2.0 mmol) dissolved in dioxane (10 mL) were added in the cold
state (+5 °C) pyridine (0.2 mL) and propionyl chloride (0.22 mL, 2.5
mmol). The flask was hermetically closed immediately, and contents
were vigorously stirred at ambient temperature for 30 min. The
solvents were then removed under reduced pressure. The solid residue
was taken up in water (12 mL), and the insoluble material was
S.
4
-Amino-2,5-dichlorothiophene-3-sulfonamide (28). 2,5-Di-
chloro-4-nitrothiophene-3-sulfonamide (26) (7.25 g, 26 mmol) was
suspended in a 1:1 ethanol/water mixture (375 mL). The suspension
was heated until the product dissolved. Then ammonium chloride (4.5
g) and powdered iron (15 g) were added. After refluxing for 10 min,
the reaction was complete. The insoluble material was removed by
filtration and rinsed with a small amount of hot ethanol. The filtrate
was concentrated under reduced pressure. The beige precipitate that
formed was collected by filtration, washed with water, and dried (3.4 g,
collected by filtration, washed with water, and dried to yield the
1
expected compound (500 mg, 82%). H NMR (DMSO-d , 500 MHz)
6
δ 1.07 (3H, t, NHCOCH CH ), 2.31 (2H, q, NHCOCH CH ), 7.58
2
3
2
3
(
2H, s, SO NH ), 9.34 (1H, s, NHCOCH CH ).
2 2 2 3
2
,5-Dichloro-4-(propylamino)thiophene-3-sulfonamide
(32a). 2,5-Dichloro-4-(propionamido)-thiophene-3-sulfonamide
31a) (450 mg, 1.8 mmol) was suspended in dry ether (20 mL)
prior to the addition of LiAlH (250 mg). After being stirred for 60
5
1
2%); mp 120 °C (dec.). IR (KBr): 3440, 3412, 3354, 3290, 1600,
557, 1336, 1166 cm . Anal. (C H Cl N O S ) C, H, N, S.
−1
4
4
2
2
2 2
(
5
,7-Dichloro-4H-thieno[3,4-e]-1,2,4-thiadiazine 1,1-Dioxide
29a). 4-Amino-2,5-dichlorothiophene-3-sulfonamide (28) (250 mg,
.97 mmol) was dissolved in triethyl orthoformate (2.5 mL). The
(
0
4
min, additional LiAlH (100 mg) was added. After 30 min of stirring,
4
the mixture was cooled in an ice bath and water was slowly added. The
mixture was then carefully adjusted to pH 4 by adding concentrated
HCl. The mixture was extracted with ethyl acetate (3 × 15 mL). The
solution was brought to boiling in an open vessel. After 1.5 h, the
suspension was allowed to cool, yielding a beige precipitate, which was
collected by filtration, washed with diethyl ether, and dried (0.21 g,
−1
combined organic layers were dried over MgSO and filtered, and the
8
0%); mp 200−203 °C. IR (KBr): 3147, 1597, 1318, 1175 cm . Anal.
4
filtrate was concentrated under reduced pressure. The dry residue was
(
C H Cl N O S ) C, H, N, S.
5
2
2
2
2 2
5
,7-Dichloro-3,4-dihydro-2H-thieno[3,4-e]-1,2,4-thiadiazine
then recrystallized from a mixture of acetone/water (1:10) (270 mg,
1
1
,1-Dioxide (30a). The suspension of 5,7-dichloro-4H-thieno[3,4-e]-
46%); mp 160 °C (dec). H NMR (DMSO-d
6
, 500 MHz) δ 0.92 (3H,
CH CH ), 3.47 (2H, qt,
), 7.68 (2H, bs, SO NH ), 8.33 (1H, s,
1,2,4-thiadiazine 1,1-dioxide (29a) (0.8 g, 3.1 mmol) in water (15 mL)
t, NHCH
NHCH CH
NHCH CH CH
2
CH
2
CH
3
), 1.60 (2H, h, NHCH
2
2
3
was supplemented under stirring with sodium borohydride (2.0 g, 53
mmol). After 10 min stirring at room temperature, the alkaline
suspension was adjusted to pH 7 with 6 N HCl and extracted 3-fold
with dichloromethane (3 × 25 mL). The combined organic layers
2
2
CH
3
).
2
2
2
2
3
5,7-Dichloro-4-propyl-4H-thieno[3,4-e]-1,2,4-thiadiazine
1,1-Dioxide (29d). 2,5-Dichloro-4-(propylamino)-thiophene-3-sulfo-
namide (32a) (310 mg, 1.1 mmol) was suspended in triethyl
orthoformate (7 mL). The mixture was heated at 120−130 °C in an
open vessel for 90 min. After the mixture was cooled, the insoluble
material that appeared was collected by filtration, washed with diethyl
were dried over MgSO and filtered. The filtrate was concentrated to
4
dryness under reduced pressure, and the residue of the title compound
was recrystallized in methanol/water 1:2 (260 mg, 32%); mp 125−130
−
1
°
C. IR (KBr): 3372, 3221, 1583, 1524, 1325, 1173 cm . Anal.
1
(
C H Cl N O S ) C, H, N, S.
ether, and dried (240 mg, 75%); mp 179−180 °C. H NMR (DMSO-
5
4
2
2
2 2
5
,7-Dichloro-4-methyl-4H-thieno[3,4-e]-1,2,4-thiadiazine
d , 500 MHz) δ 0.90 (3H, t, 4-CH CH CH ), 1.74 (2H, m, 4-
6
2
2
3
1
,1-Dioxide (29b). 5,7-Dichloro-4H-thieno[3,4-e]-1,2,4-thiadiazine
CH CH CH ), 4.17 (2H, t, 4-CH CH CH ), 7.93 (1H, s, 3-H). Anal.
2 2 3 2 2 3
1,1-dioxide (29a) (200 mg, 0.78 mmol) was dissolved in a mixture
(C H Cl N O S ) C, H, N, S.
8 8 2 2 2 2
of nitroethane (2 mL) and dimethylformamide (2 mL). The solution
was heated to 60 °C. Then potassium carbonate (0.2 g) was added,
followed by iodomethane (0.2 mL, 2.5 mmol). After stirring for 2 h,
the solvents were removed under reduced pressure. The residue was
taken up in water. The insoluble material was quickly collected by
filtration, washed with water, and dried (120 mg, 54%); mp 202−207
5,7-Dichloro-4-propyl-3,4-dihydro-2H-thieno[3,4-e]-1,2,4-
thiadiazine 1,1-Dioxide (30d). The title compound was obtained as
described for 24 starting from 5,7-dichloro-4-propyl-4H-thieno[3,4-e]-
1,2,4-thiadiazine 1,1-dioxide (29d) (180 mg, 0,6 mmol) (130 mg,
1
72%); mp 135−138 °C. H NMR (DMSO-d
, 500 MHz) δ 0.90 (3H,
CH CH ), 3.21 (2H, t, 4-
), 8.42 (1H, s, 2-H). Anal.
2
6
t, 4-CH
CH
CH
CH
), 4.50 (2H, d, 3-H
) C, H, N, S.
), 1.64 (2H, m, 4-CH
2
2
3
2
2
3
−1 1
°
C. IR (KBr): 2960, 1607, 1551, 1308, 1145 cm . H NMR (DMSO-
CH
(C
2
CH
2
3
d , 500 MHz) δ 3.74 (s, 3H, NCH ), 7.73 (s, 1H, 3-H). Anal.
8
H10Cl
2
N
2
O
2
S
2
6
3
(
C H Cl N O S ) C, H, N, S.
5,7-Dichloro-4-(2-fluoroethyl)-4H-thieno[3,4-e]-1,2,4-thia-
diazine 1,1-Dioxide (29e). To 4-amino-2,5-dichlorothiophene-3-
sulfonamide (28) (1 g, 4.0 mmol) dissolved in dry ether (20 mL) was
added in the cold state potassium carbonate (2 g). The mixture was
cooled at −30 °C, and (+5 °C) pyridine (0.2 mL) and fluoroacetyl
chloride (1 mL, 14 mmol) were added. After 90 min of stirring, the
insoluble material was collected by filtration and washed with
methanol. The combined filtrates were then concentrated under
reduced pressure. This crude residue was suspended in dry ether (20
mL) and cooled at 0 °C prior to the addition of a 1.0 M borane
solution in THF (10 mL, 10 mmol). After being stirred during 3 h at
ambient temperature, water was slowly added. The organic layer was
6
4
2
2
2 2
5
,7-Dichloro-4-methyl-3,4-dihydro-2H-thieno[3,4-e]-1,2,4-
thiadiazine 1,1-Dioxide (30b). The title compound was obtained as
described for 24 starting from 5,7-dichloro-4-methyl-4H-thieno[3,4-e]-
1
3
1
4
,2,4-thiadiazine 1,1-dioxide (28b) (500 mg, 1.8 mmol) (190 mg,
8%); mp 150−154 °C (dec.). IR (KBr): 3231, 2972, 1547, 1328,
−1 1
179 cm . H NMR (DMSO-d , 500 MHz) δ 2.91 (3H, s, 4-CH ),
6
3
.40 (2H, s, 3-H ). Anal. (C H Cl N O S ) C, H, N, S.
2
6
6
2
2
2 2
5
,7-Dichloro-4-ethyl-4H-thieno[3,4-e]-1,2,4-thiadiazine 1,1-
Dioxide (29c). 4-Amino-2,5-dichlorothiophene-3-sulfonamide (28)
4 g, 16 mmol) was suspended in triethyl orthoformate (20 mL) and
heated at 170 °C for 35 min in an open vessel. The suspension was
(
7
846
dx.doi.org/10.1021/jm400676g | J. Med. Chem. 2013, 56, 7838−7850

Journal of Medicinal Chemistry
Article
separated, dried over MgSO , and filtered, and the filtrate was
concentrated under reduced pressure. The dry residue was then
suspended in triethyl orthoformate (8 mL) and heated at 150 °C for
(36a) (250 mg, 1.1 mmol) was dissolved in a mixture of acetonitrile (5
mL) and dimethylformamide (0.5 mL). Potassium carbonate (500
mg) and methyl iodide (0.3 mL, 0.68 g, 4.8 mmol) were added, and
the mixture was heated at 55 °C for 7 h. At the end of the reaction, the
solvent was removed under reduced pressure and the residue was
taken up in water. The insoluble material was collected by filtration,
washed with water, and dried (205 mg, 77%); mp 252−254 °C. IR
4
6
0 min in an open vessel. The resulting mixture was concentrated
under reduced pressure. The residue was dissolved in acetone and
treated with charcoal. The resulting solution was concentrated and
cooled on ice. The insoluble material was collected by filtration and
−1 1
dried to yield the expected compound to give the expected title
(KBr): 3091, 1607, 1519, 1485, 1297, 1150 cm . H NMR (DMSO-
1
compound (250 mg, 20%); mp 177−179 °C. H NMR (DMSO-d ,
d , 500 MHz) δ 3.64 (s, 3H, NCH ), 7.58 (s, 1H, 5-H), 8.05 (s, 1H, 3-
6
6
3
5
00 MHz) δ 4.65 (2H, dt, NCH CH F), 4.79 (2H, dt, NCH CH F),
H). Anal. (C H ClN O S ) C, H, N, S.
2
2
2
2
6 5 2 2 2
7
.89 (1H, s, 3-H). Anal. (C H Cl FN O S ) C, H, N, S.
6-Chloro-4-methyl-3,4-dihydro-2H-thieno[3,2-e]-1,2,4-thia-
diazine 1,1-Dioxide (37b). The title compound was obtained as
described for 24 starting from 6-chloro-4-methyl-4H-thieno[3,2-e]-
1,2,4-thiadiazine 1,1-dioxide (36a) (200 mg, 0.85 mmol) (100 mg,
7
5
2
2
2 2
5
,7-Dichloro-4-(2-fluoroethyl)-3,4-dihydro-2H-thieno[3,4-e]-
1
,2,4-thiadiazine 1,1-Dioxide (30e). The title compound was
obtained as described for 24 starting from 5,7-dichloro-4-(2-
−1
fluoroethyl)-4H-thieno[3,4-e]-1,2,4-thiadiazine 1,1-dioxide (29e)
49%); mp 174−176 °C. IR (KBr): 3236, 3089, 1562, 1322, 1154 cm .
1
1
(150 mg, 0.5 mmol) (120 mg, 79%); mp 170−174 °C. H NMR
(DMSO-d , 500 MHz) δ 3.61 (2H, dt, NCH CH F), 4.56 (2H, s, 3-
H NMR (DMSO-d , 500 MHz) δ 3.00 (s, 3H, NCH ), 4.63 (d, 2H,
6
3
6
2
2
3-H ), 7.17 (s, 1H, 5-H), 8.12 (t, 1H, NH). Anal. (C H ClN O S ) C,
2
6
7
2
2 2
H ), 4.71 (2H, dt, NCH CH F), 8.47 (1H, s, N-H). Anal.
H, N, S.
2
2
2
(
C H Cl FN O S ) C, H, N, S.
6-Chloro-4-ethyl-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-Di-
oxide (36c). The title compound was obtained as described for 36b
(250 mg, 1.1 mmol) using ethyl bromide (0.4 mL, 0.58 g, 5.4 mmol)
instead of methyl iodide (190 mg, 68%); mp 189−190 °C. IR (KBr):
7
7
2
2
2 2
2,5-Dichloro-4-(2,2-difluoroacetamido)thiophene-3-sulfona-
mide (31c). To 4-amino-2,5-dichlorothiophene-3-sulfonamide (28)
(
800 mg, 3.2 mmol) dissolved in dioxane (15 mL) were added in the
cold state (+10 °C) pyridine (0.4 mL) and 2,2-difluoroacetyl chloride
0.4 mL, 3.8 mmol). The flask was hermetically closed immediately,
−1 1
3098, 2982, 1609, 1518, 1300, 1161 cm . H NMR (DMSO-d , 500
6
(
MHz) δ 1.32 (t, 3H, NCH CH ), 4.08 (q, 2H, NCH CH ), 7.68 (s,
2
3
2
3
and contents were vigorously stirred at ambient temperature for 5 min.
The solvents were then removed under reduced pressure. The
resulting oil was taken up in water (12 mL) and extracted 3-fold with
ethyl acetate (3 × 10 mL). The combined organic layers were dried
1H, 5-H), 8.12 (s, 1H, 3-H). Anal. (C H ClN O S ) C, H, N, S.
7 7 2 2 2
6-Chloro-4-ethyl-3,4-dihydro-2H-thieno[3,2-e]-1,2,4-thiadia-
zine 1,1-Dioxide (37c). The title compound was obtained as
described for 24 starting from 6-chloro-4-ethyl-4H-thieno[3,2-e]-1,2,4-
thiadiazine 1,1-dioxide (36c) (250 mg, 0.80 mmol) (90 mg, 45%); mp
119−120 °C. IR (KBr): 3235, 2980, 2931, 2872, 1558, 1324, 1154
over MgSO and filtered. The filtrate was concentrated to dryness
4
under reduced pressure, and the residue of the title compound was
1
−1 1
recrystallized in methanol/water on an ice bath (630 mg, 60%). H
cm . H NMR (DMSO-d
3.40 (q, 2H, 4-CH CH ), 4.65 (d, 2H, 3-H
(t, 1H, NH). Anal. (C ClN ) C, H, N, S.
6
, 500 MHz) δ 1.06 (t, 3H, 4-CH
CH ),
2 3
NMR (DMSO-d , 500 MHz) δ 6.42 (1H, t J = 53.09 Hz,
2
3
2
), 7.14 (s, 1H, 5-H), 7.95
6
NHCOCHF ), 7.78 (2H, s, SO NH ), 10.37 (1H, s, NHCOCHF ).
7
H
9
O S
2 2 2
2
2
2
2
2
,5-Dichloro-4-(2,2-difluoroethylamino)thiophene-3-sulfo-
6-Chloro-4-isopropyl-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-
Dioxide (36d). The title compound was obtained as described for
36b (250 mg, 1.1 mmol) using isopropyl iodide (0.5 mL, 0.92 g, 5.4
mmol) instead of methyl iodide (200 mg, 42%); mp 149−150 °C. IR
namide (32c). The title compound was obtained as described for 32a
starting from 2,5-dichloro-4-(2,2-difluoroacetamido)thiophene-3-sul-
fonamide (31c) (700 mg, 2.1 mmol) (480 mg, 72%) and was used in
the next step without further purification.
−1 1
(KBr): 3112, 2989, 1605, 1515, 1306, 1170 cm . H NMR (DMSO-
, 500 MHz) δ 1.43 (d, 6H, NCH(CH ), 4.08 (sept, 1H,
NCH(CH ) ), 7.72 (s, 1H, 5-H), 8.18 (s, 1H, 3-H). Anal.
5
,7-Dichloro-4-(2,2-difluoroethyl)-4H-thieno[3,4-e]-1,2,4-
d
6
)
3 2
thiadiazine 1,1-Dioxide (29f). 2,5-Dichloro-4-(2,2-
difluoroethylamino)thiophene-3-sulfonamide (32c) (480 mg, 1.5
mmol) was suspended in triethyl orthoformate (20 mL). The mixture
was heated at 180 °C in an open vessel for 7 h. After cooling, n-hexane
was added to the mixture, and the resulting insoluble material that
3
2
(C H ClN O S ) C, H, N, S.
8
9
2
2 2
6-Chloro-4-isopropyl-3,4-dihydro-2H-thieno[3,2-e]-1,2,4-
thiadiazine 1,1-Dioxide (37d). The title compound was obtained as
described for 24 starting from 6-chloro-4-isopropyl-4H-thieno[3,2-e]-
1,2,4-thiadiazine 1,1-dioxide (36d) (250 mg, 0.9 mmol) (130 mg,
51%); mp 100−102 °C. IR (KBr): 3267, 3241, 2970, 2954, 1545,
appeared was collected by filtration and was recrystallized in methanol
200 mg, 40%); mp 152−154 °C. H NMR (DMSO-d , 500 MHz) δ
.83 (2H, t, 4-CH CHF ), 6.53 (1H, t J = 54.20 Hz, 4-CH CHF ),
1
(
4
7
6
−1 1
1332, 1156 cm . H NMR (DMSO-d
NCH(CH ), 4.03 (sept, 1H, NCH(CH
(s, 1H, 5-H), 7.79 (t, 1H, NH). Anal. (C H11ClN O S
8 2 2 2
, 500 MHz) δ 1.15 (d, 6H,
), 4.62 (d, 2H, 3-H ), 7.21
) C, H, N, S.
2
2
2
2
6
.93 (1H, s, 3-H). Anal. (C H Cl F N O S ) C, H, N, S.
3
)
2
3
)
2
2
7
4
2
2
2
2 2
5
,7-Dichloro-4-(2,2-difluoroethyl)-3,4-dihydro-2H-thieno-
[
3,4-e]-1,2,4-thiadiazine 1,1-Dioxide (30f). The title compound
6-Chloro-4-propyl-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-Di-
oxide (36e). To 6-chloro-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-
dioxide (36a) (450 mg, 2.0 mmol) in acetonitrile (15 mL) were
added potassium carbonate (900 mg) and n-propyl iodide (0.6 mL, 6.2
mmol), and the mixture was heated at 90 °C for 150 min. At the end
of the reaction, the solvent was removed under reduced pressure and
the residue was taken up in water. The insoluble material was collected
was obtained as described for 24 starting from 5,7-dichloro-4-(2,2-
difluoroethyl)-4H-thieno[3,4-e]-1,2,4-thiadiazine 1,1-dioxide (29f)
140 mg, 0.4 mmol) (90 mg, 64%); mp 168−170 °C. H NMR
DMSO-d , 500 MHz) δ 4.58 (2H, s, 3-H ), 4.83 (2H, t, 4-
1
(
(
6
2
CH CHF ), 6.37 (1H, t, J = 55.53 Hz, 4-CH CHF ), 8.55 (1H, s,
2
2
2
2
NH). Anal. (C H Cl F N O S ) C, H, N, S.
7
6
2
2
2
2 2
6
-Chloro-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-Dioxide
by filtration, washed with water, and recrystallized in methanol (450
24
1
(
1
36a). 3-Amino-5-chlorothiophene-2-sulfonamide (35) (250 mg,
mg, 84%); mp 155−157 °C. H NMR (DMSO-d , 500 MHz) δ 0.87
6
.0 mmol) was suspended in triethyl orthoformate (2.5 mL). The
(3H, t, N-CH CH CH ), 1.68 (2H, m, N-CH CH CH ), 3.97 (2H, t,
2
2
3
2
2
3
resulting mixture was heated at about 160 °C for 30 min. A granular
yellow precipitate formed and was collected by filtration, washed with
diethyl ether, and dried (185 mg, 83%); mp 260−262 °C. IR (KBr):
N-CH CH CH ), 7.68 (1H, s, 5-H), 8.09 (1H, s, 3-H). Anal.
2 2 3
(C H ClN O S ) C, H, N, S.
8
9
2
2 2
6-Chloro-4-propyl-3,4-dihydro-2H-thieno[3,2-e]-1,2,4-thia-
diazine 1,1-Dioxide (37e). The title compound was obtained as
described for 24 starting from 6-chloro-4-propyl-4H-thieno[3,2-e]-
−
1
3
214, 3103, 3061, 1602, 1569, 1515, 1378, 1147 cm . Anal.
(
C H ClN O S ) C, H, N, S.
5
3
2
2 2
6-Chloro-3,4-dihydro-2H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-
1,2,4-thiadiazine 1,1-dioxide (36e) (200 mg, 0.76 mmol) (150 mg,
1
Dioxide (37a). The title compound was obtained as described for 30a
starting from 6-chloro-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide
74%); mp 134−136 °C. H NMR (DMSO-d , 500 MHz) δ 0.86 (3H,
6
t, N-CH CH CH ), 1.51 (2H, m, N-CH CH CH ), 3,29 (2H, t, N-
2
2
3
2
2
3
(
(
36a) (150 mg, 0,07 mmol) (110 mg, 73%); mp 154−156 °C. Anal.
CH CH CH ), 4.66 (2H, d, 3-H ), 7.14 (1H, s, 5-H), 7.95 (1H, s, N-
H). Anal. (C H ClN O S ) C, H, N, S.
8 11 2 2 2
2 2 3 2
C H ClN O S ) C, H, N, S.
5
5
2
2
2
6
-Chloro-4-methyl-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-di-
6-Chloro-4-(fluoromethyl)-4H-thieno[3,2-e]-1,2,4-thiadia-
zine 1,1-Dioxide (36f). To 6-chloro-4H-thieno[3,2-e]-1,2,4-thiadia-
oxide (36b). 6-Chloro-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide
7
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Journal of Medicinal Chemistry
Article
zine 1,1-dioxide (36a) (500 mg, 2.2 mmol) in ice-cold acetonitrile (15
mL) were added potassium carbonate (900 mg) and bromofluoro-
methane (1 mL, 16 mmol). The mixture was placed in a closed vessel
and heated at 70 °C for 8 h. At the end of the reaction, the solvent was
removed under reduced pressure and the residue was taken up in
starting from 5-chloro-3-(2,2-difluoroacetamido)thiophene-2-sulfona-
1
mide (38) (1.0 g, 3.4 mmol) (570 mg, 60%). H NMR (DMSO-d ,
6
500 MHz) δ 3.67 (2H, m, N-CH CHF ), 6.09 (1H, tt, N-CH CHF ),
2
2
2
2
7.08 (1H, s, 4-H), 7.45 (2H, bs, SO
NH
). Anal. (C
2
H
6
ClF
N
O S )
2 2 2
2
7
2
C, H, N, S.
water. The insoluble material was collected by filtration and
6-Chloro-4-(2,2-difluoroethyl)-4H-thieno[3,2-e]-1,2,4-thia-
diazine 1,1-Dioxide (36i). 5-Chloro-3-(2,2-difluoroethylamino)-
thiophene-2-sulfonamide (39) (860 mg, 3.1 mmol) was heated at 80
1
recrystallized in methanol (380 mg, 66%); mp 198−200 °C.
H
NMR (DMSO-d , 500 MHz) δ 6.10 (2H, d, N-CH F), 7.71 (1H, s, 5-
6
2
°
C in formic acid (30 mL) for 5 h. After cooling, the solvent was
removed under reduced pressure. The residue was taken up in water,
H), 8.37 (1H, s, 3-H). Anal. (C H ClFN O S ) C, H, N, S.
6
4
2
2 2
6
-Chloro-4-(isopropoxymethyl)-3,4-dihydro-2H-thieno[3,2-
e]-1,2,4-thiadiazine 1,1-Dioxide (37f). A solution of 6-chloro-4-
and the resulting insoluble material that appeared was collected by
1
(
(
fluoromethyl)-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (36f)
200 mg, 0.8 mmol) in 2-propanol (8 mL) at 50 °C was supplemented
filtration (440 mg, 49%); mp 157−158 °C. H NMR (DMSO-d
6
, 500
MHz) δ 4.60 (2H, t, N-CH CHF ), 6.46 (1H, t, N-CH CHF ), 7.70
2
2
2
2
(
1H, s, 5-H), 8.08 (1H, s, 3-H). Anal. (C H ClF N O S ) C, H, N, S.
under stirring with sodium borohydride (400 mg, 10.6 mmol). After
the mixture was stirred for 10 min, the solvent was removed by
distillation under reduced pressure and the residue was suspended in
ethyl acetate (15 mL). After removal of the solid by filtration on Celite
and evaporation of the solvent, the residue was purified on a column
using ethyl acetate/n-hexane (15/5) as a mobile phase followed by a
7 5 2 2 2 2
6-Chloro-4-(2,2-difluoroethyl)-3,4-dihydro-2H-thieno[3,2-e]-
1
,2,4-thiadiazine 1,1-Dioxide (37i). The title compound was
obtained as described for 24 starting from 6-chloro-4-(2,2-difluor-
oethyl)-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (36i) (480 mg,
1
1
5
.7 mmol) (360 mg, 75%); mp 131−133 °C. H NMR (DMSO-d ,
6
00 MHz) δ 3.86 (2H, m, N−CH CHF ), 4.78 (2H, s, 3-H ), 6.21
(1H, tt, N-CH CHF ), 7.18 (1H, s, 5-H), 8.14 (1H, bs, N-H). Anal.
2
2
2
methanol/water recrystallization on an ice bath (0.01 g, 5%); mp 65−
1
2 2
6
8 °C. H NMR (DMSO-d , 500 MHz) δ 1.10 (6H, d, OCH(CH3)2),
6
(
C H ClF N O S ) C, H, N, S.
Effect on AMPA-Evoked Membrane Depolarization. This
7 7 2 2 2 2
3
.70 (1H, m, OCH(CH3)2), 4.75 (4H, s, 3-H2/N-CH -O), 7.11 (1H,
2
s, 5-H). Anal. (C H ClFN O S ) C, H, N, S.
6
6
2
2 2
assay investigating AMPA-evoked membrane depolarization, measured
by fluorescent membrane potential dyes and an imaging based plate
reader on rat primary brain cultures, was achieved following our
6
-Chloro-4-(2-fluoroethyl)-4H-thieno[3,2-e]-1,2,4-thiadia-
zine 1,1-Dioxide (36g). The title compound was obtained as
described for 36b (600 mg, 2.7 mmol) using 1-fluoro-2-iodoethane
1
8
previously published procedure.
(
1
0.6 mL, 1.3 g, 7.4 mmol) instead of methyl iodide (440 mg, 61%); mp
1
Effect on AMPA Currents in Xenopus Laevis Oocytes.
85−187 °C. H NMR (DMSO-d , 500 MHz) δ 4.38 (2H, dt, N-
6
Electrophysiological recordings were performed at room temperature
CH CH F), 4.70 (2H, dt, N-CH CH F), 7.66 (1H, s, 5-H), 8.05 (1H,
2
2
2
2
+
on Xenopus laevis oocytes expressing rat cortex poly(A ) mRNA using
s, 3-H). Anal. (C H ClFN O S ) C, H, N, S.
7
6
2
2
2
18
a previously described procedure. S22286 (100 μM, used as positive
6
-Chloro-4-(2-fluoroethyl)-3,4-dihydro-2H-thieno[3,2-e]-
,2,4-thiadiazine 1,1-Dioxide (37g). The title compound was
obtained as described for 24 starting from 6-chloro-4-(2-fluoroethyl)-
H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (36g) (400 mg, 0.76
potent reference as AMPA potentiator with rapid wash-out) was first
bath-applied on the same oocyte for 45 s before, 30 s during, and 30 s
after the application of 10 μM AMPA in order to select for the
experiment oocytes presenting sufficient potentiation of the AMPA-
evoked current. Oocytes presenting less than 130-fold increase of the
AMPA-mediated current in presence of 100 μM S22286 were excluded
and the experiment stopped.
1
4
1
mmol) (290 mg, 65%); mp 145−147 °C. H NMR (DMSO-d , 500
MHz) δ 3.70 (2H, dt, N-CH CH F), 4.55 (2H, dt, N-CH CH F), 4.73
6
2
2
2
2
(
(
2H, s, 3-H ), 7.14 (1H, s, 5-H), 8.06 (1H, s, N-H). Anal.
2
C H ClFN O S ) C, H, N, S.
7
8
2
2 2
Effect on Excitatory Postsynaptic Response Evoked in CA1
6
-Chloro-4-(3-fluoropropyl)-4H-thieno[3,2-e]-1,2,4-thiadia-
Area on Rat Hippocampal Slices in Vitro. This assay was
zine 1,1-Dioxide (36h). To 6-chloro-4H-thieno[3,2-e]-1,2,4-thiadia-
zine 1,1-dioxide (36a) (450 mg, 2.0 mmol) in acetonitrile (20 mL)
were added potassium carbonate (900 mg) and 1-fluoro-3-
bromopropane (0.9 mL, 0.77 g, 5.5 mmol), and the mixture was
heated at 100 °C for 9 h. At the end of the reaction, the solvent was
removed under reduced pressure and the residue was taken up in
water. The insoluble material was collected by filtration, washed with
18
performed following our previously published procedure
Effect on Long-Term Potentiation (LTP) of the Postsynaptic
Response Evoked in the Dentate Gyrus on Anesthetized Rats.
Extracellular excitatory postsynaptic field potentials (EPSfP) were
recorded in the dentate gyrus using our previously published
18
procedure.
Effect on AMPA-Mediated Release of Noradrenaline on Rat
Hippocampal Slices. Male Wistar rat (200−300 g) was decapitated,
the brain was rapidly removed, and both hippocampi were carefully
isolated. Transversal hippocampal slices of 300 μm thickness were cut
water, and recrystallized in methanol (420 mg, 74%); mp 181−183 °C.
1
H NMR (DMSO-d , 500 MHz) δ 2.08 (2H, m, N−CH CH CH F,
6
2
2
2
4
.13 (2H, t, N-CH CH CH F), 4.55 (2H, dt, N-CH CH CH F), 7.64
2 2 2 2 2 2
3
(
1H, s, 5-H), 8.09 (1H, s, 3-H). Anal. (C H ClFN O S ) C, H, N, S.
8 8 2 2 2
with a tissue chopper. Hippocampal slices were incubated with [ H]-
3
6
-Chloro-4-(3-fluoropropyl)-3,4-dihydro-2H-thieno[3,2-e]-
noradrenaline ([ H]-NAD, 0.16 Ci/mL) for 30 min at 37 °C, rinsed,
1
,2,4-thiadiazine 1,1-Dioxide (37h). The title compound was
and placed in reaction chambers of a B18 Superfusion System
obtained as described for 24 starting from 6-chloro-4-(3-fluoropropyl)-
H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (36h) (400 mg, 1.4
(Brandel, USA). Slices were superfused with Krebs buffer, with (S)-
4
AMPA or with (S)-AMPA in the presence of compound. The
superfusate was collected in 5 min fractions. The quantity of
radioactivity was estimated for each fraction with a Beckman
scintillation counter and the fractional NAD release was normalized
as the percentage relative to the total radioactivity from all fractions
plus the tissue sample.
1
mmol) (320 mg, 79%); mp 125−126 °C. H NMR (DMSO-d , 500
MHz) δ 1.90 (2H, m, N-CH CH CH F), 3.44 (2H, t, N-
6
2
2
2
CH CH CH F), 4.51 (2H, dt, N-CH CH CH F), 4.68 (2H, d, 3-
2
2
2
2
2
2
H ), 7.09 (1H, s, 5-H), 7.98 (1H, t, N-H). Anal. (C H ClFN O S )
2
8
10
2
2 2
C, H, N, S.
5
-Chloro-3-(2,2-difluoroacetamido)thiophene-2-sulfona-
Effect on Object Recognition Test in Mice. The one-trial object
recognition paradigm measures a form of episodic memory in the
mouse and was achieved following our previously described
mide (38). To 3-amino-5-chlorothiophene-2-sulfonamide (35) (1.00
g, 4.7 mmol) dissolved in dioxane (30 mL) was added 2,2-
difluoroacetyl chloride (0.75 mL, 7.1 mmol). The flask was
hermetically closed immediately, and contents were vigorously stirred
at ambient temperature for 60 min. The solvents were then removed
under reduced pressure. The residue was taken up in water (15 mL).
The resulting solid material was collected by filtration and used in the
next step without further purification (1.04 g, 76%).
1
7,18
procedure.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Elemental analysis results for the new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
5
-Chloro-3-(2,2-difluoroethylamino)thiophene-2-sulfona-
mide (39). The title compound was obtained as described for 32a
7
848
dx.doi.org/10.1021/jm400676g | J. Med. Chem. 2013, 56, 7838−7850

Journal of Medicinal Chemistry
Article
(
14) Phillips, D.; Sonnenberg, J.; Arai, A. C.; Vaswani, R.; Krutzik, P.
AUTHOR INFORMATION
Corresponding Author
Phone: + 32 4 3664369. Fax: + 32 4 3664362. E-mail: Pierre.
Francotte@ulg.ac.be.
■
O.; Kleisli, T.; Kessler, M.; Granger, R.; Lynch, G.; Richard
Chamberlin, A. 5′-Alkyl-benzothiadiazides: a new subgroup of
AMPA receptor modulators with improved affinity. Bioorg. Med.
Chem. 2002, 10, 1229−1248.
*
Author Contributions
B.P. and P.d.T. equally supervised the work.
(15) Pirotte, B.; Podona, T.; Diouf, O.; de Tullio, P.; Lebrun, P.;
Dupont, L.; Somers, F.; Delarge, J.; Morain, P.; Lestage, P.; Lepagnol,
J.; Spedding, M. 4H-1,2,4-Pyridothiadiazine 1,1-dioxides and 2,3-
dihydro-4H-1,2, 4-pyridothiadiazine 1,1-dioxides chemically related to
diazoxide and cyclothiazide as powerful positive allosteric modulators
of (R/S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid
receptors: design, synthesis, pharmacology, and structure−activity
relationships. J. Med. Chem. 1998, 41, 2946−2959.
§
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
ACSF, artificial cerebrospinal fluid; ANOVA, analysis of
variance; AP/ML/DV, anterior-posterior/medial-lateral/dor-
sal-ventral; BDNF, brain-derived neurotrophic factor; CA1,
Cornu Ammonis 1; CD1, cluster of differentiation 1; EPSfP,
excitatory postsynaptic field potential; FlipR, fluorometric
imaging plate reader; MΩ, megaohm; mV, millivolt; ms,
millisecond; NAD, noradrenaline; nL, nanoliter
(16) Francotte, P.; Tullio, P.; Goffin, E.; Dintilhac, G.; Graindorge,
E.; Fraikin, P.; Lestage, P.; Danober, L.; Thomas, J. Y.; Caignard, D.
H.; Pirotte, B. Design, synthesis, and pharmacology of novel 7-
substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides as
positive allosteric modulators of AMPA receptors. J. Med. Chem.
2
(
007, 50, 3153−3157.
17) Francotte, P.; de Tullio, P.; Podona, T.; Diouf, O.; Fraikin, P.;
Lestage, P.; Danober, L.; Thomas, J. Y.; Caignard, D. H.; Pirotte, B.
Synthesis and pharmacological evaluation of a second generation of
pyridothiadiazine 1,1-dioxides acting as AMPA potentiators. Bioorg.
Med. Chem. 2008, 16, 9948−9956.
REFERENCES
■
(
1) Yamada, K. A.; Tang, C. M. Benzothiadiazides inhibit rapid
glutamate receptor desensitization and enhance glutamatergic synaptic
currents. J. Neurosci. 1993, 13, 3904−3915.
2) Yamada, K. A.; Rothman, S. M. Diazoxide blocks glutamate
desensitization and prolongs excitatory postsynaptic currents in rat
hippocampal neurons. J. Physiol. 1992, 458, 409−423.
3) Bertolino, M.; Baraldi, M.; Parenti, C.; Braghiroli, D.; DiBella, M.;
(18) 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.
(19) Mourlevat, S.; Galizzi, J. P.; Guigal-Stephan, N.; Courtade-
Gaiani, S.; Rolland-Valognes, G.; Rodriguez, M.; Barbet, F.; Bourrier,
C.; Catesson, S.; Chomel, A.; Danober, L.; Villain, N.; Caignard, D. H.;
Pirotte, B.; Lestage, P.; Lockhart, B. P. Molecular characterization of
the AMPA-receptor potentiator S70340 in rat primary cortical culture:
whole-genome expression profiling. Neurosci. Res. 2011, 70, 349−360.
(
(
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.
(
4) Bernard, K.; Danober, L.; Thomas, J. Y.; Lebrun, C.; Munoz, C.;
Cordi, A.; Desos, P.; Lestage, P.; Morain, P. DRUG FOCUS: S 18986:
a positive allosteric modulator of AMPA-type glutamate receptors
pharmacological profile of a novel cognitive enhancer. CNS Neurosci.
Ther. 2010, 16, e193−e212.
5) Staubli, U.; Rogers, G.; Lynch, G. Facilitation of glutamate
receptors enhances memory. Proc. Natl. Acad. Sci. U. S. A. 1994, 91,
77−781.
6) Black, M. D. Therapeutic potential of positive AMPA modulators
and their relationship to AMPA receptor subunits. A review of
preclinical data. Psychopharmacology (Berlin) 2005, 179, 154−163.
7) Francotte, P.; de Tullio, P.; Fraikin, P.; Counerotte, S.; Goffin, E.;
Pirotte, B. In search of novel AMPA potentiators. Recent Pat. CNS
Drug Discovery 2006, 1, 239−246.
8) Krintel, C.; Frydenvang, K.; Olsen, L.; Kristensen, M. T.; de
Barrios, O.; Naur, P.; Francotte, P.; Pirotte, B.; Gajhede, M.; Kastrup,
J. S. Thermodynamics and structural analysis of positive allosteric
modulation of the ionotropic glutamate receptor GluA2. Biochem. J.
012, 441, 173−178.
9) Goff, D. C.; Leahy, L.; Berman, I.; Posever, T.; Herz, L.; Leon, A.
C.; Johnson, S. A.; Lynch, G. A placebo-controlled pilot study of the
ampakine CX516 added to clozapine in schizophrenia. J. Clin.
Psychopharmacol. 2001, 21, 484−487.
10) O’Neill, M. J.; Witkin, J. M. AMPA receptor potentiators:
application for depression and Parkinson’s disease. Curr. Drug Targets
007, 8, 603−620.
11) Alt, A.; Nisenbaum, E. S.; Bleakman, D.; Witkin, J. M. A role for
AMPA receptors in mood disorders. Biochem. Pharmacol. 2006, 71,
273−1288.
12) Braghiroli, D.; Puia, G.; Cannazza, G.; Tait, A.; Parenti, C.; Losi,
G.; Baraldi, M. Synthesis of 3,4-dihydro-2H-1,2,4-benzo-thiadiazine
,1-dioxide derivatives as potential allosteric modulators of AMPA/
kainate receptors. J. Med. Chem. 2002, 45, 2355−2357.
13) Pirotte, B.; Francotte, P.; Goffin, E.; Fraikin, P.; Danober, L.;
(
20) Lima, L. M.; Barreiro, E. J. Bioisosterism: a useful strategy for
molecular modification and drug design. Curr. Med. Chem. 2005, 12,
3−49.
21) Nielsen, F. E.; Ebdrup, S.; Jensen, A. F.; Ynddal, L.;
(
2
(
7
(
Bodvarsdottir, T. B.; Stidsen, C.; Worsaae, A.; Boonen, H. C.;
Arkhammar, P. O.; Fremming, T.; Wahl, P.; Korno, H. T.; Hansen, J.
B. New 3-alkylamino-4H-thieno-1,2,4-thiadiazine 1,1-dioxide deriva-
tives activate ATP-sensitive potassium channels of pancreatic beta
cells. J. Med. Chem. 2006, 49, 4127−4139.
(
(
22) Topliss, J. G. Method of treating hypertension with thieno-
1,2,4]thiadiazines. U.S. Patent US3733409 A, May 15, 1973.
23) Arranz, E.; Diaz, J. A.; Vega, S.; Campos-Toimil, M.; Orallo, F.;
(
[
(
Cardelus, I.; Llenas, J.; Fernandez, A. G. Synthesis and pharmaco-
logical evaluation of 2, 3-dihydro-3-oxo-4H-thieno[3,4-e][1,2,4]-
thiadiazine 1,1-dioxides as voltage-dependent calcium channel block-
ers. Eur. J. Med. Chem. 2000, 35, 751−759.
2
(
(24) Nielsen, F. E.; Bodvarsdottir, T. B.; Worsaae, A.; MacKay, P.;
Stidsen, C. E.; Boonen, H. C.; Pridal, L.; Arkhammar, P. O.; Wahl, P.;
Ynddal, L.; Junager, F.; Dragsted, N.; Tagmose, T. M.; Mogensen, J.
P.; Koch, A.; Treppendahl, S. P.; Hansen, J. B. 6-Chloro-3-alkylamino-
(
4
H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide derivatives potently and
2
(
selectively activate ATP sensitive potassium channels of pancreatic
beta-cells. J. Med. Chem. 2002, 45, 4171−4187.
(25) Qi, J.; Wang, Y.; Jiang, M.; Warren, P.; Chen, G. Cyclothiazide
1
(
induces robust epileptiform activity in rat hippocampal neurons both
in vitro and in vivo. J. Physiol. 2006, 571, 605−618.
1
(26) Arai, A. C.; Kessler, M. Pharmacology of ampakine modulators:
from AMPA receptors to synapses and behavior. Curr. Drug Targets
2007, 8, 583−602.
(
Lesur, B.; Botez, I.; Caignard, D. H.; Lestage, P.; de Tullio, P. Ring-
fused thiadiazines as core structures for the development of potent
AMPA receptor potentiators. Curr. Med. Chem. 2010, 17, 3575−3582.
(27) Ohta, K.; Araki, N.; Shibata, M.; Komatsumoto, S.; Shimazu, K.;
Fukuuchi, Y. Presynaptic ionotropic glutamate receptors modulate in
vivo release and metabolism of striatal dopamine, noradrenaline, and
7
849
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Journal of Medicinal Chemistry
Article
5
-hydroxytryptamine: involvement of both NMDA and AMPA/
kainate subtypes. Neurosci. Res. 1994, 21, 83−89.
28) Cowen, M. S.; Beart, P. M. Cyclothiazide and AMPA receptor
desensitization: analyses from studies of AMPA-induced release of
3H]-noradrenaline from hippocampal slices. Br. J. Pharmacol. 1998,
23, 473−480.
29) Lockhart, B.; Iop, F.; Closier, M.; Lestage, P. (S)-2,3-Dihydro-
3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide: (S18986-1), a
positive modulator of AMPA receptors enhances (S)-AMPA-mediated
3H]noradrenaline release from rat hippocampal and frontal cortex
slices. Eur. J. Pharmacol. 2000, 401, 145−53.
30) Ennaceur, A.; Delacour, J. A new one-trial test for neuro-
(
[
1
(
[
[
(
biological studies of memory in rats. 1: Behavioral data. Behav. Brain
Res. 1988, 31, 47−59.
(
31) Cymerman-Craig, J.; Willis, D.; Rubbo, S. D.; Edgar, J. Mode of
action of isonicotinic hydrazide. Nature 1955, 176, 34−35.
7
850
dx.doi.org/10.1021/jm400676g | J. Med. Chem. 2013, 56, 7838−7850