Synthesis and pharmacological evaluation of a second generation of pyridothiadiazine 1,1-dioxides acting as AMPA potentiators

Article abstract of DOI:10.1016/j.bmc.2008.10.036

Taking into account structure-activity relationships obtained with our previous series, new diversely substituted 1,2,4-pyridothiadiazine 1,1-dioxides were designed to obtain novel AMPA potentiators. The aim of this work was focused on the improvement of lipophilicity, which is well known as a critical parameter to obtain in vivo active central nervous system agents. For this purpose, two positions on the pyridine ring were privileged to insert selected groups. Among the synthesized compounds emerged 7-chloro-4-ethyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide (12d), which was evaluated in two memory tests in Wistar rats and showed cognition enhancing effects after intraperitoneal injection at doses as low as 0.3 mg/kg.

Full text of DOI:10.1016/j.bmc.2008.10.036

Bioorganic & Medicinal Chemistry 16 (2008) 9948–9956
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological evaluation of a second generation
of pyridothiadiazine 1,1-dioxides acting as AMPA potentiators
Pierre Francotte a, , , Pascal de Tullio a, , Tchao Podona a, Ousmane Diouf a, Pierre Fraikin a, Pierre Lestage b,
*
Laurence Danober b, Jean-Yves Thomas b, Daniel-Henri Caignard b, Bernard Pirottea
a Drug Research Center, Laboratory of Medicinal Chemistry, University of Liège, Av. de l’Hôpital, 1, B36, 4000 Liège, Belgium
b Institut de Recherches Servier, 125, Chemin de Ronde, F-78290 Croissy-sur-Seine, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 May 2008
Revised 6 October 2008
Accepted 12 October 2008
Available online 17 October 2008
Taking into account structure-activity relationships obtained with our previous series, new diversely
substituted 1,2,4-pyridothiadiazine 1,1-dioxides were designed to obtain novel AMPA potentiators. The
aim of this work was focused on the improvement of lipophilicity, which is well known as a critical
parameter to obtain in vivo active central nervous system agents. For this purpose, two positions on
the pyridine ring were privileged to insert selected groups. Among the synthesized compounds emerged
7-chloro-4-ethyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide (12d), which was evalu-
ated in two memory tests in Wistar rats and showed cognition enhancing effects after intraperitoneal
injection at doses as low as 0.3 mg/kg.
Keywords:
AMPA potentiator
Allosteric modulation
Cognitive enhancer
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
the preparation of three series of pyridinic compounds [‘5-aza’ (7),
‘7-aza’ (8) and ‘8-aza’ (9) compounds] from which emerged the
Glutamate is the main excitatory neurotransmitter in the cen-
tral nervous system for which have been identified metabotropic
and ionotropic receptors. Among the latter and besides the KA (kai-
in vivo active 10 (S22286)10 (Fig. 1).
Knowing how lipophilicity is a critical parameter in the design of
drugs acting in the CNS, the present work was focused on the design,
the preparation and the pharmacological evaluation of ‘second gen-
eration’ pyridothiadiazine dioxides. Firstly it was envisaged to en-
hance the lipophilic character of the most potent series (‘8-aza’),
where a methyl group was introduced at the 6-position (preparation
of 6-methyl-3,4-dihydro-2H-pyrido[3,2-e]-[1,2,4]-thiadiazine 1,1-
dioxides 11, Fig. 2). Moreover, taking advantage from our last phar-
macomodulation in the benzenic series9, the 7-position of ‘5-aza’
compounds (12, Fig. 2) was selected to introduce various groups,
attempting to enhance the activity. In this context, and since the
most potent compound in the benzenic series was obtained after
introduction of a fluorine atom at the 7-position, it was tempting
to first synthesize 7-fluoro-substituted compounds belonging to
the ‘5-aza’ series. Two halogen atoms, chlorine and bromine, were
chosen to compare their impact on the activity with the benzenic
series. Finally the insertion of a methyl group was also achieved.
Substituents at the 2- and 4-positions of the heterocycle were
chosen based on previous published structure-activity relationships
(SARs): the best AMPA modulators were obtained with a hydrogen
atom (or a very short alkyl chain such as a methyl group) at the 2-
and 3-positions and a short alkyl chain (i.e., ethyl) at the 4-
position.9,10
nate) subtype, the NMDA (N-methyl-D-aspartate) and the AMPA
(a-amino-3-hydroxy-5-methylisoxazole-4-propionate) subtypes
have been shown to be involved in long term potentiation1, a phe-
nomenon believed to underlie learning and memory processes.
Many studies support the potentiation of AMPA receptors as an
innovative therapeutic strategy in the treatment of cognitive disor-
,
ders2,3 schizophrenia4, depression and Parkinson’s disease.5
Whereas AMPA agonists act by direct stimulation and may induce
severe adverse effects such as neurotoxicity, allosteric positive mod-
ulators onlypotentiatetheeffects of theendogenous neurotransmit-
ter present in the synapses; this mechanism affords in a fine tuning
of the glutamate signals and is awaited to exert less undesired ef-
fects. Several structural families are being studied for their potential
as candidate drugs in a series of central nervous system (CNS) disor-
ders.6 Pharmacomodulation studies around 1 (diazoxide), 2 (cyclo-
thiazide) and 3 (IDRA 21) led to the identification of potent
positive allosteric modulators such as the pyrrolobenzothiadiazine
4 (S18986)7 and the benzothiadiazines 58 or 6 (S29216)9 (Fig. 1).
Applying the bioisosteric concept, the switch between benzothiadi-
azine dioxide and pyridothiadiazine dioxide was tempted, and led to
The pharmacological in vitro evaluation was performed on a
model of AMPA receptors expressed in Xenopus oocytes (evaluation
of AMPA-induced current on oocytes injected with rat cortex
* Corresponding author. Tel.: +32 4 3664369; fax: +32 4 3664362.
E-mail address: Pierre.Francotte@ulg.ac.be (P. Francotte).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.036

P. Francotte et al. / Bioorg. Med. Chem. 16 (2008) 9948–9956
9949
Figure 1. AMPA potentiators.
Figure 2. Design of ‘2nd generation’ pyridinic compounds.
mRNA). Two additional memory tests were performed with the
most interesting compound to assess its potential interest as a cog-
nitive enhancer.
48 hours to complete the aromatic substitution, affording 25a in
poor yield after treatment with ammonia, and this, probably due
to the presence of the fluorine atom which exerts a deactivation
of the aromatic ring. In the four series, the ring closure using tri-
ethyl orthoformate afforded the corresponding 4H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxides (26). Then, alkylation at the 4-posi-
tion was obtained with alkyl halide in presence of potassium car-
bonate in acetonitrile giving compounds (27); the latter were
converted into their saturated counterparts (12) by reducing them
with sodium borohydride in 2-propanol.
2. Chemistry
The synthetic pathways used to prepare compounds in the 6-
methyl-3,4-dihydro-2H-pyrido[3,2-e]-[1,2,4]-thiadiazine 1,1-diox-
ide series is illustrated in scheme 1: direct nitration on 2-amino-
5-methylpyridine (13) gave intermediate 14. The resulting phenol
was then converted into the chloro-substituted derivative 15 by
action of SOCl2. Since the thiol (17) might not be obtained in one
step from 15 in an acceptable yield, this compound was prepared
in two steps, through a substitution of the chlorine atom with thio-
urea, affording 16, and a subsequent alkaline hydrolysis. Then, the
sulfenamide (18) was generated using hydroxylamine-O-sulfonic
acid, in a biphasic medium. Oxidation of the sulfenamide function
gave access to the corresponding pyridine-2-sulfonamide (19).
Reduction of the nitro group was effected routinely with iron pow-
der in presence of ammonium chloride, and the product (20) was
used in a ring closure reaction using triethyl orthoformate to ob-
tain 6-methyl-4H-pyrido[3,2-e]-[1,2,4]-thiadiazine 1,1-dioxide
(21). Alkylation of the latter was observed at the 4-position after
heating them with the appropriate alkyl halide and potassium car-
bonate in acetonitrile, resulting in (22). A subsequent reduction
with sodium borohydride in 2-propanol/chloroform gave final
compounds of interest (11) (Scheme 2).
Concerning the 3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadi-
azine 1,1-dioxides substituted at the 7-position by a fluorine atom,
it was necessary to start from 2-amino-5-nitropyridine (23), which
in four steps, was converted into 2-amino-5-fluoropyridine (24), as
described in the literature.11 Then, chlorosulfonation permitted to
access to the corresponding pyridine-3-sulfonamide (25a), as it
was realized for the 5-chloro-, 5-bromo- and 5-methyl-substituted
compounds. Concerning the fluoro-substituted compounds, it is
worth mentioning here that the reflux had to be maintained during
Alkylation at the 2-position was achieved on the final com-
pounds 11b and 12d with methyl iodide and potassium carbonate
in acetonitrile, giving 11c and 12g, respectively.
3. Results and discussion
The pyridothiadiazines from the two families of compounds [‘5-
aza’, pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxides (11); and ‘8-
aza’, pyrido[3,2-e]-[1,2,4]-thiadiazine 1,1-dioxides (12)] were
tested as AMPA potentiators using voltage-clamp recordings of
AMPA-induced current on Xenopus oocytes injected with rat cortex
poly(A+) mRNA as previously described.10 Tested compounds alone
had no effect on the holding current of Xenopus oocytes injected
with rat cortex mRNA. However, when applied in the presence of
10 lM (S)-AMPA, they increased the inward evoked current in a
concentration-dependent manner, confirming their profile as posi-
tive modulators of (S)-AMPA receptors. The activity of our com-
pounds was expressed as the EC2X and EC5X values, which are the
concentration of drug giving, respectively, a twofold and a fivefold
increase of the magnitude of the current induced by 10
AMPA. Results are reported in Tables 1 and 2.
lM (S)-
At the first glance, none of the new synthesized compounds ap-
peared to be as potent as the references. In the ‘8-aza’ series, the
introduction of a methyl group at the 6-position was clearly unfa-
vorable, as can be seen in Table 1 (compare 11a, 11b, and 11c with
9a, 10, and 9b).

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P. Francotte et al. / Bioorg. Med. Chem. 16 (2008) 9948–9956
NO2
OH
NO2
Cl
NO2
S
i
ii
iii
N
NH2
N
N
N
H2N
NH2
Cl
13
14
15
16
iv
NH2
NH2
NO2
NO2
NO2
SH
vii
vi
v
NH2
NH2
N
S
N
S
N
S
N
O
O
O
O
20
19
18
17
viii
R4
N
R4
N
H
N
xi
x
N
ix
N
N
NH
N
N
S
N
S
N
S
N
S
O
O
O
O
O
O
O
O
21
22
11a,b
11c
22a : R4=CH3
22b : R4=CH2CH3
11a : R4=CH3
11b : R4=CH2CH3
Scheme 1. Reagents: (i) HNO3, H2SO4; (ii) SOCl2; (iii) (NH2)2C@S, EtOH,
D; (iv) NaOH, H2O; (v) NH2OSO3H, Et3N, H2O/CH2Cl2; (vi) KMnO4, CH3CN, H2O; (vii) Fe, NH4Cl; (viii)
HC(OEt)3,
D
; (ix) R4-X, K2CO3, CH3CN; (x) NaBH4, 2-propanol; (xi) CH3I, K2CO3, CH3CN.
H
N
N
NH2
N
NH2
NH2
N
N
NH2
i, ii, iii, iv
v
vi
N
R7
S
R7
S
O2N
F
O
O
O
O
25a, 26a : R7=F
23
24
25
26
25b, 26b : R7=Cl
25c, 26c : R7=Br
25d, 26d : R7=CH3
vii
R4
N
R4
N
viii
N
N
NH
N
12a : R7=F, R4=CH3
R7
S
R7
S
12b : R7=F, R4=CH2CH3
12c : R7=Cl, R4=CH3
O
O
O
O
12a-f
27
12d : R7=Cl, R4=CH2CH3
12e : R7=Br, R4=CH2CH3
12f : R7=CH3, R4=CH2CH3
vii
27a : R7=F, R4=CH3
27b : R7=F, R4=CH2CH3
27c : R7=Cl, R4=CH3
27d : R7=Cl, R4=CH2CH3
27e : R7=Br, R4=CH2CH3
27f : R7=CH3, R4=CH2CH3
N
N
S
N
Cl
O
O
12g
Scheme 2. Reagents: (i) Ac2O,
CH3CN; (viii) NaBH4, 2-propanol.
D; (ii) H2, Pd/C; (iii) isoamyl nitrite, HBF4; (iv) 1—D, xylene; 2—NaOH; (v) 1—ClSO3H, SOCl2; 2—NH4OH 10%; (vi) HC(OEt)3, D
; (vii) R4-X, K2CO3,
Since the goal of the first pharmacomodulation in this series
was also the enhancement of its lipophilicity, it seemed interesting
to evaluate the influence on this parameter after the introduction
of the methyl group. The log P value of 11b was measured using
the shake flask method, and was determined at +0.41. Not surpris-
ingly, the lipophilicity increase is thus modest, since the log P value
for 10 was estimated at +0.12 (increase of 0.3 U).10
Considering Table 2, it is clear that the best substitution at the
7-position was the chlorine atom; amongst the compounds syn-
thesized in this series, 12d appeared as the most active. Since the
most potent compound in the benzenic series was obtained after
the introduction of a fluorine atom at the 7-position (6, Table 2),
it was very motivating to synthesize 7-fluoro-substituted ‘5-aza’
compounds. We have to note that the same pharmacomodulation

P. Francotte et al. / Bioorg. Med. Chem. 16 (2008) 9948–9956
9951
M) in
Table 1
Effects of 3,4-dihydro-2H-pyrido[3,2-e]-[1,2,4]-thiadiazine 1,1-dioxides substituted or not at the 6-position on the magnitude of the current induced by (S)-AMPA (10
Xenopus oocytes injected with rat cortex mRNA
l
.
Compound
R6
R2
R4
EC2X
146 20
116
86 13
1.6 0.3
(
l
M)a
EC5X
(
l
M)b
Max. increasec (%)
11a
11b
11c
2
3
4
9a
10
9b
CH3
CH3
CH3
—
—
—
H
H
H
H
H
CH3
—
—
—
H
H
CH3
CH3
> 300
> 300
> 300
9.8 1.9
509 64
78.2 8.9
nd.
nd.
nd.
CH2CH3
CH2CH3
—
—
—
CH3
CH2CH3
CH2CH3
9
844
>700
1263
nd.
134
7
24.6 2.9
21 4d
51 16
d
8.8 1.3d
38 4d
19 3d
96 8d
1328d
ndd
a,b
Concentration of drug giving a twofold and a fivefold increase of the magnitude of the current induced by (S)-AMPA (10 lM), respectively (mean SEM) (n P 3).
c
Maximum effect of the drug on the AMPA-evoked current (expressed in% of the current evoked by AMPA, taken as 100%).
d
Published results.10 nd, not determined.
Table 2
Effects of diversely 7-substituted 3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxides on the magnitude of the current induced by (S)-AMPA (10
l
M) in Xenopus oocytes
injected with rat cortex mRNA
R4
N
R4
N
N
NH
NH
R7
S
R7
S
O
O
O
O
.
12
28
Compound
R7
R2
R4
EC2X
(l
M)a
EC5X
(l
M)b
Max. increasec (%)
12a
12b
12c
12d
12e
12f
12g
7a
F
F
H
H
H
H
H
H
CH3
H
CH3
CH2CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
> 100
123
nd.
300
81 14
44
> 300
> 1000
335 35
500
nd.
> 422
nd.
1092
nd.
nd.
Cl
Cl
Br
CH3
Cl
H
21
20
4
3
4
160 29
> 1000
110 10
200
nd.
nd.
2
3
4
6
28a
28b
28c
—
—
—
F
Cl
Br
CH3
—
—
—
H
H
H
—
—
—
1.6 0.3
9.8 1.9
509 64
78.2 8.9
7.3 0.5d
14 3d
844
134
7
>700
1263
4066d
3682d
>1100d
>1300d
24.6 2.9
3.2 0.1d
5.6 0.9d
29 6d
CH2CH3
CH2CH3
CH2CH3
CH2CH3
78 16d
95 5d
H
25 8d
a,b
Concentration of drug giving a twofold and a fivefold increase of the magnitude of the current induced by (S)-AMPA (10 lM), respectively (mean SEM) (n P 3).
c
Maximum effect of the drug on the AMPA-evoked current (expressed in % of the current evoked by AMPA, taken as 100%).
d
Published results.9,10 nd, not determined.
in the ‘5-aza’ series has not the same effect on activity. The in vitro
activity observed with 12a and 12b was clearly disappointing,
regarding the difficulties encountered during their preparation.
In a general manner, we may observe here that the switch be-
tween 7-substituted benzothiadiazines to their corresponding pyr-
ido-[2,3-e]-thiadiazine analogues (‘5-aza’ compounds) negatively
influenced the in vitro activity (compare 12b, 12d, 12e, and 12f
vs 6, 28a, 28b, and 28c). This loss of activity may be explained in
part by the particular electronic distribution encountered in the
pyridinic analogues. The fact that we did not find here the same
rank order of potency than that previously observed in the benze-
nic series (fluorine > chlorine > bromine ꢀ methyl group, see Table
2) clearly shows that pyridinic derivatives have to considered as
independent series from the benzothiadiazine 1,1-dioxides. More-
over, this suggests that SARs focused on the benzene substitution
should not be taken in consideration in the future design of novel
pyridinic analogues.
Methylation of the nitrogen atom at the 2-position of com-
pound 12d resulted in a marked reduction of potency (see Table
2: 12d vs 12g) but not in the case of 11b (see Table 1: 11b vs
11c). So SARs concerning the substitution in this position remain
difficult to establish.
Based on its interesting activity observed during the screening
test and its favorable lipophilicity that should ensure distribution
to the CNS, 12d (S23310) was selected to be further evaluated
in vivo in two memory tests in Wistar rats. The first used paradigm
was the social recognition test, which was initially described by
Thor and Hollaway.12 Animals were administrated 12d at the dose

9952
P. Francotte et al. / Bioorg. Med. Chem. 16 (2008) 9948–9956
of 0.3, 1 or 3 mg/kg i.p. Significant (P < 0.01) activity was already
detected at the lowest dose of 0.3 mg/kg (see Fig. 3). The cogni-
tive-enhancing effect of 12d was also investigated in the one-trial
object recognition paradigm, which was developed by Ennaceur
and Delacour13 as a model of episodic memory. Animals received
12d at four doses: 0.3, 1, 3, 10 mg/kg i.p. The drug was found to
be active in vivo and showed dose-dependent activity after intra-
peritoneal injection (significantly different from control at 3 and
10 mg/kg, see Fig. 4), suggesting that 12d possesses the suitable
characteristics (i.e., lipophilicity) to reach the central nervous sys-
tem after i.p. administration. However, no marked effect was ob-
served after per os administration.
likely reason of 12d’s inactivity, this compound was orally admin-
istered at high doses (100 mg/kg); no effect was observed in the
treated animals. The lack of activity after such oral acute adminis-
tration combined with the absence of toxicity at this high dose sug-
gests that 12d is not orally absorbed. Taking into account this data,
it appears that the pharmacokinetic profile of 12d should be im-
proved, e.g., with the design of prodrugs. Another potential ap-
proach could rest on an in vitro metabolism study in order to
define the structural moieties sensitive to metabolic biotransfor-
mation which could be responsible for the pharmacokinetic
weakness.
Two hypotheses may explain 12d’s inactivity after oral admin-
istration: either the drug is not absorbed when administered per
os, or the compound is markedly transformed into an inactive form
by a hepatic first-pass effect. In the attempt to identify the most
4. Conclusions
A series of novel dihydro-2H-pyrido-[1,2,4]-thiadiazine 1,1-
dioxides were synthesized and evaluated as positive allosteric
Figure 3. Effect of treatment with 12d on the social recognition test in Wistar rat after intraperitoneal injection. The discrimination index (delta T2–T1) was the difference
between the investigation times during the first and the second session (with inter-sessions interval of 2 h). Under such conditions, control rats did no longer recognize the
familiar congener and spent similar times in recognizing the juvenile rat. Compound 12d significantly improved at 0.3, 1 and 3 mg/kg i.p. the recognition of the juvenile rat
(n = 8). **p = 0.01 vs control, one way ANOVA.
Figure 4. Effect of treatment with 12d on the object recognition test in Wistar rat after intraperitoneal injection. The discrimination index (delta new-fam) was the difference
between the exploration times of the new and familiar objects on the last session with inter-sessions 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 12d significantly improved at 3 and 10 mg/kg i.p. the recognition of
the familiar object as evidenced by increased exploration toward the new object (n = 12). **p = 0.01, ***p = 0.001 vs control, one way ANOVA.

P. Francotte et al. / Bioorg. Med. Chem. 16 (2008) 9948–9956
9953
modulators of the AMPA receptors, in order to improve lipophilic
parameters of previous pyridinic series, with the aim at obtaining
in vivo active compounds.
the residue was suspended in water (25 mL). The resulting insolu-
ble material, consisting of the sulfenamide 18, was collected by fil-
tration, washed with water, dried prior to its dissolution in
acetonitrile (150 mL); this solution was added to KMnO4 (2.5 g)
previously dissolved in a minimum of water. When the reaction
was complete, the mixture was filtered. The filtrate was reduced
to a small volume and adjusted to pH 3. Crystallization of the title
compound occurred at 4 °C: Crystals of 19 were collected by filtra-
tion, washed with water and dried (3.5 g, 48%): mp 160–162 °C. 1H
NMR (DMSO-d6, 500 MHz) d 2.47 (s, 3H, CH3), 7.91 (s, 2H, SO2NH2),
8.34 (s, 1H, 4-H), 8.79 (s, 1H, 6-H). Anal. (C6H7N3O4S) C, H, N, S.
In this context, the synthesis of pyrido-[2,3-e]-[1,2,4]-thiadia-
zines (‘5-aza’ compounds) substituted at the 7-position by a fluo-
rine atom was awaited to give novel potent AMPA potentiators,
as it was described with benzothiadiazines. Unfortunately, despite
all the chemical efforts invested, the resulting compounds did not
afford the expected results. Obviously, it seems that the previously
deduced SARs with benzothiadiazine 1,1-dioxides may not be fully
applied in the pyridinic series.
Amongst the newly described compounds, 12d appeared to
possess the most interesting in vitro activity with the appropriate
lipophilicity to reach the CNS. An interesting feature of this com-
pound is that it was shown to be active after i.p. administration
at doses as low as 3 mg/kg in the one-trial object recognition test,
a paradigm known as a model of episodic memory. Unfortunately
its in vivo effectiveness has not been confirmed after oral adminis-
tration, suggesting inadequate pharmacokinetic properties. This
inconvenient could be solved by the design of prodrugs character-
ized by a suitable pharmacokinetic pattern or by a novel pharma-
comodulation around the thiadiazine ring.
5.1.4. 3-Amino-5-methyl-2-pyridinesulfonamide (20)
To a solution of 19 (2.17 g, 10 mmol) in a water/ethanol mixture
(1:1, 60 mL) were added iron powder (3.46 g) and NH4Cl (0.53 g).
The mixture was then heated to reflux during 20 min. After the
hot mixture was filtered, the filtrate was reduced to a small vol-
ume. Crystallization of the title compound occurred at 4 °C: crys-
tals of 20 were collected by filtration, washed with water and
dried (1.8 g, 95%): mp 192–193 °C. 1H NMR (DMSO-d6, 500 MHz)
d 2.21 (s, 3H, CH3), 5.92 (s, 2H, NH2), 7.01 (s, 1H, 4-H), 7.28 (s,
2H, SO2NH2), 7.67 (s, 1H, 6-H). Anal. (C6H9N3O2S) C, H, N, S.
5.1.5. 6-Methyl-4H-pyrido[3,2-e]-[1,2,4]-thiadiazine 1,1-dioxide
(21)
5. Experimental
5.1. Chemistry
3-Amino-5-methyl-2- pyridinesulfonamide 20 (1.3 g, 7.0 mmol)
was added to triethyl orthoformate (13 mL). The mixture was
heated to reflux during 2 h. After cooling to room temperature,
the title compound was collected by filtration, washed with diethyl
ether and dried (1.3 g, 95%): mp 322–324 °C. 1H NMR (DMSO-d6,
500 MHz) d 2.41 (s, 3H, CH3), 7.53 (s, 1H, 5-H), 7.95 (s, 1H, 3-H),
8.49 (s, 1H, 7-H), 12.23 (s, 1H, NH). Anal. (C7H7N3O2S) C, H, N, S.
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 1H
NMR spectra were taken on a Bruker Advance 500 (500 MHz)
instrument in DMSO-d6 with TMS as an internal standard; chemi-
cal shifts are reported in d values (ppm) relative to internal TMS.
The abbreviation s = singlet, d = doublet, t = triplet, q = quadruplet,
quint = quintuplet, m = multiplet, and b = broad are used through-
out. Elemental analyses (C, H, N, S) were realized on a Carlo-Erba
EA 1108-elemental analyzer and were within 0.4% of the theoret-
ical values. All reactions were routinely checked by TLC on silica gel
Merck 60F254.
5.1.6. 4,6-Dimethyl-4H-pyrido[3,2-e]-[1,2,4]-thiadiazine 1,1-
dioxide (22a)
The mixture of 6-methyl-4H-pyrido[3,2-e]-[1,2,4]-thiadiazine
1,1-dioxide 21 (0.6 g, 3.0 mmol), K2CO3 (2.5 g) and methyl p-tolu-
enesulfonate (0.9 g) in acetonitrile (15 mL) was heated at reflux
for 16 h. The solvent was removed under reduced pressure and
the residue was suspended in water (15 mL). The resulting insolu-
ble material was collected by filtration, washed with water and
dried (0.51 g, 80%): mp 247–249 °C. 1H NMR (DMSO-d6,
500 MHz) d 2.47 (s, 3H, 6-CH3), 3.59 (s, 3H, NCH3), 7.83 (s, 1H, 5-
H), 8.03 (s, 1H, 3-H), 8.54 (s, 1H, 7-H). Anal. (C8H9N3O2S) C, H, N, S.
5.1.1. S-(5-Methyl-3-nitropyridin-2-yl)isothiouronium chloride
(16)
To a solution of 2-chloro-5-methyl-3-nitropyridine (15)14 (23 g,
0.13 mol) in ethanol (130 mL) was added thiourea (11.14 g). After
2 h heating at reflux, petroleum ether 40–60 (20 mL) was added
to the mixture. The resulting precipitate was collected by filtration,
washed with petroleum ether and dried (24.9 g, 75%): mp 205–
206 °C. 1H NMR (DMSO-d6, 500 MHz) d 2.45 (s, 3H, CH3), 8.61 (s,
1H, 4-H), 8.79 (s, 1H, 6-H), 9.76 (s, 2H, NH2), 9.89 (s, 2H, NH2). Anal.
(C7H9ClN4O2S) C, H, N, S.
5.1.7. 4-Ethyl-6-methyl-4H-pyrido[3,2-e]-[1,2,4]-thiadiazine
1,1-dioxide (22b)
The mixture of 6-methyl-4H-pyrido[3,2-e]-[1,2,4]-thiadiazine
1,1-dioxide 21 (1.2 g, 5.7 mmol), K2CO3 (2.5 g) and CH3CH2Br
(2 g) in acetonitrile (15 mL) was heated at reflux for 16 h. The sol-
vent was removed under reduced pressure and the residue was
suspended in water (15 mL). The resulting insoluble material was
collected by filtration, washed with water and dried (0.83 g,
65%): mp 185–187 °C. 1H NMR (DMSO-d6, 500 MHz) d 1.33 (t,
3H, NCH2CH3), 2.47 (s, 3H, 6-CH3), 4.20 (q, 2H, NCH2CH3), 7.92 (s,
1H, 5-H), 8.09 (s, 1H, 3-H), 8.53 (s, 1H, 7-H). Anal. (C9H11N3O2S)
C, H, N, S.
5.1.2. 5-Methyl-3-nitro-2-pyridinethiol (17)
To an aqueous solution (110 mL) of 16 (19 g, 76 mmol) was
added sodium carbonate (10.05 g). The mixture was supplemented
with NaOH (7.59 g) previously dissolved in water (10 mL). After
30 min, the mixture was adjusted to pH 1 by means of HCl 1 N.
The resulting insoluble material was collected by filtration, washed
with water and dried (10.4 g, 80%): mp 199–200 °C (lit. ꢀ200 °C15).
5.1.8. 4,6-Dimethyl-3,4-dihydro-2H-pyrido[3,2-e]-[1,2,4]-
thiadiazine 1,1-dioxide (11a)
5.1.3. 5-Methyl-3-nitro-2-pyridinesulfonamide (19)
The solution of 4,6-dimethyl-4H-pyrido[3,2-e]-[1,2,4]-thiadi-
azine 1,1-dioxide (22a) (0.50 g, 2.4 mmol) in 2-propanol-CHCl3
1:1 (25 mL) was supplemented under stirring with NaBH4 (0.4 g).
After 30 min stirring at room temperature, 5 drops of acetic acid
were added. The solvent was removed by distillation under re-
duced pressure and the residue was suspended in water (10 mL).
A solution of 17 (12 g, 71 mmol) in dichloromethane (180 mL)
with triethylamine (28.6 g) was added dropwise to hydroxyl-
amine-O-sulfonic acid (9.0 g, 80 mmol) previously dissolved in a
minimum of water. After the completion of the reaction, the sol-
vents were removed by distillation under reduced pressure and

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P. Francotte et al. / Bioorg. Med. Chem. 16 (2008) 9948–9956
The alkaline suspension was adjusted to pH 7 with 0.1 N HCl. The
resulting insoluble material was collected by filtration, washed
with water and dried (0.40 g, 80%): mp 220–221 °C. 1H NMR
(DMSO-d6, 500 MHz) d 2.30 (s, 3H, 6-CH3), 2.92 (s, 3H, N-CH3),
4.62 (s, 2H, 3-CH2), 7.17 (s, 1H, 5-H), 7.80 (s, 1H, NH), 8.13 (s, 1H,
7-H). Anal. (C8H11N3O2S) C, H, N, S.
d 8.09 (s, 1H, 3-H), 8.61 (s, 1H, 6-H), 8.79 (s, 1H, 8-H), 13.04 (s,
1H, NH). Anal. (C6H4ClN3O2S) C, H, N, S.
5.1.14. 7-Bromo-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (26c)
The title compound was obtained as described for 21 starting
from 2-amino-5-bromo-3-pyridinesulfonamide (25c)17 (3.0 g,
12 mmol) (2.7 g, 85%): mp >300 °C. 1H NMR (DMSO-d6, 500 MHz)
d 8.09 (s 1H, 3-H), 8.66 (s, 1H, 6-H), 8.85 (s, 1H, 8-H), 13.00 (s,
1H, NH). Anal. (C6H4BrN3O2S) C, H, N, S.
5.1.9. 4-Ethyl-6-methyl-3,4-dihydro-2H-pyrido[3,2-e]-[1,2,4]-
thiadiazine 1,1-dioxide (11b)
The title compound was obtained as described for 11a starting
from 4-ethyl-6-methyl-4H-pyrido[3,2-e]-[1,2,4]-thiadiazine 1,1-
dioxide (22b) (1.2 g, 5.3 mmol) (0.79 g, 65%): mp 146–148 °C. 1H
NMR (DMSO-d6, 500 MHz) d 1.08 (t, 3H, NCH2CH3), 2.29 (s, 3H,
6-CH3), 3.42 (q, 2H, NCH2CH3), 4.64 (s, 2H, 3-CH2), 7.21 (s, 1H, 5-
H) ; 7.77 (s, 1H, NH), 8.03 (s, 1H, 7-H). Anal. (C9H13N3O2S) C, H,
N, S.
5.1.15. 7-Methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (26d)
The title compound was obtained as described for 21 starting
from 2-amino-5-methyl-3-pyridinesulfonamide (25d)18 (3.0 g,
16 mmol) (2.6 g, 82%): mp >300 °C (lit. >300 °C dec.19).
5.1.10. 2,6-Dimethyl-4-ethyl-3,4-dihydro-2H-pyrido[3,2-e]-
[1,2,4]-thiadiazine 1,1-dioxide (11c)
5.1.16. 7-Fluoro-4-methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide (27a)
The solution of 4-ethyl-6-methyl-3,4-dihydro-2H-pyrido[3,2-
e]-[1,2,4]-thiadiazine 1,1-dioxide (11b) (0.2 g, 0.94 mmol) in aceto-
nitrile (10 mL) was supplemented under stirring with NaOH
(75 mg, 1.9 mmol). After 15 min stirring at reflux, methyl p-tolu-
enesulfonate was added to the mixture and agitation continued
at room temperature for 2 h. Then, the solvent was removed by
distillation under reduced pressure and the residue was suspended
in water (5 mL); the resulting insoluble material was collected by
filtration, washed with water and dried. Column chromatography
(CHCl3–diethylether 1:1) gave pure 11c (0.12 g, 55%): mp 121–
122 °C. 1H NMR (DMSO-d6, 500 MHz) d 1.11 (t, 3H, NCH2CH3),
2.32 (s, 3H, 6-CH3), 2.69 (s, 3H, 2-CH3), 3.45 (q, 2H, NCH2CH3),
4.87 (s, 2H, 3-CH2), 7.26 (s, 1H, 5-H), 7.82 (s, 1H, 7-H). Anal.
(C10H15N3O2S) C, H, N, S.
The title compound was obtained as described for 22a starting
from 7-fluoro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide
(26a) (1 g, 5.0 mmol) (0.70 g, 65%): mp 169–170 °C. 1H NMR
(DMSO-d6, 500 MHz) d 3.64 (s, 3H, NCH3), 8.30 (s, 1H, 3-H), 8.57
(d, 1H, 6-H), 8.89 (s, 1H, 8-H). Anal. (C7H6FN3O2S) C, H, N, S.
5.1.17. 4-Ethyl-7-fluoro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide (27b)
The title compound was obtained as described for 22b starting
from 7-fluoro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide
(26a) (1.0 g, 5.0 mmol) (0.71 g, 62%): mp 139–142 °C. 1H NMR
(DMSO-d6, 500 MHz)
d 1.32 (t, 3H, NCH2CH3), 4.23 (q, 2H,
NCH2CH3), 8.35 (s, 1H, 3-H), 8.56 (d, 1H, 6-H), 8.89 (s, 1H, 8-H).
Anal. (C8H8FN3O2S) C, H, N, S.
5.1.11. 2-Amino-5-fluoro-3-pyridinesulfonamide (25a)
5.1.18. 7-Chloro-4-methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide (27c)
2-Amino-5-fluoropyridine 24 (1 g, 0.89 mmol), obtained from
2-amino-5-nitropyridine 23 in four steps11 was slowly and pru-
dently added to a solution of ClSO3H (8 mL) in a dry ice-methanol
bath and then heated on an oil bath. After 48 h stirring at reflux,
the mixture was cooled to room temperature, poured into ice-
water and the resulting mixture was rapidly extracted with chloro-
form (3Â 20 mL). The combined organic layers were dried over
MgSO4 and filtered. The filtrate was concentrated to dryness under
reduced pressure. The residue, consisting of 2-amino-5-fluoro-3-
pyridinesulfonyl chloride, was dissolved in an aqueous ammonia-
cal solution (10% m/V, 30 mL). After 30 min stirring at room
temperature, the mixture was reduced to a small volume under re-
duced pressure. The resulting insoluble material was collected by
filtration, washed with water and dried (0.70 g, 41%): mp 220–
221 °C. 1H NMR (DMSO-d6, 500 MHz) d 6.47 (br s, 2H, NH2), 7.61
(br s, 2H, SO2NH2), 7.73 (d, 1H, 6-H), 8.21 (s, 1H, 4-H). Anal.
(C5H6FN3O2S) C, H, N, S.
The title compound was obtained as described for 22a starting
from 7-chloro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide
(26b) (1.0 g, 4.6 mmol) (0.78 g, 73%): mp 199–200 °C. 1H NMR
(DMSO-d6, 500 MHz) d 3.63 (s, 3H, NCH3), 8.31 (s, 1H, 3-H), 8.65
(s, 1H, 6-H), 8.89 (s, 1H, 8-H). Anal. (C7H6ClN3O2S) C, H, N, S.
5.1.19. 7-Chloro-4-ethyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide (27d)
The title compound was obtained as described for 22b starting
from 7-chloro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide
(26b) (1.0 g, 4.6 mmol) (0.76 g, 67%): mp 197–198 °C. 1H NMR
(DMSO-d6, 500 MHz)
d 1.32 (t, 3H, NCH2CH3), 4.22 (q, 2H,
NCH2CH3), 8.36 (s, 1H, 3-H), 8.67 (s, 1H, 6-H), 8.89 (s, 1H, 8-H).
Anal. (C8H8ClN3O2S) C, H, N, S.
5.1.20. 7-Bromo-4-ethyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide (27e)
5.1.12. 7-Fluoro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (26a)
The title compound was obtained as described for 22b starting
from 7-bromo-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide
(26c) (1.0 g, 3.8 mmol) (0.70 g, 63%): mp 214–215 °C. 1H NMR
The title compound was obtained as described for 21 starting
from 2-amino-5-fluoro-3-pyridinesulfonamide (25a) (3.0 g,
16 mmol) (2.8 g, 90%): mp 293–295 °C. 1H NMR (DMSO-d6,
500 MHz) d 8.07 (s, 1H, 3-H), 8.49 (d, 1H, 6-H), 8.80 (s, 1H, 8-H),
13.01 (s, 1H, NH). Anal. (C6H4FN3O2S) C, H, N, S.
(DMSO-d6, 500 MHz)
d 1.31 (t, 3H, NCH2CH3), 4.21 (q, 2H,
NCH2CH3), 8.37 (s, 1H, 3-H), 8.71 (s, 1H, 6-H), 8.95 (s, 1H, 8-H).
Anal. (C8H8BrN3O2S) C, H, N, S.
5.1.21. 4-Ethyl-7-methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide (27f)
The title compound was obtained as described for 22b starting
from 7-methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide
(26d) (1.0 g, 5.1 mmol) (0.79 g, 69%): mp 169–171 °C. 1H NMR
(DMSO-d6, 500 MHz) d 1.31 (t, 3H, NCH2CH3), 2.42 (s, 3H, 7-CH3),
5.1.13. 7-Chloro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (26b)
The title compound was obtained as described for 21 starting
from 2-amino-5-chloro-3-pyridinesulfonamide (25b)16 (3.0 g,
14 mmol) (2.7 g, 85%): mp >300 °C. 1H NMR (DMSO-d6, 500 MHz)

P. Francotte et al. / Bioorg. Med. Chem. 16 (2008) 9948–9956
9955
4.22 (q, 2H, NCH2CH3), 8.26 (s, 1H, 6-H), 8.30 (s, 1H, 3-H), 8.65 (s,
5.2. Pharmacological methods
1H, 8-H). Anal. (C9H11N3O2S) C, H, N, S.
5.2.1. AMPA-evoked current on Xenopus laevis oocytes injected
with rat cortex mRNA
5.1.22. 7-Fluoro-4-methyl-3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide (12a)
Rat cortex poly(A+) mRNA was prepared from the cerebral
cortex of male Wistar rats (15 days old) by the guanidium thio-
cyanate/cesium chloride method and was isolated with the use
of the PolyATtract mRNA isolation system (Promega, USA). Under
anaesthesia, a cluster of oocytes was removed from the abdomen
of X. Laevis and placed in Barth’s solution. Oocytes were injected
with 50.6 nl of an aqueous solution containing rat cortex
poly(A+) mRNA (1 mg/mL) by using an automatic microinjector
and were incubated at 18 °C in Barth’s solution for 3–4 days to
provide expression. They were then stored at 4 °C until use. Elec-
trophysiological recordings were performed at room temperature
on oocytes placed in a Plexiglas recording chamber continuously
superfused with ‘OR2’ solution. AMPA-evoked current was re-
corded at a holding potential of À60 mV, using standard two
electrodes voltage-clamp system. Ten micromolars of (S)-AMPA
was bath-applied during 30 s each 5 min with a constant flow
rate of 3 mL/min and the amplitude of the evoked current was
The title compound was obtained as described for 11a starting
from 7-fluoro-4-methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (27a) (0.6 g, 2.8 mmol) (0.50 g, 82%): mp 170–171 °C. 1H
NMR (DMSO-d6, 500 MHz) d 3.07 (s, 3H, NCH3), 4.73 (d, 2H, 3-
CH2), 8.02 (s, 1H, 6-H), 8.25 (t, 1H, NH), 8.41 (s, 1H, 8-H). Anal.
(C7H8FN3O2S) C, H, N, S.
5.1.23. 4-Ethyl-7-fluoro-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-
thiadiazine 1,1-dioxide (12b)
The title compound was obtained as described for 11a starting
from 4-ethyl-7-fluoro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (27b) (0.6 g, 2.6 mmol) (0.51 g, 85%): mp 135–138 °C. 1H
NMR (DMSO-d6, 500 MHz) d 1.09 (t, 3H, NCH2CH3), 3.62 (q, 2H,
NCH2CH3), 4.75 (d, 2H, 3-CH2), 8.01 (d, 1H, 6-H), 8.19 (t, 1H, NH),
8.89 (s, 1H, 8-H). Anal. (C8H10FN3O2S) C, H, N, S.
5.1.24. 7-Chloro-4-methyl-3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide (12c)
measured at the peak of the current. Applications of 10 lM
(S)-AMPA alone were done at the beginning of the experiments
until two inward currents with similar amplitude were obtained.
The mean amplitude value of these two AMPA-induced current
was taken as 100% of the response. All tested drugs were bath-
applied at successive increasing concentrations each 5 min 45 s
The title compound was obtained as described for 11a starting
from 7-chloro-4-methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (27c) (0.6 g, 2.6 mmol) (0.54 g, 89%): mp 206–208 °C. 1H
NMR (DMSO-d6, 500 MHz) d 3.07 (s, 3H, NCH3), 4.76 (s, 2H, 3-
CH2), 8.04 (s, 1H, 6-H), 8.27 (s, 1H, NH), 8.38 (s, 1H, 8-H). Anal.
(C7H8ClN3O2S) C, H, N, S.
before, during and 30 s after the application of 10 lM (S)-AMPA.
The amplitude of the AMPA-evoked current in the presence of
each concentration of the tested drug was expressed as a per-
centage of that induced in the absence of drug on the same oo-
cyte, taken as 100%. Each compound was tested on 2–3 different
oocytes. EC2X and EC5X values were calculated by interpolation as
the concentrations of the drug responsible for a twofold and five-
fold increase in the amplitude of the AMPA-evoked current,
respectively. EC50 corresponded to the concentration of the com-
pound that induced 50% of the maximal potentiation of the
AMPA-induced current.
5.1.25. 7-Chloro-4-ethyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-
thiadiazine 1,1-dioxide (12d)
The title compound was obtained as described for 11a starting
from 7-chloro-4-ethyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (27d) (0.6 g, 2.4 mmol) (0.52 g, 86%): mp 146–148 °C. 1H
NMR (DMSO-d6, 500 MHz) d 1.10 (t, 3H, NCH2CH3), 3.62 (q, 2H,
NCH2CH3), 4.78 (s, 2H, 3-CH2), 8.03 (s, 1H, 6-H), 8.23 (s, 1H, NH),
8.89 (s, 1H, 8-H). Anal. (C8H10ClN3O2S) C, H, N, S.
5.1.26. 7-Bromo-4-ethyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-
thiadiazine 1,1-dioxide (12e)
5.2.2. Social recognition test
The test, initially described by Thor and Holloway12, is based on
the natural expression of olfactory memory and on its normal
lapse: a reduction of the time spent by an adult rat in investigating
a previously exposed juvenile rat is one index of the adult rat’s
ability to recognize this particular juvenile. The test has been
adapted from the procedure described by Perio et al.20 Experiments
were carried out on Wistar adult rats, which were individually
housed for 2 days before testing. On the test day, each adult was
placed in its home cage on the observation table. After 5-min
habituation to the new environment, a juvenile rat (21 days) was
chosen randomly, and was introduced into the home cage for a first
5-min session; the social behaviour of the adult rat was recorded
through a video system, in order to measure the time spent (T1)
by the adult rat in investigating the juvenile rat (sniffing/biting/
grooming/nosing/pawing as well as jumping/crawling over or un-
der the juvenile). 12d was administered 1 min after the end of
the first 5-min session. A second 5-min session was conducted
120 min later with introduction of the same juvenile. The social
behaviour was again recorded, and its duration was measured
(T2). Adults displaying T1 values of 60 s were not examined fur-
ther. The differences in social behaviour between the two sessions
(T2–T1) were analyzed by one-way ANOVA.
The title compound was obtained as described for 11a starting
from 7-bromo-4-ethyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (27e) (0.6 g, 2.1 mmol) (0.48 g, 79%): mp 152–153 °C. 1H
NMR (DMSO-d6, 500 MHz) d1.10 (t, 3H, NCH2CH3), 3.62 (q, 2H,
NCH2CH3), 4.78 (s, 2H, 3-CH2), 8.08 (s, 1H, 6-H), 8.22 (s, 1H NH),
8.42 (s, 1H, 8-H). Anal. (C8H10BrN3O2S) C, H, N, S.
5.1.27. 4-Ethyl-7-methyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-
thiadiazine 1,1-dioxide (12f)
The title compound was obtained as described for 11a starting
from 4-ethyl-7-methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-
dioxide (27f) (0.6 g, 2.7 mmol) (0.48 g, 80%): mp 135–137 °C. 1H
NMR (DMSO-d6, 500 MHz) d 1.08 (t ; 3H, NCH2CH3), 2.19 (s, 3H,
7-CH3), 3.61 (q, 2H, NCH2CH3), 4.72 (s, 2H, 3-CH2), 7.74 (s, 1H, 6-
H), 8.01 (s, 1H, NH), 8.17 (s, 1H, 8-H). Anal. (C9H13N3O2S) C, H, N, S.
5.1.28. 7-Chloro-4-ethyl-2-methyl-3,4-dihydro-2H-pyrido[2,3-
e]-[1,2,4]-thiadiazine 1,1-dioxide (12g)
The title compound was obtained as described for 11c start-
ing from 7-chloro-4-ethyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-
thiadiazine 1,1-dioxide (12d) (0.6 g, 1.3 mmol) (0.25 g, 80%):
mp 88–89 °C. 1H NMR (DMSO-d6, 500 MHz)
d 1.11 (q, 3H,
NCH2CH3), 2.66 (s, 3H, N-CH3), 3.64 (q, 2H, NCH2CH3), 4.98 (s,
2H, 3-CH2), 8.10 (d, 1H, 6-H), 8.43 (d, 1H, 8-H). Anal.
(C9H12ClN3O2S) C, H, N, S.
5.2.3. Object recognition test in Wistar rats
The one-trial object recognition paradigm measures a form of
episodic memory in the rat.13 Recognition is measured by the

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P. Francotte et al. / Bioorg. Med. Chem. 16 (2008) 9948–9956
time spent by rats in exploring two different objects, one famil-
iar and the other new. Wistar rats were always submitted to
three sessions of the test. The first one was a session of habitu-
ation, where the rats were allowed to explore the apparatus for
a 3-min session of habituation. In the second 3-min session, the
rats were presented with two similar objects. The last 3-min ses-
sion consisted of the recognition test between the familiar and
the new object, one of the objects presented in the second ses-
sion being replaced by a new object. With an inter-trial interval
of 1 or 60 min, normal rats spend more time exploring the new
object, which demonstrates that they recognize the familiar one.
After a retention interval of 24 h, the times spent in exploring
the familiar and new objects are similar, indicating that the rats
do no longer recognize the familiar object. Since cognitive-
enhancing effect of compounds was expected, conditions of for-
getting and an inter-session interval of 24 h were used in the
present study. Compound or vehicle was administered 1 h before
each session of the test. Statistical analyses were performed on
the difference in duration of exploration of the two objects
and on the discrimination index (=delta new-fam/total explora-
tion) during the last session.
Supplementary data
Elemental analyses for the described compounds. Supplemen-
tary data associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2008.10.036.
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Acknowledgment
This study was supported in part by a grant from the Na-
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