3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides as ATP-sensitive potassium channel openers: Effect of 6,7-disubstitution on potency and tissue selectivity

Article abstract of DOI:10.1021/jm0580050

A series of 6,7-disubstituted 4H-1,2,4-benzothiadiazine 1,1-dioxides bearing a short alkylamino side chain in the 3-position were synthesized. These compounds were tested on rat pancreatic islets and on rat aorta rings. In vitro data indicated that in mos

Full text of DOI:10.1021/jm0580050

4990
J. Med. Chem. 2005, 48, 4990-5000
3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides as ATP-Sensitive
Potassium Channel Openers: Effect of 6,7-Disubstitution on Potency and Tissue
Selectivity
Pascal de Tullio,*,† Ste´phane Boverie,† Be´ne´dicte Becker,‡ Marie-He´le`ne Antoine,‡ Quynh-Anh Nguyen,‡
Pierre Francotte,† Ste´phane Counerotte,† Sophie Sebille,† Bernard Pirotte,† and Philippe Lebrun‡
Centre de Recherche en Pharmacochimie des Substances Naturelles et Synthe´tiques, Laboratoire de Chimie Pharmaceutique,
Universite´ de Lie`ge, 1 Avenue de l’Hoˆpital, B-4000 Lie`ge, Belgium, and Laboratoire de Pharmacodynamie et de The´rapeutique,
Universite´ Libre de Bruxelles, 808 Route de Lennik, B-1070 Bruxelles, Belgium
Received January 26, 2005
A series of 6,7-disubstituted 4H-1,2,4-benzothiadiazine 1,1-dioxides bearing a short alkylamino
side chain in the 3-position were synthesized. These compounds were tested on rat pancreatic
islets and on rat aorta rings. In vitro data indicated that in most cases substitution in the 6
and the 7 positions increased their activity as inhibitors of insulin secretion, while the
myorelaxant potency of the drugs was maintained or enhanced according to the nature of the
substituent in the 7-position. The presence of either chlorine or bromine atoms in the 6 and 7
positions did not improve the apparent selectivity of the drugs for the pancreatic tissue. By
contrast, the introduction of one or two fluorine atoms, as well as the presence of a methoxy
group in the 7-position, generated potent and selective inhibitors of insulin release. Radioisotopic
and fluorimetric experiments performed with the most potent compound inhibiting insulin
release (34, BPDZ 259, 6-chloro-7-fluoro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-
dioxide) confirmed that the drug activated KATP channels. 34 was found to be one of the most
potent and selective pancreatic potassium channel openers yet described.
Introduction
provoke the relaxation of smooth muscles and/or the
inhibition of endocrine releases.22,23 Because of their
broad therapeutic potential, a large variety of KATP
channel agonists has been developed.24,25 These drugs
include chromane derivatives such as cromakalim,26
cyanoguanidine compounds such as pinacidil,27 and
1,2,4-benzothiadiazine derivatives such as diazoxide28
(Figure 1). Selective activation of pancreatic KATP chan-
nels has been demonstrated to be of clinical value in
the treatment of several metabolic disorders, including
type I and type II diabetes, obesity, and hyperin-
sulinemia.29-32 Until recently, diazoxide was the only
compound reported to activate pancreatic KATP chan-
nels. Unfortunately, as a consequence of its lack of
tissue selectivity, diazoxide induces many side effects
such as hypertrichosis, edema, headache, and hypo-
tension.33
In the search for new pancreatic selective PCOs,
several years ago we developed a series of 3-alkylamino-
4H-pyrido- and -1,2,4-benzothiadiazine 1,1-dioxides.34-40
Among the drugs of this original series, BPDZ 4437 (1,
Figure 1), BPDZ 7341 (7-chloro-3-isopropylamino-4H-
1,2,4-benzothiadiazine 1,1-dioxide) (2, Figure 1), BPDZ
138 (7-fluoro-3-isopropylamino-4H-1,2,4-benzothiadiaz-
ine 1,1-dioxide)40 (3, Figure 1), and BPDZ 216 (3-
isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-
dioxide)42 (4, Figure 1) were identified as the first potent
and selective pancreatic KATP channel openers. Usually,
the benzenic derivatives appeared to exhibit a higher
potency on vascular and/or pancreatic tissue in com-
parison with their pyridinic counterparts. More recently,
3-alkylamino-6-chloro-4H-thieno[3,2-e]-1,2,4-thiadiaz-
ine 1,1-dioxides, such as NN414 (5, Figure 1), have also
ATP-sensitive potassium channels (KATP channels),
which regulate the flow of potassium ions through the
cell membrane, have been identified in a wide range of
cell types and have been found to link the metabolic
state to the electric state of the cell.1-8 KATP channels
are composed of two different protein subunits in a 4 +
4 stoichiometry.9 The KATP channel pore belongs to the
inwardly rectifying potassium channel family and is
named Kir6.x.10 The second subunit, the SUR (for
sulfonylurea receptor) subunit, contains the regulatory
sites for most drugs.10 Four variants of SUR have been
reported (SUR1, SUR2A, SUR2B, and SUR2C).11 Ac-
cording to their tissue localization, KATP channels are
composed of different subunits. For example, SUR1
combined with Kir6.2 forms the pancreatic KATP chan-
nel.12 The combination of SUR2A and Kir6.2 subunits
is found in cardiac and skeletal muscle, while the
smooth muscle KATP channel is composed of SUR2B and
Kir6.1 or Kir6.2 subunits.13 Although pancreatic KATP
channels are well-known to be involved in the insulin-
releasing process14,15 and smooth muscle KATP channels
in the control of muscle tone,16,17 the physiological roles
of the different channel subtypes have not yet been
thoroughly assessed.18,19
Several drugs, named PCOs (potassium channel
openers), have been found to activate KATP chan-
nels,20,21 leading to plasma membrane hyperpolariza-
tion and reduction in cell excitability. This may, in turn,
* To whom correspondence should be addressed.′ Phone: 32-4-366-
43-69. Fax: 32-4-366-43-62. E-mail: P.deTullio@ulg.ac.be.
† Universite´ de Lie`ge.
‡ Universite´ Libre de Bruxelles.
10.1021/jm0580050 CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/01/2005

Benzothiadiazine Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4991
thiocarbonyldiimidazole as previously described,46 led
to compounds 18-21. The 3-imidazolyl group of the
reactive intermediates was displaced by selected
branched alkylamines to give the expected 3-alkylami-
no-substituted derivatives (22-35). Such a nucleophilic
substitution occurred without any difficulties in the
cases of 6,7-dichloro (14), 7-bromo-6-chloro (15), and
6-chloro-7-fluoro (16) intermediates. However, the fluo-
rine atom in the 6-position of 6,7-difluoro-3-(1H-imida-
zol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (17) was
substituted by the alkylamine, together with the imi-
dazole group. Thus, application of this synthetic path-
way to the 6,7-difluoro intermediate (17) led to com-
pounds bearing two alkylamino side chains: the first
one, as expected, in the 3-position and the second
one, more surprisingly, in the 6-position (compounds
36-38). So to obtain the 6,7-difluoro-substituted com-
pounds (42-44), we decided to follow the route described
by Flemming et al. for obtaining 3-alkylamino-substi-
tuted thienothiadiazine dioxides.43 The action of differ-
ent alkyl isothiocyanates on 2-amino-4,5-difluoroben-
zenesulfonamide (17) generated the corresponding
sulfonylthioureas (39-41). Ring closure was then
achieved at low temperature using phosgene and led to
the expected drugs (Scheme 2, 42-44).
Figure 1. Chemical structure of diazoxide and several
analogues described as potassium channels openers.
The 6-chloro-7-methoxy derivatives (49-51) were
prepared according to Scheme 3, starting from 3-chloro-
4-methoxyaniline (45). Reaction with chlorosulfonyl
isocyanate led to the 3-oxo intermediate (46). Subse-
quent conversion of the 3-oxo function into a 3-thioxo
one, (47) followed by S-methylation, gave access to the
expected reactive 3-methylsulfanyl-substituted inter-
mediate (48). The nucleophilic substitution of the me-
thylsulfanyl group was conducted with the appropriate
alkylamine under reflux at normal pressure or in a
sealed vessel (compounds 49-51).
been proposed for the treatment of metabolic disorders
linked to insulin secretion.43
Thirty years ago, Topliss and Yudis44 demonstrated
that the introduction of a small group in the 6-position
of diazoxide improved its vasorelaxant activity, while
Wales et al.45 showed that the 6,7-dichloro analogue of
diazoxide maintained the hyperglycemic properties of
the parent compound. On this basis, it might be
expected that the disubstitution of 3-alkylamino-4H-
1,2,4-benzothiadiazine 1,1-dioxides in the 6 and 7 posi-
tions would enhance the KATP channel agonistic activity
of diazoxide analogues. In line with this proposition and
according to the structure-activity relationships (SAR)
deduced from our previous work,34,36,40 we synthesized
in the present study a series of 6- and 7-substituted 4H-
1,2,4-benzothiadiazine 1,1-dioxides bearing short and
branched alkylamino side chains in the 3-position.
These newly synthesized compounds were tested as
putative KATP channel openers on a vascular and a
pancreatic pharmacological model in order to evaluate
the impact of the 6- and 7-disubstitution on both their
potency and tissue selectivity. Moreover, further biologi-
cal investigations were conducted with a selected drug
to confirm the mechanism of action of these original
compounds.
Results and Discussion
Twenty-four new 6,7-disubstituted 4H-1,2,4-benzothi-
adiazine 1,1-dioxides bearing, in most cases, a short
alkylamino side chain in the 3-position were prepared.
To determine their putative activity on KATP channels
and also to compare their relative tissue selectivity, a
pancreatic and a vascular in vitro model were used as
pharmacological screening tools.
The ability of such compounds to inhibit glucose-
induced insulin secretion was evaluated on isolated rat
pancreatic islets and compared to that of diazoxide and
the corresponding 7-monosubsituted analogues (Tables
1 and 2). Except for compounds exhibiting a large alkyl-
amino side chain in the 3-position (n-hexyl substituent,
28) and compounds substituted by an alkylamino chain
in the 6-position (36-38) that were found to be inactive
at 50 µM, all drugs markedly inhibited insulin release
at 10 µM. At a lower concentration (1 µM), and whatever
the nature of the 6 and 7 substituent, the 3-ethylamino
and the 3-isopropylamino side chains appeared to be the
more accurate choice to elicit a marked inhibitory
activity on the insulin secreting rate (see compounds
22, 24, 29, 30, 32, 34, 42, 43, and 50).
Chemistry
The different synthetic pathways used to prepare the
6,7-disubstituted 3-alkylamino-4H-1,2,4-benzothiadiaz-
ine 1,1-dioxides described in this manuscript are il-
lustrated in Schemes 1-3. In the 6,7-dichloro, 7-bromo-
6-chloro, 6-chloro-7-fluoro, and 6,7-difluoro series (Scheme
1), a chlorosulfonation step was used to give access to
the o-aminobenzenesulfonamides key intermediates
(14-17). Starting from the appropriate aniline (6-9),
the treatment with chlorosulfonic acid led to the corre-
sponding sulfonyl chloride (10-13), which reacted with-
out further purification with diluted ammonia. Ring
closure of o-aminobenzenesulfonamides, using 1,1′-
Another interesting hydrocarbon chain on the nitro-
gen atom in the 3-position appeared to be cyclobutyl (27,
31, 35, 51). These results are in accordance with the
SAR related to the nature of the alkylamino chain in

4992 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
de Tullio et al.
Scheme 1a
a Reagents: (i) ClSO3H; (ii) NH4OH; (iii) 1,1-thiocarbonyldiimidazole; (iv) RNH2.
Scheme 2a
a Reagents: (i) K2CO3, SdCdN-R, dry acetone; (ii) TEA, 1.93 M COCl2 in toluene, THF, 0 °C.
Scheme 3a
a Reagents: (i) MeNO2, ClSO2NCO, AlCl3; (ii) P2S5, pyridine; (iii) K2CO3, CH3I; (iv) RNH2.
the 3-position and the SAR deduced from previous
studies on 7-substituted 3-alkylamino-4H-1,2,4-ben-
zothiadiazine 1,1-dioxides.40,47 Moreover, on looking at
the calculated IC50 values (Tables 1 and 2), it is noted
that these disubstituted compounds appear to be more
potent than their respective 7-monosubstituted ana-
logues (24 vs 2, 34, 43 vs 3, and 50 vs 4) and up to 150
times more potent than diazoxide. So in general the
introduction of a halogen atom in the 6-position ap-
peared to favor activity on the pancreatic tissue. The

Benzothiadiazine Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4993
Table 1. Effects of 6,7-Dichloro-Substituted or 6-Chloro-7-bromo-Substituted 3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides on
16.7 mM Glucose-Induced Insulin Release from Rat Pancreatic Islets and on Contractile Activity of K+-Depolarized Rat Aorta Rings
% of residual insulin release
(mean ( SEM (n))
IC50 (pancreas),b
EC50 (aorta),c
d
compd
22
23
24
25
26
27
28
29
30
31
diazoxide
2
10 µM
1 µM
0.1 µM
µM
µM
EC50/IC50
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Cl
Cl
Br
CH2CH3
CH2CH2CH3
CH(CH3)2
CH2CHdCH2
CH(CH2)2
CH(CH2)3
(CH2)5CH3
CH2CH3
CH(CH3)2
CH(CH2)3
9.1 ( 1.7 (18)
9.9 ( 0.9 (14)
6.3 ( 0.7 (12)a
13.5 ( 0.8 (11)
17.6 ( 2.0 (18)
8.4 ( 1.7 (18)
95.2 ( 4.7 (16)
7.9 ( 0.7 (13)
7.5 ( 0.7 (14)
9.5 ( 0.6 (14)
73.9 ( 4.4 (16)a
4.9 ( 0.4 (32) a
8.1 ( 0.8 (12)a
8.5 ( 0.6 (29)
60.2 ( 4.1 (15)
13.2 ( 1.0 (26)a
68.5 ( 4.1 (16)
92.5 ( 3.3 (14)
58.1 ( 5.4 (16)
NDg
19.7 ( 1.9 (13)
25.7 ( 2.0 (15)
57.2 ( 5.6 (16)
87.5 ( 5.0 (15)a
36.2 ( 2.4 (31)a
34.9 ( 2.8 (12)a
83.4 ( 3.8 (24)
0.25
1.37
0.28a
1.89
3.34
1.25
NDg
0.27
0.26
1.21
22.6a
0.73
0.47
1.0 ( 0.1 (8)
4.7 ( 0.5 (4)
2.3 ( 0.2 (8)
1.2 ( 0.2 (4)
>30 (4)
12.6 ( 2.0 (5)
>300 (4)
3.3 ( 0.6 (6)
5.6 ( 0.7 (6)
6.6 ( 0.5 (6)
22.4 ( 2.1 (11)a
36.3 ( 2.2 (6)a
4.8 ( 0.7 (5)a
4
3.4
8.2
0.63
>9
84.9 ( 4.5 (21)a
e
f
10.1
NDg
12.2
21.5
5.5
79.5 ( 3.8 (36)
73.3 ( 4.2 (23)
f
-
1.0a
49.7
10.2
H
H
CH(CH3)2
CH(CH3)2
90.4 ( 3.5 (23)a
91.0 ( 5.3 (13)a
52
a Published results (refs 32 and 40). b Drug concentration giving 50% inhibition of insulin release (estimated value). c Drug concentration
giving 50% relaxation of the 30 mM KCl induced contraction of rat aorta rings (mean ( SEM (n)). d Estimated selectivity ratio. e Cyclopropyl.
f Cyclobutyl. g ND: not determined.
Table 2. Effects of 6-Chloro-7-fluoro-Substituted, 6-Alkylamino-7-fluoro-Substituted, 6,7-Difluoro-Substituted, and
6-Chloro-7-methoxy-Substituted 3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides on 16.7 mM Glucose-Induced Insulin Release
from Rat Pancreatic Islets and on Contractile Activity of K+-Depolarized Rat Aorta Rings
% of residual insulin release
(mean ( SEM (n))
IC50 (pancreas),b
EC50 (aorta),c
d
compd
10 µM
1 µM
0.1 µM
µM
µM
EC50/IC50
32
33
34
35
36
37
38
42
43
44
49
50
51
3
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
F
F
F
CH2CH3
CH2CH2CH3
CH(CH3)2
CH(CH2)3
CH(CH3)2
CH2CH2CH3
CH(CH2)3
CH2CH3
CH(CH3)2
12.7 ( 0.9 (14) 7.3 ( 0.6 (14)
73.1 ( 3.0 (24)
92.0 ( 4.7 (23)
64.4 ( 3.3 (31)
74.5 ( 4.2 (23)
0.20
0.52
0.16
0.23
42.4 ( 2.8 (6)
45.3 ( 2.5 (5)
41.5 ( 1.4 (6)
39.9 ( 1.3 (6)
>200 (4)
212
7.5 ( 0.6 (12)
7.0 ( 0.6 (14)
37.6 ( 2.3 (13)
5.7 ( 0.4 (13)
87.1
259.4
173.5
NDf
e
12.8 ( 0.8 (13) 15.3 ( 1.4 (15)
NH-CH(CH3)2
NH-CH2CH2CH3
NH- CH(CH2)3
F
F
F
Cl
Cl
Cl
H
H
[70.1 ( 4.1 (20) at 50 µM]
[104.1 ( 4.1 (16) at 50 µM]
[88.6 ( 3.8 (14) at 50 µM]
NDf
NDf
e
e
NDf
NDf
10.5 ( 1.2 (15) 22.0 ( 0.9 (15)
6.1 ( 0.5 (11) 22.9 ( 2.3 (12)
94.5 ( 5.1 (16)
81.6 ( 3.4 (16)
0.37
0.30
5.68
0.48
0.24
0.51
0.76
1.75
66.7 ( 8.5 (4)
102.4 ( 15.2 (4)
>30 (4)
8.1 ( 0.9 (5)
37.0 ( 2.7 (6)
23.4 ( 1.4 (5)
43.1 ( 10.7 (5)a
274.0 ( 19.0 (5)
180.3
341.3
>5.3
16.9
154.2
45.9
56.7
156.6
CH2CH(CH3)2 42.3 ( 2.5 (15) 86.9 ( 3.2(16)
OCH3 CH2CH3
OCH3 CH(CH3)2
OCH3 CH(CH2)3
7.2 ( 0.6 (15)
7.7 ( 0.7 (15)
9.2 ( 0.8 (14)
38.0 ( 4.1 (14)
20.6 ( 1.9 (13)
38.1 ( 3.0 (14)
85.3 ( 4.7 (21)
73.8 ( 4.1 (19)
90.0 ( 4.6 (16)
e
F
CH(CH3)2
3.7 ( 0.6 (13)a 47.3 ( 3.7 (23)a 96.9 ( 4.9 (16)a
4
OCH3 CH(CH3)2
8.5 ( 0.9 (24)
67.6 ( 4.3 (20)
a Published results (ref 40). b Drug concentration giving 50% inhibition of insulin release (estimated value). c Drug concentration giving
50% relaxation of the 30 mM KCl induced contraction of rat aorta rings (mean ( SEM (n)). d Estimated selectivity ratio. e Cyclobutyl.
f ND: not determined.
replacement of the chlorine atom in the 7-position by a
fluorine, a bromine atom, or a methoxy group had little
impact on the effect of the drugs. Among these newly
described drugs, compounds belonging to the 6-chloro-
7-fluoro-4H-1,2,4-benzothiadiazine 1,1-dioxide series
(32-35), and more specifically the 3-ethyl (32), the
3-isopropyl (34), and the cyclobutyl (35) derivatives, can
be considered as among the most potent inhibitors of
insulin release reported to date.
The myorelaxant effect of these compounds was
evaluated on the contractile activity of KCl-depolarized
rat aorta rings. Results are presented in Tables 1 and
2 as the EC50 values, together with the EC50/IC50 ratio,
which reflects the apparent tissue selectivity (vascular
versus pancreatic) of the drugs. As previously noted with
3-alkyl-4H-1,2,4-benzothiadiazine 1,1-dioxides,44 the in-
troduction of two chlorine atoms in the 6 and 7 positions
(compounds 22-27) led to a sizable increase in myore-

4994 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
de Tullio et al.
laxant activity, in comparison with their previously
reported 7-chloro-subtituted analogues,40 except for the
hindered 3-hexylamino-substituted compound (28). A
marked enhancement in vasorelaxant activity was also
noticed in the 6-chloro-7-methoxy series (49-51) (com-
pare 50 to its 7-methoxy counterpart 4). By contrast,
in the 6-chloro-7-fluoro and in the 6-chloro-7-bromo
series, the introduction of a chlorine atom in the
6-position failed to exert any obvious impact (30 vs its
7-bromo counterpart 52 and 34 vs 3). As reported
previously with the 3-alkylamino-7-fluoro-4H-1,2,4-ben-
zothiadiazine 1,1-dioxides,40 the presence of two fluorine
atoms on the benzene ring (compounds 42-44) was
deleterious for the myorelaxant activity of the drugs.
The presence of a hindered group in the 3-position, such
as n-hexyl (compound 28), was also clearly unfavorable
to myorelaxant activity.
Comparison of these in vitro results by means of the
EC50/IC50 ratio allows us to assess the apparent tissue
selectivity (vascular versus pancreatic) of the com-
pounds. Drugs of the 6,7-dichloro series (22-27), and
more specifically the 3-ethylamino and the 3-isopro-
pylamino derivatives (22 and 24), highly potent on
pancreatic B-cells, were characterized by a decrease in
tissue selectivity in comparison with compound 2. The
7-bromo derivatives (29-31) expressed a moderate
selectivity for the pancreatic tissue. As previously noted
with monosubstituted 7-fluoro- and 7-methoxy-3-alkyl-
aminobenzothiadiazine 1,1-dioxides,40,42 the presence of
at least one fluorine atom or one methoxy group in the
7-position of the benzothiadiazine ring led to drugs more
selective for the pancreatic tissue. 6-Chloro-7-fluoro-3-
ethylamino (32), 6-chloro-7-fluoro-3-isopropylamino (34),
6-chloro-7-fluoro-3-cyclobutylamino (35), and 6,7-dif-
luoro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-
dioxides (43) were identified as the most promising
compounds in terms of insulin inhibitory activity (IC50
e 0.3 µM) and pancreatic selectivity (EC50/IC50 ratio up
to 340).
Figure 2. Effect of 10 µM BPDZ 259 (34) on 86Rb outflow from
rat pancreatic islets perifused throughout in the absence (b)
or presence (O) of 10 µM glibenclamide. Basal media contained
5.6 mM glucose and extracellular Ca2+. Mean values ((SEM)
are from six individual experiments.
Taken as a whole, such in vitro results indicate that
a halo substitution in the 6-position of the 7-substituted
3-alkylamino-1,2,4-benzothiadiazine ring yielded an
increase in inhibitory activity on the insulin-releasing
process, while the myorelaxant potency of the drugs was
maintained or enhanced according to the nature of the
substituent group in the 7-position.
Compound 34 (6-chloro-7-fluoro-3-isopropylamino-4H-
1,2,4-benzothiadiazine 1,1-dioxide), namely, BPDZ 259,
was found to be the most potent compound acting on
pancreatic B-cells. Therefore, further radioisotopic and
fluorimetric experiments were performed in order to
characterize its mechanism of action. The drug (10 µM)
provoked a rapid, sustained, and marked increase in
86Rb outflow (42K substitute) from prelabeled and peri-
fused rat pancreatic islets (Figure 2). The stimulatory
effect of 34 on 86Rb FOR (fractional outflow rate) was
reversible and totally abolished by the presence of
glibenclamide (10 µM), a hypoglycemic sulfonylurea
known to block KATP channels48,49 (Figure 2). Such data
indicate that BPDZ 259 induces an increase in mem-
brane K+ permeability through the activation of ATP-
sensitive K+ channels.22,41,50
Figure 3. (Left panels) Effect of 100 nM (O) and 1 µM (b)
BPDZ 259 (34) on 45Ca outflow (upper) and insulin release
(lower) from pancreatic islets perifused throughout in the
presence of 16.7 mM glucose. Basal media contained extra-
cellular Ca2+. (Right panels) Effect of 10 µM BPDZ 259 (34)
on 45Ca outflow (upper) and insulin release (lower) from
pancreatic islets perifused throughout in the presence of 16.7
mM glucose. Basal media contained extracellular Ca2+ (b) or
were deprived of Ca2+ and enriched with EGTA (O). Mean
values are from four to six individual experiments.
Ca2+ inflow through voltage-dependent Ca2+ channels.
In agreement with this, Figure 3 (upper panels) clearly
documents an inhibitory effect of 34 on 45Ca outflow
from rat pancreatic islets exposed to 16.7 mM glucose
and extracellular Ca2+. Under such experimental condi-
tions, a decrease in 45Ca FOR is known to result from a
Activation of KATP channels might be expected to
hyperpolarize the plasma membrane and to restrict

Benzothiadiazine Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4995
Figure 4. Effect of 1 µM BPDZ 259 (34) on glucose (20 mM)
induced increase in [Ca2+]i. Basal media contained 2.8 mM
glucose and extracellular Ca2+. Each graph is a representative
experiment conducted on a single cell.
Figure 5. Effect of a rise in the extracellular K+ concentration
from 5 to 50 mM on 45Ca outflow from rat pancreatic islets
perifused throughout in the absence (O) or presence (b) of 10
µM BPDZ 259 (34). Basal media contained 2.8 mM glucose
and extracellular Ca2+. Mean values ((SEM) are from four
individual experiments.
reduction in 40Ca entry through voltage-sensitive Ca2+
channels.50 This proposal is further confirmed by the
lack of effect of 34 on 45Ca outflow from pancreatic islets
perifused in the absence of extracellular Ca2+ (Figure
3, right upper panel).
The inhibitory effect of 34 on 45Ca FOR was sustained,
reversible, and concentration-dependent. The paired
difference in 45Ca FOR before (40-44 min) and during
(60-68 min) exposure to 100 nM, 1 µM, and 10 µM 34
averaged 0.40 ( 0.02, 0.78 ( 0.04, and 0.80 ( 0.03
%/min, respectively.
the 6 and 7 positions by different halogen atoms or by
a methoxy group. The effects of these compounds were
characterized in two different models: rat insulin-
secreting cells and rat aorta rings. The in vitro data
indicate that halo-substitution in the 6-position of the
7-substituted 3-alkylamino-4H-1,2,4-benzothiadiazine
ring enhanced the inhibitory effect on insulin release,
while the myorelaxant properties of the drugs were
unaffected or increased according to the nature of the
substituent in the 7-position. A fluorine atom and a
methoxy group were found to be substituents leading
to marked tissue selectivity (pancreatic vs aortic).
Radioisotopic and fluorimetric experiments performed
with the most potent drug inhibiting the insulin secre-
tory rate, compound 34 (6-chloro-7-fluoro-3-isopropyl-
amino-4H-1,2,4-benzothiadiazine 1,1-dioxide), also named
BPDZ 259, confirmed that the drug activated the
Moreover, the effect of 34 on 45Ca outflow was
accompanied by a concentration-dependent reduction in
insulin output. The effect of 34 on the insulin-releasing
process can be viewed as the result of the decrease in
40Ca2+ entry because the dynamic measurement of the
insulin secretory rate displayed a time course parallel
to that of the 45Ca FOR response.
Incidentally, the cationic and secretory responses to
34 were clearly reversible, indicating that the drug did
not damage the pancreatic B-cells.
A decrease in Ca2+ entry as mediated by 34 should
provoke a reduction in the cytosolic Ca2+ concentration.
Such an effect of 34 is attested by calcium fluorimetry
experiments, indicating that the drug was able to
counteract the rise in cytosolic Ca2+ provoked by an
insulinotropic glucose concentration (Figure 4).
Altogether, these radioisotopic and fluorimetric data
suggest that in insulin-secreting cells compound 34
activates KATP channels. This in turn reduces Ca2+
entry, decreases the cytosolic Ca2+ concentration, and
ultimately inhibits insulin output. Such a view is further
supported by the failure of 34 to affect the increase in
45Ca outflow from pancreatic islets exposed to 50 mM
extracellular K+ (Figure 5). Indeed, the 45Ca response
to high K+ is known to be sensitive to Ca2+ channel
blockers but resistant to potassium channel open-
ers.22,41,51
K
ATP channels. Thus, BPDZ 259, a potent and selective
pancreatic KATP channel opener, might be considered
as a valuable substitute for diazoxide in the treatment
of glucose homeostasis disorders.
Materials and Methods
Chemistry. Melting points were determined on a Bu¨chi-
Tottoli capillary apparatus and are uncorrected. IR spectra
were recorded as KBr pellets on a Perkin-Elmer 1000 FT
spectrophotometer (Perkin-Elmer). The 1H NMR spectra were
taken on a Bruker AW-80 instrument, on a Bruker AM-400,
and on a Bruker AM-500 (Bruker Belgium, Brussel, Belgium)
in DMSO-d6 with HMDS (hexamethyldisiloxane) or TMS
(tetramethylsilane) as internal standard. Chemical shifts are
reported in δ units (ppm) relative to internal HMDS. In the
data presentation, s ) singlet, d ) doublet, t ) triplet, q )
quadruplet, m ) multiplet, b ) broad are used. Elemental
analyses (C, H, N, S) were realized on a Carlo-Erba EA 1108-
elemental analyzer and were within (0.4% of the theoretical
values. All reactions were routinely checked by TLC on Merck
60F 254 silica gel (Merck, Darmstad, Germany).
Conclusions
Having previously explored the 7-monosubstituted
3-alkylamino-1,2,4-benzothiadiazine 1,1-dioxides as pu-
tative KATP channel openers, we synthesized and ex-
amined several 3-alkylamino derivatives substituted in
2-Amino-4,5-dichlorobenzenesulfonamide (14).52 3,4-
Dichloroaniline (6, 20 g, 0.0123 mol) was added portionwise

4996 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
de Tullio et al.
to chlorosulfonic acid (70 mL) cooled in an ice/water bath. The
suspension was then refluxed and supplemented after 1 h with
thionyl chloride (20 mL). The reflux was maintained for
another 30 min. After cooling, the mixture was poured on ice
and extracted with AcOEt. The organic layer was dried over
anhydrous MgSO4, and the solvent was removed under
reduced pressure. The crude residue of 2-amino-4,5-dichlo-
robenzenesulfonyl chloride (10) was dissolved in dioxane (50
mL) and added dropwise to a 10% w/v aqueous solution of
ammonia (100 mL). After 10 min of being stirred, the solution
was concentrated under reduced pressure to a small volume.
The yellow-brown precipitate obtained was collected by filtra-
tion and washed with water. The crude product was suspended
in water, and this suspension was adjusted to pH 12 with 2.5
M NaOH. The resulting solution was treated with charcoal
and filtered. The filtrate was adjusted to pH 3-4 with
concentrated HCl. The precipitate that appeared was collected
by filtration, washed with water, dried, and crystallized in
MeOH/water (44%). Mp 175-178 °C (lit., 175-178 °C52).
2-Amino-5-bromo-4-chlorobenzenesulfonamide (15).53
4-Bromo-3-chloroaniline (7, 2 g, 0.0097 mol) was added por-
tionwise to chlorosulfonic acid (8 mL) cooled in an ice/water
bath. The suspension was then heated at 150 °C for 1 h. After
cooling, the mixture was poured onto ice and extracted with
AcOEt. The organic layer was dried over anhydrous MgSO4,
and the solvent was removed under reduced pressure. The
crude residue of 2-amino-5-bromo-4-chlorobenzenesulfonyl
chloride (11) was dissolved in dioxane (10 mL) and added
dropwise to a 10% w/v aqueous solution of ammonia (50 mL).
After 30 min of being stirred, the solution was treated with
charcoal, filtered, and concentrated under reduced pressure
to a small volume. The yellow-brown precipitate that appeared
was collected by filtration, washed with water, and dried (42%).
Mp 177-180 °C; IR (KBr); 1H NMR (DMSO-d6, 400 MHz).
Anal. (C6H6BrClN2O2S) C, H, N, S.
2-Amino-4-chloro-5-fluorobenzenesulfonamide (16).
3-Chloro-4-fluoroaniline (8, 2 g, 0.0137 mol) was added por-
tionwise to chlorosulfonic acid (6 mL) cooled on an ice/water
bath. The suspension was heated at 150 °C and supplemented
after 1 h with thionyl chloride (1 mL). The reflux was
maintained for another 30 min. After cooling, the mixture was
poured onto ice and the precipitate was filtered off. The crude
residue of 2-amino-4-chloro-5-fluorobenzenesulfonyl chloride
(12) was dissolved in dioxane (10 mL) and added dropwise to
a 10% w/v aqueous solution of ammoniac (50 mL). After the
mixture was stirred for 30 min, concentration under reduced
pressure to a small volume gave rise to a precipitate of the
title compound (45%). Mp 178-180 °C; IR (KBr); 1H NMR
(DMSO-d6, 400 MHz). Anal. (C6H6ClFN2O2S) C, H, N, S.
2-Amino-4,5-difluorobenzenesulfonamide (17). 3,4-Di-
fluoroaniline (9, 8.7 g, 0.067 mol) was added portionwise to
chlorosulfonic acid (26 mL) cooled on an ice/water bath. The
mixture was refluxed for 90 min. After cooling, the mixture
was supplemented with thionyl chloride (10 mL) and refluxed
for another 90 min. The mixture was poured onto ice and ex-
tracted with AcOEt. The organic layer was dried over anhy-
drous MgSO4, and the solvent was removed under reduced
pressure. The crude residue of 2-amino-4,5-difluorobenzene-
sulfonyl chloride (13) was dissolved in dioxane (30 mL) and
added dropwise to a 10% w/v aqueous solution of ammonia
(100 mL). After the mixture was stirred for 30 min, concentra-
tion under reduced pressure to a small volume gave rise to a
precipitate of the title compound (57%). Mp 129-130 °C; IR
(KBr); 1H NMR (DMSO-d6, 400 MHz). Anal. (C6H6F2N2O2S)
C, H, N, S.
solvent was removed under reduced pressure, and the residue
was triturated with water. The resulting solution was adjusted
to pH 12 with 2.5 M NaOH, treated with charcoal, and filtered.
The filtrate was adjusted to pH 3-4 with concentrated HCl.
The precipitate that appeared was collected by filtration,
washed with water, and crystallized in acetone/n-hexane
(59%). Mp 261-264 °C; IR (KBr); 1H NMR (DMSO-d6, 400
MHz). Anal. (C10H6BrClN4O2S) C, H, N, S.
6-Chloro-7-fluoro-3-(1H-imidazol-1-yl)-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide Monohydrate (20). 2-Amino-4-
chloro-5-fluorobenzenesulfonamide (16, 4 g, 0.0178 mol) and
1,1′-thiocarbonyldiimidazole (8.84 g, 0.05 mol) were dissolved
in dioxane (60 mL). The mixture was heated under reflux for
3 h. The solvent was removed under reduced pressure, and
the residue was triturated with water. The resulting solution
was adjusted to pH 12 with 2.5 M NaOH, treated with
charcoal, and filtered. The filtrate was adjusted to pH 3-4
with concentrated HCl. The precipitate that appeared was
collected by filtration, washed with water, and dried (48%).
Mp 271-275 °C; IR (KBr); 1H NMR (DMSO-d6, 400 MHz).
Anal. (C10H6ClFN4O2S‚H2O) C, H, N, S.
6,7-Difluoro-3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (21). 2-Amino-4,5-difluorobenzenesulfona-
mide (17, 5.5 g, 0.026 mol) and 1,1′-thiocarbonyldiimidazole
(15 g, 0.084 mol) were dissolved in dioxane (50 mL). The
mixture was heated under reflux for 4 h. The solvent was
removed under reduced pressure, and the residue was tritu-
rated with water. The resulting solution was adjusted to pH
3-4 with 12 M HCl, and the precipitate that appeared was
collected by filtration, washed with water, dried, and crystal-
lized in acetone/diethyl ether (35%). Mp 264-266 °C; IR (KBr);
1H NMR (DMSO-d6, 400 MHz). Anal. (C10H6F2N4O2S) C, H,
N, S.
6,7-Dichloro-3-(ethylamino)-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (22). A mixture of 6,7-dichloro-3-(1H-
imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (18, 0.5
g, 0.001 57 mol) and a 70% w/v aqueous solution of ethylamine
(5 mL) was heated in a sealed vessel for 5 h at 150 °C. After
the mixture was cooled, the excess amine was eliminated by
distillation under reduced pressure. The residue was sus-
pended in water and stirred for 1 h. The precipitate was
collected by filtration, washed with water, dried, and crystal-
lized in MeOH/water (60%). Mp 308-310 °C (lit., 302-304
°C53); IR (KBr); 1H NMR (DMSO-d6, 400 MHz). Anal.
(C9H9Cl2N3O2S) C, H, N, S.
6,7-Dichloro-3-(propylamino)-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide Monohydrate (23). The title compound
was obtained as described for 22, starting from 6,7-dichloro-
3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (18,
0.5 g, 0.001 57 mol) and propylamine (5 mL). The final product
was crystallized in MeOH/water (65%). Mp >300 °C (lit., 314-
315 °C53); IR (KBr); 1H NMR (DMSO-d6, 400 MHz) Anal.
(C10H11Cl2N3O2S‚H2O) C, H, N, S.
6,7-Dichloro-3-(isopropylamino)-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide Monohydrate (24). The title compound
was obtained as described for 22 starting from 6,7-dichloro-
3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (18,
0.5 g, 0.001 57 mol) and isopropylamine (5 mL). The final
product was crystallized from hot MeOH (58%). Mp 296-298
°C (lit., 289-291 °C53); IR (KBr); 1H NMR (DMSO-d6, 80 MHz).
Anal. (C10H11Cl2N3O2S‚H2O) C, H, N, S.
3-Allylamino-6,7-dichloro-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (25). A mixture of 6,7-dichloro-3-(1H-imidazol-1-
yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (18, 0.5 g, 0.00157
mol) and allylamine (5 mL) was heated in a sealed vessel for
3 h at 140 °C. The excess amine was eliminated by dis-
tillation under reduced pressure. The residue was suspended
in water, and the pH was adjusted to 3-4 with concentrated
HCl. The precipitate was collected by filtration, washed
with water, dried, and crystallized in MeOH/water (29%). Mp
6,7-Dichloro-3-(1H-imidazol-1-yl)-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (18). The title compound was obtained
as described in the literature,46 starting from 2-amino-4,5-
dichlorobenzenesulfonamide (14).
7-Bromo-6-chloro-3-(1H-imidazol-1-yl)-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (19). 2-Amino-5-bromo-4-chlo-
robenzenesulfonamide (15, 0.2 g, 0.7 mmol) and 1,1′-thiocar-
bonyldiimidazole (0.45 g, 0.0025 mol) were dissolved in dioxane
(3 mL). The mixture was heated under reflux for 2 h. The
1
298-301 °C; IR (KBr); H NMR (DMSO-d6, 400 MHz). Anal.
(C10H9Cl2N3O2S) C, H, N, S.
3-Cyclopropylamino-6,7-dichloro-4H-1,2,4-benzothia-
diazine 1,1-dioxide (26). A mixture of 6,7-dichloro-3-(1H-

Benzothiadiazine Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4997
imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (18, 0.5
g, 0.001 57 mol) and cyclopropylamine (5 mL) was heated in a
sealed vessel for 5 h at 150 °C. The excess amine was
eliminated by distillation under reduced pressure. The residue
was suspended in water, and the pH was adjusted to 12 with
2.5 M NaOH. The solution was treated with charcoal and
filtered, and the pH of the filtrate was adjusted to pH
3-4 with concentrated HCl. The precipitate that appeared
was collected by filtration, washed with water, dried, and
crystallized in hot MeOH (62%). Mp 295-297 °C (lit., 282-
285 °C53); IR (KBr); 1H NMR (DMSO-d6, 400 MHz). Anal.
(C10H9Cl2N3O2S) C, H, N, S.
tained as described for 22, starting from 6-chloro-7-fluoro-3-
(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (20,
0.5 g, 0.001 67 mol) and isopropylamine (5 mL) (59%). Mp
228-232 °C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz). Anal.
(C10H11ClFN3O2S) C, H, N, S.
6-Chloro-3-(cyclobutylamino)-7-fluoro-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (35). The title compound was
obtained as described for 27, starting from 6-chloro-7-fluoro-
3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (20,
0.5 g, 0.001 67 mol) and cyclobutylamine (1 mL) in dioxane (5
1
mL) (55%). Mp >290 °C; IR (KBr); H NMR (DMSO-d6, 400
MHz). Anal. (C11H11ClFN3O2S) C, H, N, S.
3-Cyclobutylamino-6,7-dichloro-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (27). 6,7-Dichloro-3-(1H-imidazol-1-yl)-4H-
1,2,4-benzothiadiazine 1,1-dioxide (18, 0.5 g, 0.001 57 mol) was
dissolved in dioxane (4 mL) and supplemented with cyclobu-
tylamine (1 mL). The mixture was heated in a sealed vessel
for 8 h at 140 °C. The excess amine was eliminated by
distillation under reduced pressure. The residue was sus-
pended in water (20 mL), and the pH was adjusted to 12 with
2.5 M NaOH. The solution was treated with charcoal and
filtered, and the pH was adjusted to pH 3-4 with concen-
trated HCl. The precipitate was collected by filtration, washed
with water, dried, and crystallized in hot MeOH (60%). Mp
7-Fluoro-3,6-di(isopropylamino)-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (36). The title compound was obtained as
described for 22, starting from 6,7-difluoro-3-(1H-imidazol-1-
yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (21, 0.5 g, 0.001 76
mol) and isopropylamine (5 mL) (65%). Mp 234-237 °C; IR
(KBr); 1H NMR (DMSO-d6, 80 MHz). Anal. (C13H19FN4O2S)
C, H, N, S.
7-Fluoro-3,6-di(propylamino)-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (37). The title compound was obtained as
described for 22, starting from 6,7-difluoro-3-(1H-imidazol-1-
yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (21, 0.5 g, 0.001 76
mol) and n-propylamine (5 mL) (62%). Mp 236 °C, then
256 °C; IR (KBr); 1H NMR (DMSO-d6, 400 MHz). Anal.
(C13H19FN4O2S) C, H, N, S.
1
320-326 °C; IR (KBr); H NMR (DMSO-d6, 400 MHz). Anal.
(C11H11Cl2N3O2S) C, H, N, S.
6,7-Dichloro-3-(hexylamino)-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (28). A mixture of 6,7-dichloro-3-(1H-
imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (18, 0.5
g, 0.001 57 mol) and n-hexylamine (5 mL) was refluxed for 4
h. The excess amine was eliminated by distillation under
reduced pressure. The residue was suspended in water and
stirred for 1 h. The precipitate was collected by filtration,
washed with water, dried, and crystallized in hot MeOH (64%).
Mp 282-286 °C; IR (KBr); 1H NMR (DMSO-d6, 400 MHz).
Anal. (C13H17Cl2N3O2S) C, H, N, S.
3,6-Di(cyclobutylamino)-7-fluoro-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (38). The title compound was obtained
as described for 27, starting from 6,7-difluoro-3-(1H-imidazol-
1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (21, 0.5 g, 0.001 76
mol) and cyclobutylamine (1 mL) in dioxane (5 mL) (45%); IR
1
(KBr); H NMR (DMSO-d6, 400 MHz). Mp 258 °C, then 297
°C. Anal. (C15H19FN4O2S) C, H, N, S.
N-Ethyl-N′-(2-amino-4,5-difluorobenzenesulfonyl)thio-
urea (39). 2-Amino-4,5-difluorobenzenesulfonamide (17, 0.4
g, 0.0019 mol) was dissolved in dry acetone (3 mL) and
supplemented with K2CO3 (0.32 g) and ethyl isothiocyanate
(0.3 mL). The mixture was heated at 60 °C for 4 h. The solvent
was removed under reduced pressure, and the residue was
suspended in water (25 mL). The solution was adjusted to pH
3-4 with concentrated HCl and stirred at room temperature
for a few hours. The precipitate was collected by filtration,
washed with water, and dried. The crude product was used
without further purification (52%).
N-Isopropyl-N′-(2-amino-4,5-difluorobenzenesulfonyl)-
thiourea (40). The title compound was obtained as described
for 39, starting from 2-amino-4,5-difluorobenzenesulfonamide
(17) and isopropyl isothiocyanate. The crude product was used
without further purification (48%).
7-Bromo-6-chloro-3-(ethylamino)-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (29). The title compound was obtained
as described for 22, starting from 7-bromo-6-chloro-3-(1H-
imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (19, 0.5
g, 0.001 38 mol) and an aqueous solution of ethylamine (70%,
5 mL). The final product was crystallized from hot MeOH
1
(60%). Mp >290 °C; IR (KBr); H NMR (DMSO-d6, 80 MHz).
Anal. (C9H9BrClN3O2S) C, H, N, S.
7-Bromo-6-chloro-3-(isopropylamino)-4H-1,2,4-benzo-
thiadiazine 1,1-Dioxide (30). The title compound was ob-
tained as described for 22, starting from 7-bromo-6-chloro-3-
(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (19,
0.5 g, 0.001 38 mol) and isopropylamine (5 mL) (62%). IR
(KBr); 1H NMR (DMSO-d6, 400 MHz). Mp >290 °C. Anal.
(C10H11BrClN3O2S) C, H, N, S.
N-Isobutyl-N′-(2-amino-4,5-difluorobenzenesulfonyl)-
thiourea (41). The title compound was obtained as described
for 39, starting from 2-amino-4,5-difluorobenzenesulfonamide
(17) and isobutyl isothiocyanate. The crude product was used
without further purification (37%).
7-Bromo-6-chloro-3-(cyclobutylamino)-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (31). The title compound was
obtained as described for 27, starting from 7-bromo-6-chloro-
3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (19,
0.5 g, 0.001 38 mol) and cyclobutylamine (1 mL) in dioxane (5
6,7-Difluoro-3-(ethylamino)-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (42). N-Ethyl-N′-(2-amino-4,5-difluoroben-
zenesulfonyl)thiourea (39, 0.2 g, 0.68 mmol) and triethylamine
(0.2 mL) were dissolved in dry THF (5 mL). The mixture was
cooled in an ice/water bath and slowly supplemented with a
20% phosgene solution in toluene (0.5 mL). After 2 h at 0 °C,
the solvent was removed under reduced pressure and the
residue was triturated with water, adjusted to pH 12 with 2.5
M NaOH, treated with charcoal, and filtered. The filtrate was
adjusted to pH 3-4 with concentrated HCl, and the precipi-
tate that appeared was collected by filtration, washed with
1
mL) (58%). Mp >290 °C; IR (KBr); H NMR (DMSO-d6, 400
MHz). Anal. (C11H11BrClN3O2S) C, H, N, S.
6-Chloro-3-(ethylamino)-7-fluoro-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (32). The title compound was obtained
as described for 22, starting from 6-chloro-7-fluoro-3-(1H-
imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (20, 0.5
g, 0.001 67 mol) and a 70% w/v aqueous solution of ethylamine
(5 mL) (65%). Mp >290 °C; IR (KBr); 1H NMR (DMSO-d6, 400
MHz). Anal. (C9H9ClFN3O2S) C, H, N, S.
6-Chloro-7-fluoro-3-(propylamino)-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (33). The title compound was ob-
tained as described for 22, starting from 6-chloro-7-fluoro-
3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide
(20, 0.5 g, 0.001 67 mol) and propylamine (5 mL) (59%). Mp
1
water, and dried (75%). Mp 246-252 °C; IR (KBr); H NMR
(DMSO-d6, 400 MHz). Anal. (C9H9F2N3O2S) C, H, N, S.
6,7-Difluoro-3-isopropylamino-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (43). The title compound was obtained as
described for 42, starting from 2N-isopropyl-N′-(2-amino-4,5-
difluorobenzenesulfonyl)thiourea (40) (68%). Mp 242-244 °C;
IR (KBr); 1H NMR (DMSO-d6, 400 MHz). Anal. (C10H11F2N3O2S)
C, H, N, S.
1
287-289 °C; IR (KBr); H NMR (DMSO-d6, 400 MHz). Anal.
(C10H11ClFN3O2S) C, H, N, S.
6-Chloro-7-fluoro-3-(isopropylamino)-4H-1,2,4-benzo-
thiadiazine 1,1-Dioxide (34). The title compound was ob-

4998 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
de Tullio et al.
1
6,7-Difluoro-3-isobutylamino-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (44). The title compound was obtained as
described for 42, starting from 2N-isobutyl-N′-(2-amino-4,5-
difluorobenzenesulfonyl)thiourea (41) (65%). Mp >270 °C; IR
(KBr); 1H NMR (DMSO-d6, 400 MHz). Anal. (C11H13F2N3O2S)
C, H, N, S.
(KBr); H NMR (DMSO-d6, 80 MHz). Anal. (C11H14ClN3O3S)
C, H, N, S.
6-Chloro-3-cyclobutylamino-7-methoxy-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (51). The title compound was
obtained as described for 49, starting from 6-chloro-7-methoxy-
3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide mono-
hydrate (48) and cyclobutylamine (1 mL) in dioxane (5 mL)
(62%). Mp >284 °C; IR (KBr); 1H NMR (DMSO-d6, 400 MHz).
Anal. (C12H14ClN3O3S) C, H, N, S.
Biological Assays. Measurement of Insulin Release
from Incubated Rat Pancreatic Islets. Pancreatic islets
were isolated by the collagenase method from fed Wistar rats
(180-220 g). Groups of 10 islets, each derived from the same
batch of islets, were preincubated for 30 min at 37 °C in 1 mL
of a physiological salt medium (in mM: NaCl 115, KCl 5, CaCl2
2.56, MgCl2 1, NaHCO3 24), supplemented with 2.8 mM
glucose and 0.5% (w/v) dialyzed albumin (fracteom V, Sigma),
and equilibrated against a mixture of O2 (95%) and CO2 (5%).
The islets were then incubated at 37 °C for 90 min in 1 mL
of the same medium containing 16.7 mM glucose and the
reference compound or the benzothiadiazine derivative. The
release of insulin was measured radioimmunologically using
rat insulin as a standard.41
Residual insulin release was expressed as a percentage of
the value recorded in control experiments (100%), i.e., in the
absence of drug and presence of 16.7 mM glucose.
Measurement of the Contractile Activity in Rat Aorta.
All experiments were performed with aortae removed from fed
Wistar rats (180-220 g). A section of the aorta was cleared of
adhering fat and connective tissue and was cut into transverse
rings (3-4 mm long). The endothelium was removed by
rubbing the intimal surface with forceps. The segments were
suspended under 1.5 g of tension by means of steel hooks in
an organ bath containing 20 mL of a Krebs bicarbonate
buffered solution of the following composition (in mM): NaCl
118, KCl 4.7, CaCl2 2.5, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2,
glucose 5. The physiological solutions were maintained at 37
°C and bubbled continuously with a mixture of O2 (95%) and
CO2 (5%). The isometric contractions of the aortic rings were
measured with a force-displacement transducer. After 60 min
of equilibration, the rings were exposed to 30 mM KCl. When
the tension had stabilized, the drugs were added to the bath
at increasing concentrations until maximal relaxation (or until
0.3 mM). The relaxation response was expressed as the
percentage of the contractile response to KCl. The ED50 values
(drug concentration evoking 50% inhibition of the plateau
phase induced by KCl) were assessed from dose-response
curves using Datanalyst software (EMKA Technologies,
France).55
Measurement of 86Rb and 45Ca Outflow and Insulin
Release from Perifused Rat Pancreatic Islets. Experi-
ments were performed with pancreatic islets isolated from fed
Wistar rats (180-220 g). The media used for incubating,
washing, and perifusing the islets consisted of a physiological
salt medium (in mM: NaCl 115, KCl 5, CaCl2 2.56, MgCl2 1,
NaHCO3 24) supplemented with 0.5% (w/v) dialyzed albumin
(fraction V, Sigma) and gassed with O2 (95%)/CO2 (5%).
The methods used to measure 86Rb (42K substitute) outflow,
45Ca outflow, and insulin release from perifused pancreatic
islets have been described previously.22,51 Briefly, groups of 100
islets were incubated for 60 min in the physiological medium
containing 16.7 mM glucose and either 86 Rb (0.15-0.25 mM,
50 µCi/mL) or 45Ca (0.02-0.04 mM, 100 µCi/mL). After
incubation, the islets were washed four times with a nonra-
dioactive medium and then placed in a perifusion chamber.
The perifusate was delivered at a constant rate (1.0 mL/min).
From 31 to 90 min, the effluent was continuously collected over
successive periods of 1 min each. An aliquot of the effluent
(0.5 mL) was used for scintillation counting, while the remain-
der was stored at -20 °C for insulin radioimmunoassay. At
the end of the perifusion, the radioactive content of the islets
was also determined. The outflow of 86Rb or 45Ca (cpm/min)
was expressed as a fractional outflow rate (% of instantaneous
6-Chloro-7-methoxy-3-oxo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (46). Chlorosulfonyl isocyanate
(5 mL, 0.057 mol) and nitroethane (50 mL) were mixed
together in a closed, dried vessel. The mixture was cooled at
-40 °C (acetone and carbogene) and protected from moisture
during the slow addition, under vigorous stirring, of 3-chloro-
4-methoxyaniline (45, 4.8 g, 0.03 mol) dissolved in nitroethane
(10 mL). At the end of the addition, anhydrous AlCl3 (5 g,
0.0375 mol) was added to the resulting suspension and the
mixture was heated at 110 °C for 20 min. The hot solution
was poured onto ice (200 g), and after the mixture was stirred
and after complete melting of the ice, the resulting precipitate
was collected by filtration and dissolved in an aqueous solution
of sodium hydrogenocarbonate (5 g/150 mL). The solution was
treated with charcoal and filtered, and the filtrate was
adjusted to pH 1 by means of 12 N HCl. The white precipitate
that appeared was collected by filtration, washed with
1
water, and dried (28%). Mp 277-279 °C; IR (KBr); H NMR
(DMSO-d6, 400 MHz). Anal. (C8H7ClN2O4S) C, H, N, S.
6-Chloro-7-methoxy-3-thioxo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (47). A mixture of 6-chloro-7-
methoxy-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-di-
oxide (46, 2.62 g, 0.01 mol) and phosphorus pentasulfide (4.5
g) in anhydrous pyridine (25 mL) was refluxed for 3 h. The
solvent was removed under reduced pressure, and the residue
was dissolved in an aqueous solution of NaOH (5 g/100 mL).
The solution was treated with charcoal and filtered, and the
filtrate was adjusted to pH 1 by means of 12 N HCl. The
precipitate was collected by filtration, washed with water, and
suspended in an aqueous solution of sodium hydrogenocar-
bonate (3 g/100 mL). The suspension was heated until most
of the insoluble material dissolved and was then treated with
charcoal and filtered. The filtrate was adjusted to pH 1 with
12 N HCl, and the precipitate was collected by filtration,
1
washed with water, and dried. Mp 233-235 °C; IR (KBr); H
NMR (DMSO-d6, 400 MHz). Anal. (C8H7ClN2O3S2) C, H, N, S.
6-Chloro-7-methoxy-3-methylsulfanyl-4H-1,2,4-benzo-
thiadiazine 1,1-Dioxide Monohydrate (48). 6-Chloro-7-
methoxy-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxide (47, 0.47 g, 0.0017 mol) was dissolved in a hydrometh-
anolic 1:1 solution of sodium hydrogenocarbonate (1 g/10 mL),
and an excess of methyl iodide (1.8 mL) was added. After 30
min of being stirred, the resulting suspension was adjusted
to pH 5-6 by means of 6 N HCl. The suspension was
concentrated under reduced pressure to half of the volume,
and the white precipitate was collected by filtration, washed
with water, and dried. Mp 271-275 °C; IR (KBr); 1H NMR
(DMSO-d6, 400 MHz). Anal. (C9H9ClN2O3S2‚H2O) C, H, N, S.
6-Chloro-3-(ethylamino)-7-methoxy-4H-1,2,4-benzo-
thiadiazine 1,1-Dioxide (49). A mixture of 6-chloro-7-meth-
oxy-3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide
monohydrate (48, 0.5 g,0.017 mol) and a 70% w/v aqueous
solution of ethylamine (5 mL) was heated in a sealed vessel
for 4 h at 120 °C. The excess amine was eliminated by
distillation under reduced pressure, and the residue was
dissolved in an aqueous 2% w/v solution of NaOH (20 mL).
The alkaline solution was treated with charcoal and was
filtered, and the filtrate was adjusted to pH 4-5 with
concentrated HCl. The precipitate was collected by filtration,
washed with water, and dried. The compound was recrystal-
lized in methanol/water (75%). Mp >300 °C; IR (KBr); 1H NMR
(DMSO-d6, 400 MHz). Anal. (C10H12ClN3O3S) C, H, N, S.
6-Chloro-3-isopropylamino-7-methoxy-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (50). The title compound was
obtained as described for 49, starting from 6-chloro-7-methoxy-
3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide mono-
hydrate (48) and isopropylamine (70%). Mp 281-283 °C; IR

Benzothiadiazine Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4999
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A transmembrane domain of the sulfonylurea receptor mediates
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islet content/min, FOR). Some media contained no CaCl2 and
were enriched with 0.5 mM EGTA (Sigma).
When high concentrations (50 mM) of extracellular K+ were
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keep the osmolarity constant.
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Results are expressed as the mean ((SEM) together with
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perifused in the presence of 16.7 mM glucose was taken as
the difference between the mean value for 45Ca outflow or
insulin output recorded in each individual experiment between
the 40-44 and 60-68 min of perifusion.
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The magnitude of the increase in 45Ca outflow was estimated
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45Ca observed during stimulation (45-68 min) after correction
for the basal value (40-44 min).
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Acknowledgment. This study was supported by
grants from the National Fund for Scientific Research
(F.N.R.S., Belgium) from which P. de Tullio is Research
Associate and P. Lebrun is Research Director. The
authors gratefully acknowledge the technical assistance
of D. Dewalque, M. P. Fraikin, J. Sergooris, and A. Van
Praet.
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Clayton, P.; McCloy, R.; De Lonlay, P.; Nihoul-Fe´ke´te´, C.; Robert,
J.; Saudubray, J.-M.; Rahier, J.; Lindley, K.; Hussain, K.;
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Supporting Information Available: Elemental analysis
results and IR and 1H NMR spectra of the newly synthesized
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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