Toward tissue-selective pancreatic B-cells KATP channel openers belonging to 3-alkylamino-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides

Article abstract of DOI:10.1021/jm021117w

3-(Alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides were synthesized, and their activity on rat-insulin-secreting cells and rat aorta rings was compared to that of the K_ATP channel activators diazoxide and pinacidil. Structure-activity relationships indicated that an improved potency and selectivity for the pancreatic tissue was obtained by introducing a fluorine atom in the 7-position and a short linear (preferably ethyl) or cyclic (preferably cyclobutyl) hydrocarbon chain on the nitrogen atom in the 3-position. By contrast, strong myorelaxant activity was gained by the introduction of a halogen atom different from the fluorine atom in the 7-position and a bulky branched alkylamino chain in the 3-position. Thus, 3-(ethylamino)-7-fluoro-4H-1,2,4-benzothiadiazine 1,1-dioxide (11) expressed a marked inhibitory activity on pancreatic B-cells (IC₅₀ = 1 μM) associated with a weak vasorelaxant effect (ED₅₀ > 300 μM), whereas 7-chloro-3-(1,1-dimethylpropyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide (27), which was only slightly active on insulin-secreting cells (IC₅₀ > 10 μM), was found to be very potent on vascular smooth muscle cells (ED₅₀ = 0.29 μM). Radioisotopic and electrophysiological investigations performed with 7-chlorinated, 7-iodinated, and 7-fluorinated 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides confirmed that the drugs activated K_ATP channels. The present data revealed that subtle structural modifications of 3-(alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides can generate original compounds activating K_ATP channels and exhibiting different in vitro tissue selectivity profiles.

Full text of DOI:10.1021/jm021117w

3342
J . Med. Chem. 2003, 46, 3342-3353
Tow a r d Tissu e-Selective P a n cr ea tic B-Cells KATP Ch a n n el Op en er s Belon gin g to
3-Alk yla m in o-7-h a lo-4H-1,2,4-ben zoth ia d ia zin e 1,1-Dioxid es
Pascal de Tullio,† Be´ne´dicte Becker,# Ste´phane Boverie,† Michael Dabrowski,‡ Philip Wahl,§
Marie-He´le`ne Antoine,# Fabian Somers,† Sophie Sebille,† Raogo Ouedraogo,# J ohn Bondo Hansen,§
Philippe Lebrun,# and Bernard Pirotte*,†
Centre de Recherche en Pharmacochimie des Substances Naturelles et Synthe´tiques, Laboratoire de Chimie Pharmaceutique,
Universite´ de Lie`ge, 1, Avenue de l’Hoˆpital, CHU, Tour 4, B-4000 Lie`ge, Belgium, University Laboratory of Physiology,
Parks Road, Oxford, OX1 3PT, U.K., Laboratoire de Pharmacodynamie et de The´rapeutique, Universite´ Libre de Bruxelles,
808, Route de Lennik, B-1070 Bruxelles, Belgium, and Discovery, Novo Nordisk A/ S, Novo Nordisk Park,
DK-2760 Malov, Denmark
Received December 16, 2002
3-(Alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides were synthesized, and their
activity on rat-insulin-secreting cells and rat aorta rings was compared to that of the KATP
channel activators diazoxide and pinacidil. Structure-activity relationships indicated that an
improved potency and selectivity for the pancreatic tissue was obtained by introducing a fluorine
atom in the 7-position and a short linear (preferably ethyl) or cyclic (preferably cyclobutyl)
hydrocarbon chain on the nitrogen atom in the 3-position. By contrast, strong myorelaxant
activity was gained by the introduction of a halogen atom different from the fluorine atom in
the 7-position and a bulky branched alkylamino chain in the 3-position. Thus, 3-(ethylamino)-
7-fluoro-4H-1,2,4-benzothiadiazine 1,1-dioxide (11) expressed a marked inhibitory activity on
pancreatic B-cells (IC50 ) 1 µM) associated with a weak vasorelaxant effect (ED50 > 300 µM),
whereas 7-chloro-3-(1,1-dimethylpropyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide (27), which
was only slightly active on insulin-secreting cells (IC50 > 10 µM), was found to be very potent
on vascular smooth muscle cells (ED50 ) 0.29 µM). Radioisotopic and electrophysiological
investigations performed with 7-chlorinated, 7-iodinated, and 7-fluorinated 3-alkylamino-4H-
1,2,4-benzothiadiazine 1,1-dioxides confirmed that the drugs activated KATP channels. The
present data revealed that subtle structural modifications of 3-(alkylamino)-7-halo-4H-1,2,4-
benzothiadiazine 1,1-dioxides can generate original compounds activating KATP channels and
exhibiting different in vitro tissue selectivity profiles.
In tr od u ction
Kir6.2 or Kir6.1 combinations form the cardiac type and
the smooth muscle type KATP channels, respectively.6
Potassium channels sensitive to intracellular levels
of adenosine triphosphate (ATP-sensitive K+ channels
or KATP channels), which link the membrane potential
to the metabolic state of the cell, have been described
in a wide range of cell types including pancreatic B-cells1
and smooth muscle cells.2 In pancreatic B-cells (insulin-
secreting cells), these channels have been shown to be
involved in the insulin secretory process.3,4 In the
vascular tissue, the KATP channels play an important
role in controlling muscle tone and contractility.5
Recent progress in molecular biology indicated that
KATP channels consist of octameric complexes of two
unrelated subunits: a pore-forming subunit (Kir6.x) and
a regulatory subunit, the sulfonylurea receptor (SURx).6
According to their tissue localization, KATP channels
exist in different isoforms resulting from the assembly
of the SUR and Kir subunits in multiple combinations.
The combination of the SUR1 with the Kir6.2 subunits
(SUR1/Kir6.2) forms the pancreatic B-cell type KATP
channels, whereas the SUR2A/Kir6.2 and the SUR2B/
A variety of compounds have been reported to activate
KATP channels. Those drugs, named potassium channel
openers/activators (or PCOs), are potentially of great
value as new therapeutic agents.7 Indeed, pancreatic
B-cells KATP channel activators could find a considerable
therapeutic interest in the prevention or treatment
of several diseased states involving altered endo-
crine pancreatic function, such as type 1 and type 2
diabetes.8-10 However, because of the ubiquitous dis-
tribution of KATP channels, and in order to reduce the
side effects of PCOs, the development of novel com-
pounds should be linked to a high selectivity for a single
channel subtype. Although the KATP channel activator
diazoxide was found to preserve endogenous insulin
production in patients with newly manifested autoim-
mune diabetes8 and was found to reduce body fat in
hyperinsulinaemic obese adults,10 the drug was also
responsible for marked side effects such as hypertri-
chosis, oedema, headache, and hypotension as a result
of its lack of tissue selectivity. Those side effects
considerably limit the interest of diazoxide in clinical
practice and encourage the development of pancreatic
B-cells selective PCOs for such therapeutic indications.
* To whom correspondence should be addressed. Phone: +32 4 366
43 65. Fax: +32 4 366 43 62. E-mail: B.Pirotte@ulg.ac.be.
† Universite´ de Lie`ge.
#
Universite´ Libre de Bruxelles.
We recently reported that 3-(alkylamino)-4H-pyrido-
[4,3-e]-1,2,4-thiadiazine 1,1-dioxides such as BPDZ 44
‡ University Laboratory of Physiology.
§ Novo Nordisk A/S.
10.1021/jm021117w CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/19/2003

Benzothiadiazine Dioxides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3343
F igu r e 1. Chemical structure of diazoxide and several analogues described as potassium channel openers: BPDZ 44, BPDZ 83,
BPDZ 73 and its quinazolinone isostere (5) in two possible tautomeric forms.
(2), structurally related to the KATP channel activator
diazoxide (1) (Figure 1), were powerful inhibitors of
insulin release from rat pancreatic B-cells.11,12 Com-
investigations were performed in order to elucidate the
mechanism of action of the newly synthesized com-
pounds.
pound 2 was identified as a pancreatic B-cell KATP
13,14
Ch em istr y
channel activator
and was also found to be far less
potent than diazoxide at relaxing smooth muscle tis-
3-(Alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-
dioxides (10-25, 28-46) were obtained in four steps
starting from the appropriate para halo-substituted
aniline (Scheme 1). The first step was the synthesis of
7-halo-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxides (7a -d ) from the reaction of different anilines
(6a -d ) with chlorosulfonyl isocyanate, according to a
previously described procedure.19 The 3-oxo compounds
(7a -d ) were converted into the corresponding 3-thioxo
derivatives (8a -d ) by means of phosphorus pentasulfide
in pyridine, as previously described.8 7-Halo-3-thioxo-
3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides (8a -
d ) reacted with methyl iodide in a hydromethanolic
solution of sodium bicarbonate to give the corresponding
7-halo-3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-
dioxides (9a -d ). The 3-methylsulfanyl-substituted in-
termediates (9a -d ) were heated in a sealed vessel with
an excess of the appropriate alkyl/cycloalkylamine to
give the desired 3-(alkylamino)-7-halo-4H-1,2,4-
benzothiadiazine 1,1-dioxides (10-25, 28-46).
sue.12,15
In the search for a novel series of pyridothiadiazine
dioxides acting as PCOs, we also developed 3-alkyl-
amino-7-chloro-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1,1-
16
dioxides such as BPDZ 83 (3)
(Figure 1). These
compounds may be regarded as isomers of 3-alkylamino-
4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides (“7-aza”
compounds) bearing the nitrogen atom of the pyridine
ring in the 5- instead of the 7-position of the heterocycle
(“5-aza” compounds) and bearing a chlorine atom in the
7-position such as the PCO prototype diazoxide (7-
chlorobenzothiadiazine dioxide) (1, Figure 1). Compound
3 was found to be much more potent as a vasodilator
than as an inhibitor of insulin secretion, revealing that
this “5-aza” (3) and related compounds exhibited an
opposite tissue selectivity.16 Moreover, further investi-
gations indicated that compound 3 expressed, on the
vascular tissue, the pharmacological profile of a typical
KATP channel activator.16
Recently, we have prepared 7-chloro-3-isopropylamino-
4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 73) (4) and
its 7-methoxy analogue, 7-methoxy-3-isopropylamino-
4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 216), which
were revealed to be potent and quite tissue-selective
pancreatic B-cell KATP channel activators.8,17 In addition,
we also synthesized quinazolinone isosteres of BPDZ 73
(for example, compound 5). The latter compounds lacked
tissue selectivity and were active on pancreatic and
vascular tissues.18
The present work aimed at developing new analogues
of BPDZ 73 (4) by varying the nature of the halogen
atom in the 7-position and the size as well as the
branching of the alkylamino chain in the 3-position.
Particular attention was paid to the identification of
structural requirements leading to improvement of
potency and pancreatic B-cell selectivity. The new
compounds were examined as putative potassium chan-
nel openers on rat pancreatic islets (inhibition of
glucose-induced insulin release) as well as on rat aorta
(myorelaxant effect on 30 mM KCl-precontracted aorta
rings). From the biological data collected, tissue selec-
tivity linked to structure-activity relationships was
discussed. Finally, radioisotopic and electrophysiological
Compounds with a more bulky alkylamino group in
the 3-position, such as 7-chloro-3-(tert-butylamino)-4H-
1,2,4-benzothiadiazine 1,1-dioxide (26) and 7-chloro-3-
(1,1-dimethylpropylamino)-4H-1,2,4-benzothiadiazine 1,1-
dioxide (27), could not be obtained from the reaction
between 9b and the amine because of the steric hin-
drance of the alkyl moiety and because of the poor
reactivity of the 3-methylsulfanyl intermediate. Reactiv-
ity toward nucleophilic substitution was improved by
converting 7-chloro-3-methylsulfanyl-4H-1,2,4-benzo-
thiadiazine 1,1-dioxide (9b) into the corresponding
3-methylsulfinyl intermediate (49),20 which reacted with
the bulky alkylamines to give the expected final com-
pounds (26, 27) (Scheme 2).
Last, 7-chloro-3-(isopropylamino)-4-methyl-4H-1,2,4-
benzothiadiazine 1,1-dioxide (48) (or “4-methyl-substi-
tuted BPDZ 73”) was obtained from the reaction of the
4-methyl-substituted 3-methylsulfanyl intermediate (47)
with isopropylamine.
Resu lts a n d Discu ssion
Biological results revealed that most compounds were
powerful inhibitors of the glucose-induced insulin re-

3344 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
de Tullio et al.
Sch em e 1a
a
(i) ClSO2NCO, CH3NO2, AlCl3; (ii) P2S5, pyridine, ∆; (iii) NaHCO3, CH3I, CH3OH/H2O; (iv) RNH2, ∆.
Sch em e 2a
a
(i) K2CO3, CH3I, CH3CN/DMF; (ii) (CH3)2CHNH2, ∆; (iii) Na2CO3, Br2, H2O; (iv) RNH2, ∆.
lease from incubated rat pancreatic islets (see Tables
1-4), some of them (22, 34, 36) being 25-50 times more
potent than the reference compound diazoxide. Except
for the 7-fluoro-substituted compounds (Table 1), the
size and the branching of the alkyl chain introduced on
the exocyclic nitrogen atom in the 3-position markedly
affected the activity on insulin-secreting cells. Thus, for
the 7-chloro- (Table 2), 7-bromo- (Table 3), and 7-iodo-
substituted (Table 4) benzothiadiazine dioxides, the
inhibitory activity on the insulin-releasing process
decreased with an increase in the size of the hydro-
carbon chain. More precisely, the rank order of potency
appeared to be the following: methyl < ethyl e iso-
propyl > propyl g sec-butyl > 1,2-dimethylpropyl. The
most potent drugs had a short linear or branched
hydrocarbon chain such as ethyl or isopropyl (see 22,
4, 34, 36, and 42). For the 7-fluoro-substituted deriva-
tives, the influence of the size and the branching of the
alkyl chain was less pronounced (see 10-15) although
the best choice for the hydrocarbon moiety was again
ethyl or isopropyl (see 11 and 13). Bulky hydrocarbon
chains such as tert-butyl and 1,1-dimethylpropyl (qua-
ternary carbon atoms) were responsible for a marked
decrease of activity on B-cells (compare 26 with 4 and
27 with 24).
than with the cyclopropyl moiety (compare 17, 30, and
40 with 16, 29, and 39). The activity of the 3-cyclobu-
tylamino-substituted derivatives was close to that of the
ethylamino- and isopropylamino-substituted compounds
in their respective series of 7-halobenzothiadiazine
dioxides.
The nature of the halogen atom in the 7-position was
found to have little influence on the activity on B-cells,
although 7-iodo-substituted compounds were less potent
than the 7-fluoro-, 7-chloro-, and 7-bromo-substituted
drugs.
The myorelaxant activity of the benzothiadiazine
dioxides was affected by the nature of the hydrocarbon
chain on the exocyclic nitrogen atom in the 3-position
but also by the nature of the halogen atom in the
7-position.
In marked contrast to the biological effects on B-cells,
a more pronounced myorelaxant activity was observed
when the size and the branching of the hydrocarbon
chain in the 3-position were increased (see compounds
10-15 in Table 1, compounds 21-27 in Table 2,
compounds 33-38 in Table 3, and compounds 42-44
in Table 4). Thus, whatever the nature of the halogen
atom in the 7-position, the weaker vasorelaxing effect
was obtained in each series with a methylamino sub-
stituent in the 3-position (see 10, 21, and 33), whereas
the stronger myorelaxant potency was observed with a
When cycloalkyl groups were present, the most potent
compounds were obtained with the cyclobutyl rather

Benzothiadiazine Dioxides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3345
Ta ble 1. Effects of 3-Alkylamino-7-fluoro-4H-1,2,4-benzothiadiazine 1,1-Dioxides on Insulin Secretion from Rat Pancreatic Islets and
on Contractile Activity of K+-Depolarized Rat Aorta Rings
a
% RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ( SEM
(n)). IC50: drug concentration giving 50% inhibition of insulin release (estimated value). c ED50: drug concentration giving 50% relaxation
b
d
of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n)). ED50/IC50
:
estimated selectivity ratio. e Cyclopropyl.
f Cyclobutyl. Cyclopentyl. Cyclopropylmethyl. Not determined.
g
h
i
Ta ble 2. Effects of 3-Alkylamino-7-chloro-4H-1,2,4-benzothiadiazine 1,1-Dioxides on Insulin Secretion from Rat Pancreatic Islets and
on Contractile Activity of K+-Depolarized Rat Aorta Rings
a
% RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ( SEM
(n)). IC50: drug concentration giving 50% inhibition of insulin release (estimated value). c ED50: drug concentration giving 50% relaxation
b
d
of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n)). ED50/IC50
:
estimated selectivity ratio. e Cyclopropyl.
f Cyclobutyl. Cyclopropylmethyl. Published results (ref 8). Not determined.
g
h
i
1,2-dimethylpropylamino (see 15, 25, 38, and 44), a tert-
butylamino (see 26), or a 1,1-dimethylpropylamino (see
27) group. As a result, compound 27 with an ED50 value
below 0.5 µM was found to be as potent as the reference
PCO pinacidil. Such an observation is in accordance
with the structure-activity relationships previously

3346 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
de Tullio et al.
Ta ble 3. Effects of 3-Alkylamino-7-bromo-4H-1,2,4-benzothiadiazine 1,1-Dioxides on Insulin Secretion from Rat Pancreatic Islets and
on Contractile Activity of K+-Depolarized Rat Aorta Rings
a
% RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ( SEM
(n)). IC50: drug concentration giving 50% inhibition of insulin release (estimated value). c ED50: drug concentration giving 50% relaxation
b
d
of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n)). ED50/IC50
:
estimated selectivity ratio. e Cyclopropyl.
f Cyclobutyl. Not determined.
g
Ta ble 4. Effects of 3-Alkylamino-7-iodo-4H-1,2,4-benzothiadiazine 1,1-Dioxides on Insulin Secretion from Rat Pancreatic Islets and
on Contractile Activity of K+-Depolarized Rat Aorta Rings
a
% RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ( SEM
(n)). IC50: drug concentration giving 50% inhibition of insulin release (estimated value). c ED50: drug concentration giving 50% relaxation
b
of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n)). ED50/IC50: estimated selectivity ratio. e Cyclobutyl. f Not
d
determined.
established for pinacidil analogues for which the 1,2,2-
trimethylpropyl and the 1,1-dimethylpropyl side chains
were recognized as the most interesting hydrocarbon
chains for improving myorelaxant activity.21,22
The nature of the halogen atom in the 7-position also
strongly affected the activity on the vascular tissue. The
introduction of a fluorine atom in the 7-position de-
creased the myorelaxant activity of benzothiadiazine
dioxides. The rank order of potency was defined as
follows: 7-fluoro compounds < 7-chloro compounds e
7-bromo compounds e 7-iodo compounds.
The introduction of a short cycloalkylamino group in
the 3-position provoked a marked decrease of activity
in rat aorta rings. The cyclopropyl derivatives (see 16,
29, and 39) were systematically found to be less active
than their corresponding cyclobutyl analogues (see 17,
30, and 40).
An ED50/IC50 ratio can be calculated in order to assess
the tissue selectivity (vascular versus pancreatic tissue)
of the new compounds (see Tables 1-4). Diazoxide,

Benzothiadiazine Dioxides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3347
which is known to be poorly selective,12 was found to
exhibit a selectivity ratio of about 1 (see Tables 1-4),
indicating that the compound was almost equipotent on
both tissues. Not surprisingly, pinacidil was found to
be clearly selective for the vascular tissue with a
selectivity ratio lower than 0.005 (see Tables 1-4).
The 7-fluoro-substituted benzothiadiazine dioxides
(see Table 1) may be regarded as the most interesting
series of drugs in terms of tissue selectivity. These
compounds were very potent as inhibitors of the insulin-
releasing process (all drugs were more potent than
diazoxide), while their myorelaxant activity was weak
(all drugs were less potent than diazoxide). In the linear
hydrocarbon side chain series, the 7-methylamino- and
the 7-(ethylamino)-substituted derivatives 10 and 11
markedly inhibited insulin release (IC50 ) 2.2 and 1.0
µM, respectively) and were found to be essentially
inactive as myorelaxants (ED50 > 300 µM). As a result,
these drugs were at least 135- to 300-fold more potent
on B-cells than on vascular smooth muscle cells. Like-
wise, the cyclobutyl derivative 17 exhibited a pro-
nounced activity on pancreatic B-cells (IC50 ) 1.3 µM)
associated with a weak myorelaxant activity on vascular
smooth muscle cells (ED50 ) 72 µM). Although the allyl
derivative 20 also expressed a marked tissue selectivity,
its efficiency on the pancreatic tissue was less pro-
nounced (IC50 ) 2.7 µM).
As expected, the selectivity of the 7-fluoro-substituted
compounds for the pancreatic versus the vascular
smooth muscle tissue decreased with an increase in the
size and the branching of the hydrocarbon side chain
in the 3-position (see selectivity ratio in Table 1). Such
an observation can also be made for the other series of
7-halo-substituted benzothiadiazine dioxides (see Tables
2-4). The selectivity ratio of the 7-chloro-substituted
compounds was between 129 for the methylamino
derivative 21 and <0.03 for the 1,1-dimethylpropy-
lamino derivative 27. With a selectivity ratio greater
than 40 and an IC50 value on pancreatic B-cells near or
below 1 µM, compounds 21 (methyl), 22 (ethyl), and 4
(isopropyl) are examples of 7-chloro compounds express-
ing an attractive pancreatic B-cell selectivity and po-
tency. In this series of chlorinated derivatives, com-
pound 27, with a bulky quaternary carbon atom located
on the 3-alkylamino side chain, was the most potent and
vascular smooth muscle selective drug.
F igu r e 2. Effect of compound 42 (50 µM) on 86Rb outflow from
rat pancreatic islets perifused in the absence (b) and presence
(O) of glibenclamide (10 µM) throughout. Mean values ((SEM)
refer to four individual experiments.
presence of a hydrogen atom in the 4-position of the
heterocycle.
Taken as a whole, the present pharmacological data
give important insights into the structural requirements
leading to potency and tissue selectivity (pancreatic
versus vascular smooth muscle). Looking at the nature
of the halogen atom in the 7-position and the alkylamino
side chain in the 3-position, selectivity for the pancreatic
tissue is obtained by introducing a fluorine atom in the
7-position and a short linear (preferably ethyl) or cyclic
(preferably cyclobutyl) hydrocarbon chain on the nitro-
gen atom in the 3-position. An improvement in myore-
laxant activity is gained by the introduction of a halogen
atom different from the fluorine atom in the 7-position
and a bulky branched alkylamino chain in the 3-posi-
tion. The presence of a hydrogen atom in the 4-position
is also required for biological activity.
Chlorinated, iodinated, and fluorinated compounds
exhibiting a marked inhibitory effect on the insulin
secretory rate were selected for further pharmacological
investigations. The aim of these studies was to ascertain
that the biological effects of the drugs (and their
analogues) on pancreatic B-cells were related to KATP
channel activation.
In the series of 7-bromo- and 7-iodo-substituted ben-
zothiadiazine dioxides, as a result of their more pro-
nounced myorelaxant activity, only a few drugs ex-
pressed selectivity for the pancreatic tissue (see Tables
3 and 4). However, the cyclobutylamino side chain in
the 3-position again conferred a marked effect on B-cells
associated with a weak activity on aorta rings (see
compound 40).
In the first series of experiments, using the efflux of
86Rb (42K substitute) as an index of K+ permeabil-
ity,8,13,14,23 we have characterized the effects of com-
pounds 4, 24, and 42 on 86Rb outflow from prelabeled
and perifused rat pancreatic islets. Figure 2 illustrates
the effects of compound 42, but all three drugs provoked
86
The introduction of a methyl group in the 4-position
of compound 4 (leading to compound 48) was responsible
for a complete loss of activity on pancreatic B-cells and
on vascular smooth muscle cells [% RIS at 50 µM is 96.5
( 5.1% (n ) 14); ED50 on aorta rings is >100 µM (n )
4)]. Such a feature is in accordance with our previous
data reporting the effect of 3-(alkylamino)-4-methyl-4H-
pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides12 and reflects
the critical importance, for biological activity, of the
a pronounced and sustained increase in the rate of
-
Rb outflow. When the same experiments were repeated
in the presence of the KATP channel blocker glibencla-
mide in the perifusing medium, the stimulatory effects
of compound 4, 24, and 42 were completely abolished
(Figure 2 and data not shown). Such data suggest that
the drugs activated KATP channels in rat islet cells.
In a second set of experiments, we investigated the
effect of 18 (BPDZ 176) on KATP channels heterologously

3348 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
de Tullio et al.
F igu r e 3. Effects of BPDZ 176 (18) on SUR1/Kir6.2, SUR2A/Kir6.2, SUR2B/Kir6.2 or Kir6.2∆C36 currents. Macroscopic currents
were recorded from inside-out patches in response to a series of voltage ramps from -110 to +100 mV from oocytes coexpressing
Kir6.2 and either SUR1 (A) or SUR2A (B). 100 µM MgATP and 100 µM BPDZ 176 were added to the intracellular solution as
indicated by the bars. (C) Mean macroscopic slope conductances (G) in the presence of 100 µM MgATP (white columns) or 100 µM
BPDZ 176 and 100 µM MgATP (black columns) in excised patches from oocytes expressing either Kir6.2 and SUR1, SUR2A,
SUR2B, or Kir6.2∆C36 alone. Data are expressed as a fraction of the mean slope conductance in control solution (no additions)
(GC). The vertical bars indicate 1 SEM (n ) 3-6 in each column).
expressed in Xenopus oocytes. Figure 3 shows that the
compound activated the pancreatic B-cell type KATP
channel (Kir6.2/SUR1) but not the cardiac (Kir6.2/
SUR2A) or the smooth muscle (Kir6.2/SUR2B) type. The
mean data are presented in Figure 3C showing that, in
the presence of 100 µM MgATP, 100 µM BPDZ 176 only
activated SUR1 comprising KATP channels. A small
inhibition on SUR2A or SUR2B comprising KATP chan-
nels was also observed, suggesting that the compound
interacted with SUR2A and SUR2B without activating
any currents. Moreover, the effects seen on the KATP
channels expressed in oocytes were likely to be mediated
by the SUR rather than the Kir6.2 subunits because
compound 18 had no effect on Kir6.2 expressed in the
absence of SUR (Figure 3C).
As performed with 3-(alkylamino)-4H-pyrido[4,3-e]-
1,2,4-thiadiazine 1,1-dioxides such as 212 and 3-(alkyl-
amino)-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1,1-dioxides
such as 3,16 the pKa value of representative 7-halo-
substituted 3-(alkylamino)-4H-1,2,4-benzothiadiazine
1,1-dioxides was determined in order to predict the
ionization state of the drugs at the physiological pH of
7.4. By analogy with pyridothiadiazine dioxides, the
acidic character of the benzothiadiazine dioxides should
also be linked to the lability of the hydrogen atom in
the 4-position of the heterocycle. Previous studies on
pyridothiadiazine dioxides indicated that the active form
of the drugs in aqueous solution was the nonionized
species and that a partial ionization of the drugs at
physiological pH could be responsible for a loss of
biological activity.12,16
of 7-halobenzothiadiazine dioxides appears to be cor-
related with the electronic impact of the substituent in
the 7-position (para position relative to the nitrogen
atom bearing the labile proton), as reflected by its
Hammett σp constant ( σp ) 0.15, 0.24, 0.26, and 0.28
for the fluorine, the chlorine, the bromine, and the
iodine atoms, respectively).25 Taking into account their
pKa values, the 7-halo-substituted benzothiadiazine
dioxides are expected to be present as nonionized species
in aqueous solution at physiological pH.
Crystallographic studies conducted with typical ex-
amples of 3-alkylaminopyridothiadiazine dioxides12,16,26
led to the conclusion that those compounds, whatever
the position of the nitrogen atom in the pyridine ring,
adopted in the solid state a 4H-tautomeric form. In the
latter form, the two N-H groups of the “guanidine”
moiety were in the same spatial configuration as the
guanidinic N-H groups of pinacidil. X-ray data previ-
ously obtained with the 3-isopropylamino-substituted
benzothiadiazine 4227 suggested that in the solid state
3-(alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-
dioxides also adopted the same 4H-tautomeric confor-
mation and the same spatial disposition of the guani-
dinic N-H groups as 3-(alkylamino)-4H-1,2,4-pyrido-
thiadiazine 1,1-dioxides.
Con clu sion s
Several 3-(alkylamino)-7-halo-4H-1,2,4-benzothiadi-
azine 1,1-dioxides were synthesized and examined as
putative PCOs on two different tissues: rat pancreatic
B-cells and rat aorta rings. The nature of the alkylamino
side chain in the 3-position and the nature of the
halogen atom in the 7-position were found to consider-
ably affect both the potency and the in vitro tissue
selectivity. Selectivity for the pancreatic tissue was
obtained by introducing a fluorine atom in the 7-position
and a short linear (preferably ethyl) or cyclic (preferably
cyclobutyl) hydrocarbon chain on the nitrogen atom in
the 3-position. By contrast, strong myorelaxant activity
was reached after the introduction of a halogen atom
different from the fluorine atom in the 7-position and
the addition of a bulky branched alkylamino chain in
the 3-position.
The pKa values of 13, 4, 36, and 42 (the 3-isopropyl-
amino-substituted derivatives in each series of 7-
halobenzothiadiazine dioxides) determined by UV spec-
trophotometry amounted to 9.63, 9.51, 9.41, and 9.28,
respectively. By comparison, the pKa value of diazoxide
has been reported to be 8.62.24 As observed with
pyridothiadiazine dioxides,12,16 the replacement of the
alkyl chain in the 3-position with an alkylamino chain
(compare diazoxide and 4) provoked an increase in the
pKa value. Such an effect probably results from the lone
pair delocalization of the exocyclic nitrogen atom into
the guanidinic system. Incidentally, the acidic character

Benzothiadiazine Dioxides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3349
and phosphorus pentasulfide (40 g) in anhydrous pyridine (250
mL) was refluxed for 4 h. The solvent was removed under
reduced pressure, and the residue was dissolved in an aqueous
solution of NaOH (50 g/500 mL). The alkaline solution was
treated with charcoal and was filtered, and the filtrate was
adjusted to pH 1 by means of 12 N HCl. The compound, which
precipitated, was collected by filtration, washed with water,
and suspended in an aqueous solution of sodium bicarbonate
(20 g/400 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 white precipitate that appeared was
collected by filtration, washed with water, and dried (16.4 g,
75%): mp 193-195 °C; 1H NMR (DMSO-d6, 80 MHz) δ 7.25-
7.80 (m, 3H, 5-H + 6-H + 8-H). Anal. (C7H5FN2O2S2) C, H, N,
S.
Radioisotopic and electrophysiological investigations
performed with different 3-(alkylamino)-7-halo-4H-1,2,4-
benzothiadiazine 1,1-dioxides indicated that the drugs
activated KATP channels and further revealed the chan-
nel subtype selectivity of the fluorinated compound 18
(Kir6.2/SUR1 versus Kir6.2/SUR2A and KIR6.2/SUR2B).
Pancreatic B-cells selective KATP channel activators
could be of high therapeutic value for the prevention
and/or treatment of type 1 and type 2 diabetes. Thus,
the present data contribute to the identification of new
compounds expected to become appropiate substitutes
for diazoxide in the treatment of glucose homeostasis
disorders.
Exp er im en ta l Section
7-Ch lor o-3-th ioxo-3,4-d ih yd r o-2H-1,2,4-ben zoth ia d ia -
zin e 1,1-Dioxid e (8b). The title compound was obtained from
7-chloro-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-di-
oxide (7b, 22.0 g, 95 mmol) by following the experimental
conditions described for 8a (17.7 g, 75%): mp 229-232 °C (lit.,
217-218 °C30).
7-Br om o-3-t h ioxo-3,4-d ih yd r o-2H-1,2,4-b en zot h ia d ia -
zin e 1,1-Dioxid e (8c). The title compound was obtained from
7-bromo-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-di-
oxide (7c, 26.0 g, 95 mmol) by following the experimental
conditions described for 8a . After acidification of the alkaline
solution, the insoluble material was suspended in a hy-
dromethanolic 1:1 solution of sodium bicarbonate (10 g/400
mL) and the mixture was heated until most of the insoluble
material dissolved. The suspension was treated with charcoal
and filtered. The filtrate was adjusted to pH 0-1 with 12 N
HCl, and the white precipitate that appeared was collected
by filtration, washed with water, and dried (18.9 g, 68%): mp
209-211 °C (lit., 223-225 °C31).
Ch em istr y. Melting points were determined on a Bu¨chi-
Tottoli capillary apparatus and are uncorrected. IR spectra
were recorded as KBr pellets on a FTIR Perkin-Elmer Spec-
trum 1000 spectrophotometer. UV data and spectra were
obtained with a Hitachi U 3010 spectrophotometer. The 1H
NMR spectra were taken on a Bruker AW-80 (80 MHz)
instrument in DMSO-d6 with HMDS as an internal standard;
chemical shifts are reported in δ values (ppm) relative to
internal HMDS. The abbreviations s ) singlet, d ) doublet, t
) triplet, q ) quadruplet, quint. ) quintuplet, m ) multiplet,
and b ) broad are used throughout. 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 silica gel Merck
60F 254.
7-F lu or o-3-oxo-3,4-d ih yd r o-2H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (7a ). Chlorosulfonyl isocyanate (16.8 mL,
0.193 mol) and nitromethane (160 mL) were mixed together
in a closed dried vessel. The mixture was cooled at -5 °C (ice
and salt bath) and protected from moisture during the slow
addition, under vigorous stirring, of 4-fluoroaniline (17.8 g,
0.16 mol) dissolved in nitromethane (50 mL). At the end of
addition (∼15 min), anhydrous AlCl3 (28.0 g, 0.21 mol) was
added to the resulting suspension and the mixture was
refluxed for 30-45 min. The hot solution was poored onto ice
(800 g), and after stirring and complete melting of ice, the
resulting precipitate was collected by filtration and washed
twice with water (75 mL). The insoluble crude material was
suspended in an aqueous solution of sodium bicarbonate (10
g/200 mL) and heated until most of the precipitate was
solubilized. The suspension was treated with charcoal and was
filtered, and the filtrate was adjusted to pH 1 by means of 12
N HCl. The white product, which precipitated, was collected
by filtration, washed with water, and dried (21.8 g, 63%): mp
7-Iod o-3-t h ioxo-3,4-d ih yd r o-2H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (8d ). The title compound was obtained from
7-iodo-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide
(7d , 30.8 g, 95 mmol) by following the experimental conditions
described for 8c (25.5 g, 79%): mp 219-222 °C (lit., 217-218
°C30).
7-F lu o r o -3-m e t h y lsu lfa n y l-4H -1,2,4-b e n zo t h ia d ia -
zin e 1,1-Dioxid e Mon oh yd r a te (9a ). 7-Fluoro-3-thioxo-3,4-
dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (8a , 12 g, 52
mmol) was dissolved in a 1:1 hydromethanolic solution of
sodium bicarbonate (7 g/280 mL), and an excess of methyl
iodide (12 mL) was added. After the mixture was stirred for
30 min, 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
1
1
dried (12.2 g, 89%): mp 265-268 °C; H NMR (DMSO-d6, 80
>300 °C; H NMR (DMSO-d6, 80 MHz) δ 7.10-7.70 (m, 3H,
MHz) δ 2.45 (s, 3H, SCH3), 7.10-7.50 (m, 2H, 5-H + 6-H),
7.60 (d, 1H, 8-H), 12.50 (bs, 1H, N-H). Anal. (C8H7FN2O2S2‚
H2O) C, H, N, S.
5-H + 6-H + 8-H), 9.55 (b, 1H, N-H), 11.25 (bs, 1H, SO2NH).
Anal. (C7H5FN2O3S) C, H, N, S.
7-Ch lor o-3-oxo-3,4-d ih yd r o-2H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (7b). The title compound was obtained from
4-chloroaniline (20.4 g, 0.16 mol) by following the experimental
conditions described for 7a (24.2 g, 65%): mp >300 °C (lit.,
316-318 °C19).
7-Br om o-3-oxo-3,4-d ih yd r o-2H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (7c). The title compound was obtained from
4-bromoaniline (27.7 g, 0.16 mol) by following the experimental
conditions described for 7a except that the crude material was
dissolved in a hydromethanolic 1:1 solution of sodium bicar-
bonate (10 g/400 mL) instead of an aqueous solution (31.0 g,
70%): mp >300 °C (lit., 335 °C 28).
7-Iodo-3-oxo-3,4-dih ydr o-2H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e (7d ). The title compound was obtained from 4-iodo-
aniline (35.3 g, 0.16 mol) by following the experimental
conditions described for 7c (29.6 g, 57%): mp >300 °C (lit.,
311-313 °C29).
7-F lu or o-3-t h ioxo-3,4-d ih yd r o-2H-1,2,4-b en zot h ia d ia -
zin e 1,1-Dioxid e (8a ). A mixture of 7-fluoro-3-oxo-3,4-dihy-
dro-2H-1,2,4-benzothiadiazine 1,1-dioxide (7a , 20.3 g, 95 mmol)
7-Ch lor o-3-m e t h ylsu lfa n yl-4H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (9b). The title compound was obtained from
7-chloro-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxide (8b, 12 g, 48 mmol) by following the experimental
conditions described for 9a (12.6 g, 85%): mp 287-290 °C (lit.,
290-291 °C30).
7-Br om o-3-m eth ylsu lfa n yl-4H-1,2,4-ben zoth ia d ia zin e
1,1-Dioxid e (9c). The title compound was obtained from
7-bromo-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxide (8c, 12 g, 41 mmol) by following the experimental
conditions described for 9a (12.6 g, 82%): mp 282-284 °C; 1H
NMR (DMSO-d6, 80 MHz) δ 2.45 (s, 3H, SCH3), 7.15 (d, 1H,
5-H), 7.75 (d, 1H, 6-H), 7.85 (s, 1H, 8-H), 12.55 (bs, 1H, N-H).
7-Iod o-3-m eth ylsu lfa n yl-4H-1,2,4-ben zoth ia d ia zin e 1,1-
Dioxid e (9d ). The title compound was obtained from 7-iodo-
3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (8d ,
12 g, 35 mmol) by following the experimental conditions
described for 9a (12.4 g, 79%): mp 271-273 °C; 1H NMR
(DMSO-d6, 80 MHz) δ 2.45 (s, 3H, SCH3), 7.00 (d, 1H, 5-H),

3350 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
de Tullio et al.
1504, 1494 (CdC, CdN, N-H), 1258, 1168 (SdO) cm-1 1H
NMR (DMSO-d6, 80 MHz) δ 0.80 (d, 6H, CH(CH3)CH(CH3)2),
1.00 (d, 3H, CH(CH3)CH(CH3)2), 1.65 (m, 1H, CH(CH3)-
CH(CH3)2), 3.60 (m, 1H, NHCH), 6.90 (bd, 1H, NHCH), 7.15
(d, 1H, 5-H), 7.30 (d, 1H, 6-H), 7.45 (s, 1H, 8-H), 10.15 (s, 1H,
NH). Anal. (C12H16FN3O2S) C, H, N, S.
3-Cyclop r op yla m in o-7-flu or o-4H -1,2,4-b en zot h ia d ia -
zin e 1,1-Dioxid e (16). Met h od A. Yield 0.34 g (70%); mp
242-245 °C; IR (KBr) 3274 (N-H), 1620, 1530, 1483 (CdC,
CdN, N-H), 1245, 1158 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 0.40-1.00 (m, 4H, CH(CH2)2), 2.60 (m, 1H, NHCH),
7.00 (b, 1H, NHCH), 7.25 (d, 1H, 5-H), 7.40 (d, 1H, 6-H), 7.70
(s, 1H, 8-H), 10.50 (s, 1H, NH). Anal. (C10H10FN3O2S) C, H,
N, S.
3-Cyclob u t yla m in o-7-flu or o-4H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (17). Meth od B. Yield 0.36 g (70%); mp
252-253 °C; IR (KBr) 3288 (N-H), 1631, 1580, 1557, 1474
(CdC, CdN, N-H), 1258, 1153 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 1.00-2.35 (bm, 6H, CH(CH2)3), 4.15 (m, 1H,
NHCH), 6.95-7.80 (m, 4H, 5-H + 6-H + 8-H + NHCH), 10.40
(bs, 1H, NH). Anal. (C11H12FN3O2S) C, H, N, S.
3-Cyclop en t yla m in o-7-flu or o-4H -1,2,4-b en zot h ia d ia -
zin e 1,1-Dioxid e (18). Meth od B. Yield 0.41 g (76%); mp
237-239 °C; IR (KBr) 3299 (N-H), 1639, 1594, 1568, 1492
(CdC, CdN, N-H), 1257, 1161 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 1.00-2.15 (bm, 8H, CH(CH2)4), 4.00 (bm, 1H,
NHCH), 6.95-7.55 (m, 4H, 5-H + 6-H + 8-H + NHCH), 10.15
(bs, 1H, NH). Anal. (C12H14FN3O2S) C, H, N, S.
3-Cyclop r op ylm et h yla m in o-7-flu or o-4H -1,2,4-b en zo-
th ia d ia zin e 1,1-Dioxid e (19). Meth od A. Yield 0.31 g (60%);
mp 221-222 °C; IR (KBr) 3295 (N-H), 1641, 1592, 1495
(CdC, CdN, N-H), 1240, 1155 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 0.30-1.30 (m, 5H, CH(CH2)2 + CH(CH2)2), 3.05
(t, 2H, NHCH2), 7.00-7.60 (m, 4H, NH + 5-H + 6-H + 8-H),
10.50 (bs, 1H, NH). Anal. (C11H12FN3O2S) C, H, N, S.
7.90 (d + s, 2H, 6-H + 8-H), 12.50 (bs, 1H, N-H). Anal. (C8H7-
IN2O2S2) C, H, N, S.
;
Gen er a l P r oced u r es for t h e Syn t h esis of 3-(Alk yl-
am in o)-7-h alo-4H-1,2,4-ben zoth iadiazin e 1,1-Dioxides (10-
25, 28-46). Meth od A. A mixture of the appropriate 7-halo-
3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (9a -
d) (0.5 g) and the appropriate alkylamine (or if not commercially
available, a concentrated aqueous solution of the amine) (5
mL) was heated in a sealed vessel for 4 h at 140 °C. The excess
of amine was eliminated by distillation under reduced pres-
sure, 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 6 N HCl. The precipitate was collected by
filtration, washed with water, and dried. The compound was
recrystallized in methanol/water.
Meth od B. A mixture of the appropriate 7-halo-3-methyl-
sulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (9a -d ) (0.5 g),
the appropriate alkylamine (1 mL), and dioxane (5 mL) was
heated in a sealed vessel for 4 h at 140 °C. After removal of
the solvent and the excess of amine under reduced pressure,
the compound was isolated and purified as described in method
A.
Meth od C. A mixture of the appropriate 7-halo-3-methyl-
sulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (9a -d ) (0.5 g)
and the appropriate alkylamine (5-10 mL) was refluxed for
72 h. After removal of the excess of amine under reduced
pressure, the compound was isolated and purified as described
in method A.
The following compounds were obtained according to the
synthetic procedures described above.
7-Flu or o-3-m eth ylam in o-4H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e Mon oh yd r a te (10). Meth od A Usin g a 40% w /v
Aqu eou s Solu tion of Meth yla m in e. Yield 0.35 g (75%); mp
275-277 °C; IR (KBr) 3324 (N-H), 1637, 1597, 1494 (CdC,
CdN, N-H), 1271, 1148 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 2.75 (d, 3H, NHCH3), 7.10 (m, 2H, 5-H + NHCH3),
7.30 (dd, 1H, 6-H), 7.45 (d, 1H, 8-H), 9.65 (s, 1H, NH). Anal.
(C8H8FN3O2S‚H2O) C, H, N, S.
3-Allyla m in o-7-flu or o-4H-1,2,4-ben zoth ia d ia zin e 1,1-
Dioxid e (20). Meth od A. Yield 0.30 g (62%); mp 209-210
°C; IR (KBr) 3353 (N-H), 1615, 1595, 1570, 1493 (CdC,
CdN, N-H), 1259, 1164 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 3.80 (bm, 2H, CH2CHdCH2), 4.90-5.30 (m, 2H, CH2-
CHdCH2), 5.55-6.05 (m, 1H, CH2CH ) CH2), 6.90-7.60 (m,
7-Flu or o-3-(eth yla m in o)-4H-1,2,4-ben zoth ia d ia zin e 1,1-
Dioxid e (11). Meth od A Usin g a 70% w /v Aqu eou s Solu -
tion of Eth yla m in e. Yield 0.36 g (79%); mp 235-237 °C; IR
(KBr) 3289 (N-H), 1636, 1593, 1491 (CdC, CdN, N-H), 1260,
1144 (SdO) cm-1; 1H NMR (DMSO-d6, 80 MHz) δ 1.05 (t, 3H,
CH2CH3), 3.15 (m, 2H, NHCH2CH3), 6.80-7.55 (m, 4H, 5-H +
6-H + 8-H + NHCH2). Anal. (C9H10FN3O2S) C, H, N, S.
7-Flu or o-3-pr op yla m in o-4H-1,2,4-ben zoth ia dia zin e 1,1-
Dioxid e (12). Meth od A. Yield 0.39 g (80%); mp 199-202
°C; IR (KBr) 3304 (N-H), 1636, 1595, 1494 (CdC, CdN, N-H),
4H, NH + 5-H + 6-H + 8-H), 10.60 (s, 1H, NH). Anal. (C10H10
FN3O2S) C, H, N, S.
-
7-Ch lor o-3-m eth ylam in o-4H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e (21). Meth od A Usin g a 40% w /v Aqu eou s Solu -
tion of Meth yla m in e. Yield 0.30 g (65%); mp >300 °C (lit.,
318-320 °C32); IR (KBr) 3311 (N-H), 1636, 1596, 1477
(CdC, CdN, N-H), 1260, 1138 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 2.55 (d, 3H, NHCH3), 7.55 (bd, 1H, 5-H), 7.60
(d, 1H, 6-H), 7.65 (s, 1H, 8-H), 8.45 (bm, 1H, NHCH3), 10.55
(s, 1H, NH). Anal. (C8H8ClN3O2S) C, H, N, S.
7-Ch lor o-3-(eth ylam in o)-4H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e (22). Meth od A Usin g a 70% w /v Aqu eou s Solu -
tion of Eth yla m in e. Yield 0.30 g (60%); mp 252-253 °C (lit.,
246-250 °C32); IR (KBr) 3306 (N-H), 1635, 1585, 1483
(CdC, CdN, N-H), 1267, 1170 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 1.05 (t, 3H, CH2CH3), 3.15 (m, 2H, NHCH2),
7.15 (d, 2H, 5-H + NHCH2), 7.50 (d + s, 2H, 6-H + 8-H), 10.60
(bs, 1H, NH). Anal. (C9H10ClN3O2S) C, H, N, S.
1
1257, 1144 (SdO) cm-1; H NMR (DMSO-d6, 80 MHz) δ 0.80
(t, 3H, CH2CH2CH3), 1.50 (m, 2H, CH2CH2CH3), 3.15 (m, 2H,
NHCH2CH2CH3), 6.80-7.55 (m, 4H, 5-H + 6-H + 8-H +
NHCH2), 10.55 (bs, 1H, NH). Anal. (C10H12FN3O2S) C, H, N,
S.
7-F lu or o-3-isop r op yla m in o-4H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (13). Met h od A. Yield 0.36 g (74%); mp
242-244 °C; IR (KBr) 3304 (N-H), 1639, 1568, 1509, 1493
(CdC, CdN, N-H), 1267, 1148 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 1.10 (d, 6H, CH(CH3)2), 3.90 (m, 1H, NH-
CH(CH3)2), 7.00 (bs, 1H, NHCH(CH3)2), 7.10-7.50 (m, 3H, 5-H
+ 6-H + 8-H), 10.25 (bs, 1H, NH). Anal. (C10H12FN3O2S) C,
H, N, S.
7-Ch lor o-3-pr opylam in o-4H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e (23). Meth od A. Yield 0.36 g (70%); mp 211-218 °C
(lit., 215-216 °C32); IR (KBr) 3296 (N-H), 1631, 1584, 1482
(CdC, CdN, N-H), 1262, 1166 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 0.80 (t, 3H, CH2CH2CH3), 1.45 (m, 2H, CH2CH2-
R,S-3-(2-Bu tyla m in o)-7-flu or o-4H-1,2,4-ben zoth ia d ia -
zin e 1,1-Dioxid e Mon oh yd r a te (14). Meth od A. Yield 0.41
g (75%); mp 203-205 °C; IR (KBr) 3492 (N-H), 1651, 1595,
1568, 1498 (CdC, CdN, N-H), 1267, 1159 (SdO) cm-1 1H
;
CH3), 3.10 (m, 2H, NHCH2CH2CH3), 7.10 (d, 2H, NHCH2
5-H), 7.50 (d + s, 2H, 6-H + 8-H), 10.55 (bs, 1H, NH). Anal.
(C10H12ClN3O2S) C, H, N, S.
+
NMR (DMSO-d6, 80 MHz) δ 0.80 (t, 3H, CH(CH3)CH2CH3),
1.05 (d, 3H, CH(CH3)CH2CH3), 1.40 (m, 2H, CH(CH3)CH2CH3),
3.60 (m, 1H, NHCH), 7.00 (bd, 1H, NHCH), 7.10 (d, 1H, 5-H),
7.30 (d, 1H, 6-H), 7.40 (s, 1H, 8-H), 10.35 (s, 1H, NH). Anal.
(C11H14FN3O2S‚H2O) C, H, N, S.
7-Ch lor o-3-(3-p e n t yla m in o)-4H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (28). Meth od C. Yield 0.34 g (60%); mp
233-236 °C; IR (KBr) 3290 (N-H), 1627, 1582, 1482 (CdC,
CdN, N-H), 1242, 1158 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 0.75 (t, 6H, CH(CH2CH3)2), 1.40 (m, 4H,CH(CH2CH3)2),
3.55 (m, 1H,NHCH), 6.95 (bd, 1H, NHCH), 7.10 (d, 1H, 5-H),
R,S-7-F lu or o-3-(3-m eth yl-2-bu tyla m in o)-4H-1,2,4-ben -
zoth ia d ia zin e 1,1-Dioxid e (15). Meth od C. Yield 0.38 g
(70%); mp 193-198 °C; IR (KBr) 3298 (N-H), 1636, 1593,

Benzothiadiazine Dioxides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3351
7.50 (d, 1H, 6-H), 7.55 (s, 1H, 8-H), 10.30 (bs, 1H, NH). Anal.
(C12H16ClN3O2S) C, H, N, S.
(69%); mp 254-256 °C; IR (KBr) 3319 (N-H), 1618, 1578, 1468
(CdC, CdN, N-H), 1252, 1151 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 0.90 (d, 6H, CH(CH3)CH(CH3)2), 1.00 (d, 3H,
CH(CH3)CH(CH3)2), 1.65 (m, 1H, CH(CH3)CH(CH3)2), 3.60 (m,
1H, NHCH), 6.95 (bs, 1H, NHCH), 7.10 (d, 1H, 5-H), 7.65 (d,
7-Ch lor o-3-cyclop r op yla m in o-4H -1,2,4-b en zot h ia d ia -
zin e 1,1-Dioxid e (29). Met h od A. Yield 0.36 g (69%); mp
261-262 °C (lit., 246-249 °C32); IR (KBr) 3307 (N-H), 1616,
1590, 1521, 1484 (CdC, CdN, N-H), 1263, 1144 (SdO) cm-1
;
1H, 6-H), 7.70 (s, 1H, 8-H), 10.30 (bs, 1H, NH). Anal. (C12H16
-
1H NMR (DMSO-d6, 80 MHz) δ 0.40-0.90 (m, 4H, CH(CH2)2),
2.60 (m, 1H, NHCH(CH2)2), 7.30 (d, 2H, NHCH(CH2)2 + 5-H),
BrN3O2S) C, H, N, S.
7-Br om o-3-cyclop r op yla m in o-4H -1,2,4-b en zot h ia d ia -
zin e 1,1-Dioxid e (39). Met h od A. Yield 0.36 g (70%); mp
248-253 °C; IR (KBr) 3274 (N-H), 1621, 1528, 1483 (CdC,
CdN, N-H), 1251, 1158 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 0.35-0.95 (m, 4H, CH(CH2)2), 3.60 (bs, 1H, NHCH),
7.20 (d, 1H, 5-H), 7.65 (m, 2H, 6-H + 8-H), 10.50 (bs, 1H, NH).
Anal. (C10H10BrN3O2S) C, H, N, S.
7.50 (d + s, 2H, 6-H + 8-H), 10.50 (bs, 1H, NH). Anal. (C10H10
ClN3O2S) C, H, N, S.
-
7-Ch lor o-3-cyclob u t yla m in o-4H -1,2,4-b en zot h ia d ia -
zin e 1,1-Dioxid e (30). Meth od B. Yield 0.35 g (65%); mp
275-278 °C; IR (KBr) 3284 (N-H), 1633, 1575, 1479 (CdC,
CdN, N-H), 1238, 1155 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 1.10-2.35 (bm, 6H, CH(CH2)3), 4.15 (m, 1H, NHCH),
7.15 (d, 1H, 5-H), 7.30-7.65 (m, 3H, 6-H + 8-H + NHCH),
10.25 (bs, 1H, NH). Anal. (C11H12ClN3O2S) C, H, N, S.
7-Ch lor o-3-cyclop r op ylm et h yla m in o-4H -1,2,4-b en zo-
th ia d ia zin e 1,1-Dioxid e (31). Meth od A. Yield 0.34 g (62%);
mp 288-290 °C; IR (KBr) 3298 (N-H), 1635, 1582, 1483
(CdC, CdN, N-H), 1267, 1160 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 0.30-1.30 (m, 5H, CH(CH2)2 + CH(CH2)2), 3.05
(m, 2H, NHCH2), 7.05 (s, 1H, NH), 7.15 (d, 1H, 5-H), 7.50 (d,
1H, 6-H), 7.60 (s, 1H, 8-H). Anal. (C11H12ClN3O2S) C, H, N, S.
3-Allyla m in o-7-ch lor o-4H-1,2,4-b en zot h ia d ia zin e 1,1-
Dioxid e (32). Meth od A. Yield 0.32 g (62%); mp 220-222
°C; IR (KBr) 3310 (N-H), 1651, 1630, 1584, 1483 (CdC,
CdN, N-H), 1239, 1164 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 3.80 (bm, 2H, CH2CHdCH2), 4.95-5.25 (m, 2H, CH2-
CHdCH2), 5.55-6.05 (m, 1H, CH2CHdCH2), 7.15 (d, 1H, 5-H),
7.30 (b, 1H, NH), 7.50 (d, 1H, 6-H), 7.60 (s, 1H, 8-H), 10.65 (s,
1H, NH). Anal. (C10H10ClN3O2S) C, H, N, S.
7-Br om o-3-cyclob u t yla m in o-4H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (40). Meth od B. Yield 0.34 g (63%); mp
279-281 °C; IR (KBr) 3288 (N-H), 1631, 1580, 1563, 1480
(CdC, CdN, N-H), 1258, 1153 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 1.00-2.35 (bm, 6H, CH(CH2)3), 4.15 (m, 1H,
NHCH), 7.10 (d, 1H, 5-H), 7.55 (dd, 1H, 6-H), 6.90-7.80 (b,
NHCH), 8.70 (d, 1H, 8-H), 10.50 (bs, 1H, NH). Anal. (C11H12
-
BrN3O2S) C, H, N, S.
3-Allyla m in o-7-b r om o-4H-1,2,4-b en zot h ia d ia zin e 1,1-
Dioxid e (41). Meth od A. Yield 0.33 g (65%); mp 243-245
°C; IR (KBr) 3361 (N-H), 1631, 1582, 1477 (CdC, CdN, N-H),
1
1252, 1162 (SdO) cm-1; H NMR (DMSO-d6, 80 MHz) δ 3.80
(bm, 2H, CH2CHdCH2), 4.95-5.25 (m, 2H, CH2CHdCH2),
5.55-6.05 (m, 1H, CH2CHdCH2), 7.10 (d, 1H, 5-H), 7.30 (bs,
1H, NH), 7.65 (m, 2H, 6-H + 8-H), 10.70 (s, 1H, NH). Anal.
(C10H10BrN3O2S) C, H, N, S.
7-Iodo-3-isopr opylam in o-4H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e (42). Meth od A. Yield 0.34 g (67%); mp 306-308
°C; IR (KBr) 3286 (N-H), 1617, 1571, 1476 (CdN, CdC, N-H),
7-Br om o-3-m eth ylam in o-4H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e Mon oh yd r a te (33). Meth od A Usin g a 40% w /v
Aqu eou s Solu tion of Meth yla m in e. Yield 0.35 g (70%); mp
305-307 °C; IR (KBr) 3355 (N-H), 1641, 1581, 1547, 1477
(CdC, CdN, N-H), 1263, 1145 (SdO) cm-1; 1H NMR (DMSO-
1
1245, 1174 (SdO) cm-1; H NMR (DMSO-d6, 80 MHz) δ 1.15
(d, 6H, CH(CH3)2), 3.90 (m, 1H, NHCH), 7.00 (d, 1H, 5-H), 7.15
(bd, 1H, NHCH), 7.75 (d, 1H, 6-H), 7.85 (s, 1H, 8-H), 10.45
(bs, 1H, NH)). Anal. (C10H12IN3O2S) C, H, N, S.
d6, 80 MHz) δ 2.70 (d, 3H, NHCH3), 7.10 (d, 2H, NHCH3
+
R ,S -7-Iod o-3-(2-b u t yla m in o)-4H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (43). Met h od A. Yield 0.37 g (70%); mp
253-254 °C; IR (KBr) 3318 (N-H), 1618, 1573, 1476 (CdN,
5-H), 7.50-7.80 (m, 2H, 6-H + 8-H), 9.70 (s, 1H, NH). Anal.
(C8H8BrN3O2S‚H2O) C, H, N, S.
1
CdC, N-H), 1247, 1149 (SdO) cm-1; H NMR (DMSO-d6, 80
7-Br om o-3-(eth yla m in o)-4H-1,2,4-ben zoth ia dia zin e 1,1-
Dioxid e (34). Meth od A Usin g a 70% w /v Aqu eou s Solu -
tion of Eth yla m in e. Yield 0.31 g (63%); mp 267-268 °C; IR
(KBr) 3305, 3189 (N-H), 1630, 1583, 1478 (CdC, CdN, N-H),
MHz) δ 0.80 (t, 3H, CH(CH3)CH2CH3), 1.10 (d, 3H, CH-
(CH3)CH2CH3), 1.40 (m, 2H, CH(CH3)CH2CH3), 3.60 (m, 1H,
NHCH), 6.80-7.10 (m, 2H, 5-H + NHCH), 7.60-7.90 (m, 2H,
6-H + 8-H), 10.30 (bs, 1H, NH). Anal. (C11H14IN3O2S) C, H,
N, S.
1
1249, 1159 (SdO) cm-1; H NMR (DMSO-d6, 80 MHz) δ 1.00
(t, 3H, CH2CH3), 3.10 (m, 2H, NHCH2CH3), 7.15 (m, 2H,
NHCH2CH3 + 5-H), 7.60 (d, 1H, 6-H), 7.70 (s, 1H, 8-H), 10.60
(bs, 1H, NH). Anal. (C9H10ClN3O2S) C, H, N, S.
R,S-7-Iodo-3-R,S-(3-m eth yl-2-bu tylam in o)-4H-1,2,4-ben -
zoth ia d ia zin e 1,1-Dioxid e (44). Meth od C. Yield 0.33 g
(60%); mp 267-270 °C; IR (KBr) 3317 (N-H), 1616, 1573, 1475
(CdN, CdC, N-H), 1248, 1156 (SdO) cm-1; 1H NMR (DMSO-
d6, 80 MHz) δ 0.75 (d, 6H, CH(CH3)CH(CH3)2), 1.00 (d, 3H,
CH(CH3)CH(CH3)2), 1.65 (m,1H, CH(CH3)CH(CH3)2), 3.60 (m,-
1H, NHCH), 6.80-7.05 (bd, 2H, 5-H + NHCH), 7.60-7.90 (bd,
2H, 6-H + 8-H), 10.25 (bs, 1H, NH). Anal. (C12H16IN3O2S) C,
H, N, S.
7-Br om o-3-pr op yla m in o-4H-1,2,4-ben zoth ia dia zin e 1,1-
Dioxid e (35). Meth od A. Yield 0.39 g (75%); mp 234-235
°C; IR (KBr) 3295 (N-H), 1631, 1580, 1567, 1479 (CdC,
CdN, N-H), 1260, 1164 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 0.85 (t, 3H, CH2CH2CH3), 1.50 (m, 2H, CH2CH2CH3),
3.15 (m, 2H, NHCH2CH2CH3), 7.00 (s, 1H, NHCH2), 7.15 (s,
1H, 5-H), 7.55 (s, 1H, 6-H), 7.70 (s, 1H, 8-H), 10.60 (bs, 1H,
NH). Anal. (C10H12BrN3O2S) C, H, N, S.
7-Br om o-3-isop r op yla m in o-4H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (36). Met h od A. Yield 0.21 g (40%); mp
220-221 °C; IR (KBr) 3293 (N-H), 1619, 1578, 1480 (CdC,
CdN, N-H), 1276, 1146 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 1.20 (d, 6H, CH(CH3)2), 3.95 (m, 1H, NHCH), 7.15 (bd,
1H, NHCH), 7.20 (d, 1H, 5-H), 7.75 (m, 2H, 6-H + 8-H), 10.45
(bs, 1H, NH)). Anal. (C10H12BrN3O2S) C, H, N, S.
7-Iodo-3-(3-pen tylam in o)-4H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e (45). Meth od C. Yield 0.33 g (60%); mp 210-220
°C; IR (KBr) 3314 (N-H), 1617, 1574, 1475 (CdC, CdN, N-H),
1
1277, 1151 (SdO) cm-1; H NMR (DMSO-d6, 80 MHz) δ 0.75
(t, 6H, CH(CH2CH3)2), 1.40 (m, 4H,CH(CH2CH3)2), 3.60 (m,
1H,NHCH), 6.95 (bd, 2H, NHCH + 5-H), 7.75 (d, 1H, 6-H),
7.85 (s, 1H, 8-H), 10.25 (bs, 1H, NH). Anal. (C12H16IN3O2S) C,
H, N, S.
R,S-7-Br om o-3-(2-bu tyla m in o)-4H-1,2,4-ben zoth ia d ia -
zin e 1,1-Dioxid e (37). Met h od A. Yield 0.30 g (56%); mp
224-226 °C; IR (KBr) 3320 (N-H), 1622, 1579, 1480 (CdC,
CdN, N-H), 1277, 1158 (SdO) cm-1; 1H NMR (DMSO-d6, 80
MHz) δ 0.80 (t, 3H, CH(CH3)CH2CH3), 1.10 (d, 3H, CH-
(CH3)CH2CH3), 1.40 (m, 2H, CH(CH3)CH2CH3), 3.70 (m, 1H,
NHCH), 6.95 (s, 1H, NHCH), 7.10 (d, 1H, 5-H), 7.60 (d, 1H,
7-Iodo-3-cyclobu tylam in o-4H-1,2,4-ben zoth iadiazin e 1,1-
Dioxid e (46). Meth od B. Yield 0.32 g (60%); mp 295-297
°C; IR (KBr) 3297 (N-H), 1633, 1556, 1476 (CdC, CdN, N-H),
1241, 1148 (SdO) cm-1; 1H NMR (DMSO-d6, 80 MHz) δ 1.30-
2.30 (m, 6H, CH(CH2)3), 4.10 (m, 1H, NHCH), 6.95 (d, 1H, 5-H),
7.50 (d, 1H, NHCH), 7.75 (d, 2H, 6-H + 8-H), 10.35 (bs, 1H,
NH). Anal. (C11H12IN3O2S) C, H, N, S.
6-H), 7.65 (s, 1H, 8-H), 10.35 (s, 1H, NH). Anal. (C11H14
BrN3O2S) C, H, N, S.
R,S-7-Br om o-3-(3-m eth yl-2-bu tyla m in o)-4H-1,2,4-ben -
zoth ia d ia zin e 1,1-Dioxid e (38). Meth od C. Yield 0.39 g
-
7-Ch lor o-4-m et h yl-3-m et h ylsu lfa n yl-4H -1,2,4-b en zo-
th ia d ia zin e 1,1-Dioxid e (47). K2CO3 (0.96 g, 6.9 mmol) and
an excess of methyl iodide (5 mL) were added to a solution of
7-chloro-3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-diox-

3352 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
de Tullio et al.
ide (9b, 2.0 g, 7.6 mmol) in acetonitrile/DMF, 4:1 (25 mL). The
suspension was stirred for 10 h at room temperature. The
solvent was removed by distillation under reduced pressure,
and the solid residue was taken up by water (40 mL). The
resulting aqueous suspension was adjusted to pH 2 by means
of formic acid, and the solid was collected by filtration and
washed with water. The crude compound was recrystallized
in methanol/water (1.87 g, 89%): mp 211-213 °C (lit., 219-
220 °C).33 Anal. (C9H9ClN2O2S2) C, H, N, S.
Biologica l Assa ys. Mea su r em en ts of In su lin Relea se
fr om In cu ba ted P a n cr ea tic 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 (Sigma) and equilibrated
against a mixture of O2 (95%) and CO2 (5%).
7-Ch lor o-3-isop r op yla m in o-4-m et h yl-4H -1,2,4-b en zo-
th ia d ia zin e 1,1-Dioxid e Hem ih yd r a te (48). A mixture of
7-chloro-4-methyl-3-methylsulfanyl-4H-1,2,4-benzothiadia-
zine 1,1-dioxide (47, 0.4 g, 1.45 mmol) in isopropylamine (4
mL) was refluxed for 90 min. The amine was removed by
distillation under reduced pressure, and the residue was
suspended in water (20 mL). After the mixture was stirred
for 1 h at room temperature, the precipitate was collected by
filtration, washed with water, and recrystallized in methanol/
water (0.27 g, 63%): mp 176-183 °C; IR (KBr) 3321 (N-H),
The islets were then incubated at 37 °C for 90 min in 1 mL
of the same medium containing 16.7 mM glucose and, in
addition, the reference compound or the benzothiadiazine
derivative. The release of insulin was measured radioimmu-
nologically using rat insulin as a standard.34
Residual insulin secretion 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.
Mea su r em en ts of Con tr a ctile Activity in Ra t Aor ta .
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).
1603, 1554, 1470 (CdN, CdC, N-H), 1283, 1171 (SdO) cm-1
;
1H NMR (DMSO-d6, 80 MHz) δ 1.10 (d, 6H, CH(CH3)2), 3.20
(s, H2O), 3.35 (s, 3H, N-CH3), 3.95 (m, 1H, NHCH), 7.30-
7.70 (m, 4H, NH + 5-H + 6-H + 8-H). Anal. (C11H14ClN3O2S‚
0.5H2O) C, H, N, S.
7-Ch lor o-3-m et h ylsu lfin yl-4H -1,2,4-b en zot h ia d ia zin e
1,1-Dioxid e (49). The suspension of 7-chloro-3-methylsulf-
anyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (9b, 5.0 g, 19 mmol)
in an aqueous solution of Na2CO3 (2.2 g/25 mL) was supple-
mented with 2 N NaOH until complete dissolution. Bromine
(1.0 mL) was added under stirring at room temperature, and
after the mixture was vigorously stirred for 10 min, the
resulting suspension was adjusted to pH 2-3 by means of 6
N HCl. The insoluble portion was collected by filtration,
washed with water, and suspended under stirring in methanol
(20 mL). The precipitate was collected by filtration, washed
with methanol, and dried (4.5 g, 85%): mp 260-261 °C; 1H
NMR (DMSO-d6, 80 MHz) δ 2.95 (s, 3H, SOCH3), 7.80-8.00
(m, 3H, 5-H + 6-H + 8-H). Anal. (C8H7ClN2O3S2) C, H, N, S.
7-Ch lor o-3-t er t -b u t yla m in o-4H -1,2,4-b e n zot h ia d ia -
zin e 1,1-Dioxid e (26). A mixture of 7-chloro-3-methylsulfinyl-
4H-1,2,4-benzothiadiazine 1,1-dioxide (49, 0.5 g, 1.79 mmol)
and tert-butylamine (8 mL) was heated in a sealed vessel for
2 h at 160 °C. The excess of amine was eliminated by
distillation under reduced pressure, and the residue was
dissolved in a hydromethanolic (1:1) 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 6
N HCl. The precipitate was collected by filtration, washed with
water, and dried. The compound was recrystallized in methanol/
water (0.28 g, 55%): mp 264-266 °C; IR (KBr) 3328 (N-H),
Mea su r em en ts of 86Rb Ou tflow fr om Ra t P a n cr ea tic
Islets. The method used for measuring 86Rb (42K substitute)
outflow from prelabeled and perifused rat pancreatic islets was
previously decribed in detail.8,13,23 Experiments were conducted
in the absence and presence of glibenclamide in the basal
medium.
Electr op h ysiology. Mouse Kir6.2 (Genbank D50581), rat
SUR1 (Genbank L40624), SUR2A (Genbank D83598), and
SUR2B (Genbank D86038) cDNAs were cloned in the pBF
vector. A truncated form of Kir6.2 (Kir6.2∆C36), which lacks
the C-terminal 36 amino acids and forms functional channels
in the absence of SUR, was prepared as described previously.35
Capped mRNA was prepared using the mMESSAGE large
scale in vitro transcription kit (Ambion, Austin, TX), as
previously described.36 Female Xenopus laevis were anaesthe-
tized with MS222 (2 g/L added to the water). One ovary was
removed via a minilaparotomy, the incision was sutured, and
the animal was allowed to recover. Once the wound had
completely healed, the second ovary was removed in a similar
operation and the animal was then killed by decapitation while
under anaesthesia. Immature stages V-VI oocytes were
incubated for 60 min with 1.0 mg/mL collagenase (Sigma, type
V) and manually defolliculated. Oocytes were either injected
with ∼1 ng of Kir6.2∆C36 mRNA or co-injected with ∼0.1 ng
Kir6.2 mRNA and ∼2 ng of mRNA encoding wild-type SUR.
The final injection volume was 50 nL/oocyte. Isolated oocytes
were maintained in Barth’s solution and studied 1-5 days
after injection. Patch pipets were pulled from thick-walled
borosilicate glass and had resistances of 250-500 kΩ when
filled with pipet solution. Macroscopic currents were recorded
from giant excised inside-out patches at a holding potential
of 0 mV and at 20-24 °C. Currents were evoked by repetitive
3 s voltage ramps from -110 to +100 mV and recorded using
an EPC7 patch-clamp amplifier (List Electronik, Darmstadt,
Germany). They were filtered at 0.2 kHz, digitized at 0.4 kHz
1627, 1576, 1481 (CdN, CdC, N-H), 1280, 1141 (SdO) cm-1
;
1H NMR (DMSO-d6, 80 MHz) δ 1.30 (s, 9H, C(CH3)3), 6.75 (bs,
1H, NH C(CH3)3), 7.05 (d, 1H, 5-H), 7.50 (d, 1H, 6-H), 7.60 (s,
1H, 8-H), 10.20 (bs, 1H, NH). Anal. (C11H14ClN3O2S) C, H, N,
S.
7-Ch lor o-3-(1,1-d im et h ylp r op yla m in o)-4H -1,2,4-b en -
zoth ia d ia zin e 1,1-Dioxid e (27). The compound was obtained
as described for 26, starting from 7-chloro-3-methylsulfinyl-
4H-1,2,4-benzothiadiazine 1,1-dioxide (49, 0.5 g, 1.79 mmol)
and using 1,1-dimethylpropylamine (8 mL) instead of tert-
butylamine (0.22 g, 40%): mp 258-259 °C; IR (KBr) 3305 (N-
H), 1629, 1566, 1479 (CdN, CdC, N-H), 1250, 1149 (SdO)
cm-1 1H NMR (DMSO-d6, 80 MHz) δ 0.75 (t, 3H, C(CH3)2-
;
CH2CH3), 1.20 (s, 6H, C(CH3)2CH2CH3), 1.70 (q, 2H, C(CH3)2-
CH2CH3), 6.65 (bs, 1H, NH C(CH3)2CH2CH3), 7.05 (d, 1H, 5-H),
7.50 (d, 1H, 6-H), 7.60 (s, 1H, 8-H), 10.20 (bs, 1H, NH). Anal.
(C12H16ClN3O2S) C, H, N, S.
Ion iza tion Con sta n ts. The pKa values of compounds 4, 13,
36, and 42 were determined spectroscopically by means of a
Perkin-Elmer UV/vis 554 spectrophotometer at 20 °C. UV
spectra were taken in different aqueous buffers of pH ranging
from
3 to 12. The pKa values were calculated by the
Debye-Hu¨ckel equation at the maximum basic form absor-
bance.16

Benzothiadiazine Dioxides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3353
Structurally Related to Diazoxide and Pinacidil as Potassium
Channel Openers Acting on Vascular Smooth Muscle Cells:
Design, Synthesis and Pharmacological Evaluation. J . Med.
Chem. 2000, 43, 1456-1466.
using a Digidata 1200 interface, and analyzed using pClamp
software (Axon Instruments Inc., Foster City, CA). The pipet
(external) solution contained the following (mM): 140 KCl, 1.2
MgCl2, 2.6 CaCl2, 10 HEPES (pH 7.4 with KOH). The intra-
cellular (bath) solution contained the following (mM): 107 KCl,
2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES (pH 7.2 with KOH;
final [K+] ≈ 140 mM). Compound 18 (BPDZ 176) was prepared
as a 100 mM stock solution in DMSO. In control experiments,
the maximal DMSO concentration applied (0.1%) had no effect
on the KATP current. Solutions containing ATP were made up
fresh each day.
(17) Dabrowski, M.; Ashcroft, F. M.; Ashfield, R.; Lebrun, P.; Pirotte,
B.; Egebjerg, J .; Hansen, J . B.; Wahl, P. The novel diazoxide
analog 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine
1,1-dioxide is a selective Kir6.2/SUR1 channel opener. Diabetes
2002, 51, 1896-1906.
(18) Somers, F.; Ouedraogo, R.; Antoine, M.-H.; de Tullio, P.; Becker,
B.; Fontaine, J .; Damas, J .; Dupont, L.; Rigo, B.; Delarge, J .;
Lebrun, P.; Pirotte, B. Original 2-Alkylamino-6-halogenoquinazo-
lin-4(3H)-ones and KATP Channel Activity. J . Med. Chem. 2001,
44, 16, 2575-2585.
(19) Girard, Y.; Atkinson, J . G.; Rokach, J . A New Synthesis of 1,2,4-
Benzothiadiazines and a Selective Preparation of o-Aminoben-
zenesulphonamides. J . Chem. Soc., Perkin Trans. 1 1979, 1043-
1047.
(20) Wollweber, H.; Horstmann, H.; Stoepel, K.; Garthoff, B.; Puls,
W.; Krause, H. P.; Thomas, G. 3-Amino-2H-benzothiadiazine-
1,1-dioxide mit Antihypertensiver und Potentieller Diabetogener
Wirkung (3-Amino-2H-benzothiadiazine 1,1-dioxide with anti-
hypertensive and potential diabetogenic effect). Arzneim.-Forsch.
1981, 31, 279-288.
(21) Manley, P. W.; Quast, U. Structure-Activity Studies of Potas-
sium Channel Opening in Pinacidil-Type Cyanoguanidines,
Nitroethenediamines, Thioureas, and Ureas. J . Med. Chem.
1992, 35, 2327-2340.
Ack n ow led gm en t. This study was supported by
grants from the National Fund for Scientific Research
(F.N.R.S., Belgium) from which P. de Tullio is a Re-
search Associate, B. Pirotte is a Senior Research As-
sociate, and P. Lebrun is a Research Director. The
technical assistance of S. Counerotte, D. Dewalque, P.
Neven, M. L. Pirard, A.-M. Vanbellinghen, and A. Van
Praet is gratefully acknowledged.
Refer en ces
(22) Quast, U.; Bray, K. M.; Andres, H.; Manley, P. W.; Baumlin, Y.;
Dosogne, J . Binding of the K+ Channel Opener [3H]P1075 in Rat
Isolated Aorta: Relationship to Functional Effects of Openers
and Blockers. Mol. Pharmacol. 1993, 43, 474-481.
(23) Lebrun, P.; Antoine, M.-H.; Ouedraogo, R.; Dunne, M.; Kane,
C.; Hermann, M.; Herchuelz, A.; Masereel, B.; Delarge, J .; de
Tullio, P.; Pirotte, B. Activation of ATP-Dependent K+ Channels
and Inhibition of Insulin Release: Effect of BPDZ 62. J .
Pharmacol. Exp. Ther. 1996, 277, 156-162.
(24) de Tullio, P.; Pirotte, B.; Dupont, L.; Masereel, B.; Laeckmann,
D.; Podona, T.; Diouf, O.; Lebrun, P.; Delarge, J . Synthesis and
Structural Studies of a New Class of Heterocyclic Compounds:
1,2,4-Pyridothiadiazine 1,1-Dioxides, Pyridyl Analogues of 1,2,4-
Benzothiadiazine 1,1-Dioxides. Tetrahedron 1995, 51, 3221-
3234.
(1) Cook, D. L.; Hales, C. N. Intracellular ATP Directly Blocks K+
Channels in Pancreatic B Cells. Nature 1984, 311, 271-273.
(2) Standen, N. B.; Quayle, J . M.; Davies, N. W.; Brayden, J . E.;
Huang, Y.; Nelson, M. T. Hyperpolarizing Vasodilators Activate
ATP-Sensitive K+ Channels in Arterial Smooth Muscle. Science
1989, 245, 177-180.
(3) Ashcroft, F. M. Adenosine 5′-Triphosphate-Sensitive Potassium
Channels. Annu. Rev. Neurosci. 1988, 11, 97-118.
(4) Petersen, O. H.; Dunne, M. J . Regulation of K+ Channels Plays
a Crucial Role in the Control of Insulin Secretion. Pfluegers Arch.
1989, 414, S115-S120.
(5) Kolb, H. A. Potassium Channels in Excitable and Non-Excitable
Cells. Rev. Physiol. Biochem. Pharmacol. 1990, 15, 51-79.
(6) Babenko, A. P.; Aguilar-Bryan, L.; Bryan, J . A View of SUR/Kir
6.X KATP Channels. Annu. Rev. Physiol. 1998, 60, 667-687.
(7) Robertson, D. W.; Steinberg, M. I. Potassium Channel Modula-
tors: Scientific Applications and Therapeutic Promise. J . Med.
Chem. 1990, 33, 1529-1541.
(8) Lebrun, P.; Arkhammar, P.; Antoine, M.-H.; Nguyen, Q.-A.;
Hansen, J . B.; Pirotte, B. A potent diazoxide analogue activating
ATP-sensitive K+ channels and inhibiting insulin release. Dia-
betologia 2000, 43, 723-732.
(9) Bjo¨rk, E.; Berne, C.; Ka¨mpe, O.; Wibell, P.; Oskarsson, P.;
Karlsson, F. A. Diazoxide Treatment at Onset Preserves Re-
sidual Insulin Secretion in Adults with Autoimmune Diabetes.
Diabetes 1996, 45, 1427-1430.
(10) Alemzadeh, R.; Langley, G.; Upchurch, L.; Smith, P.; Slonim,
A. E. Beneficial Effect of Diazoxide in Obese Hyperinsulinemic
Adults. J . Clin. Endocrinol. Metab. 1998, 83, 1911-1915.
(11) Pirotte, B.; de Tullio, P.; Lebrun, P.; Antoine, M.-H.; Fontaine,
J .; Masereel, B.; Schynts, M.; Dupont, L.; Herchuelz, A.; Delarge,
J . 3-(Alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides
as Powerful Inhibitors of Insulin Release from Rat Pancreatic
B-Cells: A New Class of Potassium Channel Openers? J . Med.
Chem. 1993, 36, 3211-3213.
(12) de Tullio, P.; Pirotte, B.; Lebrun, P.; Fontaine, J .; Dupont, L.;
Antoine, M.-H.; Ouedraogo, R.; Khelili, S.; Maggetto, C.; Ma-
sereel, B.; Diouf, O.; Podona, T.; Delarge, J . 3- and 4-Substituted
4H-Pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides as Potassium
Channel Openers: Synthesis, Pharmacological Evaluation, and
Structure-Activity Relationships. J . Med. Chem. 1996, 39, 937-
948.
(13) Pirotte, B.; Antoine, M.-H.; de Tullio, P.; Hermann, M.; Her-
chuelz, A.; Delarge, J .; Lebrun, P. A Pyridothiadiazine (BPDZ
44) as a New and Potent Activator of ATP-Sensitive K+ Chan-
nels. Biochem. Pharmacol. 1994, 47, 1381-1386.
(14) Antoine, M.-H.; Pirotte, B.; Hermann, M.; de Tullio, P.; Delarge,
J .; Herchuelz, A.; Lebrun, P. Cationic and Secretory Effects of
BPDZ 44 and Diazoxide in Rat Pancreatic Islets. Experientia
1994, 50, 830-832.
(25) March, J . Advanced Organic Chemistry, 3rd ed.; Wiley: New
York, 1985.
(26) Dupont, L.; de Tullio, P.; Tinant, B.; Pirotte, B. (R/S)-3-(3-Methyl-
2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxide Mono-
hydrate. Acta Crystallogr. 2001, E57, o1050-o1051.
(27) Dupont, L.; Pirotte, B.; de Tullio, P. 7-Iodo-3-isopropylamino-
4H-1,2,4-benzothiadiazine 1,1-Dioxide. Acta Crystallogr. 1999,
C55, 1152-1154.
(28) Di Bella, M.; Monzani, A.; Albasini, A.; Conti, G. Ricerche Nel
Campo della 1,2,4-Benzotiadiazina (Searches in the Field of
1,2,4-Benzothiadiazines). Farmaco 1966, 12, 829-838.
(29) Di Bella, M.; Rinaldi, M.; Coppi, G.; Fabio, U.; Manicardi, G.
Azione Antimicrobica di Derivati dell’ 1,2,4-Benzotiadiazin-1,1-
diossido (Antimicrobial action of derivatives of 1,2,4-benzothia-
diazine 1,1-dioxide). Farmaco 1974, 29, 915-922.
(30) Di Bella, M.; Rinaldi, M.; Fabio, U.; Manicardi, G. Azione
Antimicrobica di Derivati dell’1,2,4-Benzotiadiazin-1,1-diossido
(Antimicrobial action of derivatives of 1,2,4-benzothiadiazine 1,1-
dioxide). Farmaco 1972, 27, 990-998.
(31) Parenti, C.; Constantino, L.; Di Bella, M.; Raffa, L.; Baggio, G.
G.; Rossi, T. Heteroaryl-alkanoic Acids with Possible Anti-
inflammatory Activities IV. Boll. Chim. Farm. 1987, 126, 239-
243.
(32) Raffa, L.; Di Bella, M.; Ferrari, P.; Rinaldi, M.; Ferrari, N. Azione
Cardiovasculare di Derivati dell’1,2,4-Benzothiadiazin-1,1-di-
ossido. Nota IV (Cardiovascular Action of Derivatives of 1,2,4-
Benzothiadiazine 1,1-Dioxide. Part IV). Farmaco, Ed. Sci. 1974,
29, 411-423.
(33) Di Bella, M.; Rinaldi, M.; Fabio, U.; Manicardi, G. Azione
Antimicrobica di Derivati dell’1,2,4-Benzotiadiazin-1,1-diossido
(Antimicrobial Action of Derivatives of 1,2,4-Benzothiadiazine
1,1-Dioxide). Farmaco 1973, 28, 411-417.
(34) Leclercq-Meyer, V.; Marchand, J .; Woussen-Colle, M. C.; Giroix,
M.-H.; Malaisse, W. J . Multiple Effects of Leucine on Glucagon,
Insulin, and Somatostatin Secretion from the Perfused Rat
Pancreas. Endocrinology 1985, 116, 1168-1174.
(35) Tucker, S. J .; Gribble, F. M.; Zhao, C.; Trapp, S.; Ashcroft, F.
M. Truncation of Kir6.2 Produces ATP-sensitive K-channels in
the Absence of the Sulphonylurea Receptor. Nature 1997, 387,
179-181.
(15) Khelili, S.; de Tullio, P.; Lebrun, P.; Fillet, M.; Antoine, M.-H.;
Ouedraogo, R.; Dupont, L.; Fontaine, J .; Felekidis, A.; Leclerc,
G.; Delarge, J .; Pirotte, B. Preparation and Pharmacological
Evaluation of the R- and S-Enantiomers of 3-(2′-Butylamino)-
4H- and 3-(3′-Methyl-2′-butylamino)-4H-pyrido[4,3-e]-1,2,4-thia-
diazine 1,1-Dioxide, Two Tissue Selective ATP-Sensitive Potas-
sium Channel Openers. Bioorg. Med. Chem. 1999, 7, 1513-1520.
(16) Pirotte, B.; Ouedraogo, R.; de Tullio, P.; Khelili, S.; Somers, F.;
Boverie, S.; Dupont, L.; Fontaine, J .; Damas, J .; Lebrun, P.
3-Alkylamino-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1,1-Dioxides
(36) Gribble, F. M.; Ashfield, R.; A¨ mma¨la¨, C.; Ashcroft, F. M.
Properties of Cloned ATP-Sensitive K-Currents Expressed in
Xenopus Oocytes. J . Physiol. 1997, 498, 87-98.
J M021117W