Effect on KATP channel activation properties and tissue selectivity of the nature of the substituent in the 7- and the 3-position of 4H-1,2,4-benzothiadiazine 1,1-dioxides

Article abstract of DOI:10.1021/jm0311339

The present work explored 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides diversely substituted in the 7-position. Those compounds, structurally related to previously described potassium channel openers such as the benzothiadiazine dioxide BPDZ 73, we

Full text of DOI:10.1021/jm0311339

3492
J. Med. Chem. 2005, 48, 3492-3503
Effect on KATP Channel Activation Properties and Tissue Selectivity of the
Nature of the Substituent in the 7- and the 3-Position of
4H-1,2,4-Benzothiadiazine 1,1-Dioxides
Ste´phane Boverie,† Marie-He´le`ne Antoine,‡ Fabian Somers,† Be´ne´dicte Becker,‡ Sophie Sebille,†
Raogo Ouedraogo,‡ Ste´phane Counerotte,† Bernard Pirotte,† Philippe Lebrun,‡ and Pascal de Tullio*,†
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, and Laboratoire de Pharmacodynamie et de
the´rapeutique, Universite´ Libre de Bruxelles, 808, route de Lennik, B-1070 Bruxelles, Belgium
Received December 11, 2003
The present work explored 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides diversely
substituted in the 7-position. Those compounds, structurally related to previously described
potassium channel openers such as the benzothiadiazine dioxide BPDZ 73, were tested as
putative KATP channel activators on the pancreatic endocrine tissue and on the vascular smooth
muscle tissue. The nature of the substituent introduced in the 7-position as well as the nature
of the alkylamino side chain in the 3-position strongly affected both potency and tissue selectivity
of 4H-1,2,4-benzothiadiazine 1,1-dioxides. Thus, compounds bearing in the 7-position a methyl
or a methoxy group or devoid of a substituent in this position, and bearing an ethyl, an isopropyl,
or a cyclobutylamino group in the 3-position were found to be potent and selective inhibitors
of insulin release from rat pancreatic B-cells (i.e. 10a, 10b, 12b, 12d, 22c). In contrast,
3-alkylamino-7-trifluoromethyl- (20a-c) and 3-alkylamino-7-pentyl-4H-1,2,4-benzothiadiazine
1,1-dioxides (11a,b) expressed a marked myorelaxant activity on rat aorta ring. Among the
latter compounds, the 3-alkylamino-7-pentyl derivative (11a) showed a clear selectivity for
the vascular smooth muscle tissue. The present work gives new insights into the role of the
substituent in both the 7- and the 3-position for the design of 4H-1,2,4-benzothiadiazine 1,1-
dioxide potassium channel openers exhibiting different tissue selectivity profiles.
Introduction
ATP-sensitive potassium channels (KATP channels) are
involved in the transmembrane movement of potassium
ions and the control of the cell membrane potential.1
These channels are able to link the membrane excit-
ability to the metabolic state of the cell. They have been
characterized in numerous cell types such as cardiac,
pancreatic B (insulin-secreting)-cells, skeletal muscles
cells, smooth muscle cells, and central neurons.2-8 Over
the past few years, an increasing interest in drugs
(diazoxide (1), pinacidil (2), cromakalim (3), etc.) acti-
vating KATP channels has been observed (Figure 1). Such
compounds, named potassium channel openers (PCOs),
mainly exert their biological effects by promoting mem-
brane hyperpolarization.9-11 They are able to interfere
with several physiological processes such as insulin
release from pancreatic B-cells and contractile activity
from smooth muscle cells.12,13 The therapeutic interest
of potassium channel openers is of significance since
they provide efficient tools to interfere with the cell
excitability. Clinical studies have shown that short-term
treatment with the KATP channel activator diazoxide
preserved endogenous insulin production in patients
with newly manifested autoimmune diabetes and reduce
body fat in hyperinsulinemic obese adults.14,15 However,
the poor tissue selectivity of diazoxide impairs its
Figure 1. Chemical structure of several KATP channel openers.
clinical use due to marked side effects such as hyper-
trichosis, edema, and headache.16 KATP channels are
large heteromultimers composed of two different pro-
teinic subunits coassembled in a 4:4 stoichiometry to
form an octameric channel.17 One subunit, the Kir6.x,
is a traditional channel protein belonging to the inward
rectifying potassium channel superfamily (Kir) while the
second subunit refers to the sulfonylurea receptor
(SUR). Tissue selectivity properties of PCOs are cor-
related to the identity of the SUR isoform expressed in
the tissue. For instance, the Kir6.2/SUR1 channel,
which is found in neuronal cells and pancreatic B-cells,
is activated by diazoxide but only slightly by pinaci-
dil.9,18 The Kir6.2/SUR2A channel, predominant in
skeletal cardiac muscle cells, is activated by pinacidil
but not by diazoxide while the Kir6.2/SUR2B channel,
* To whom correspondence should be addressed. Tel: + 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/jm0311339 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/22/2005

4H-1,2,4-Benzothiadiazine 1,1-Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3493
Scheme 1a
derivatives (7a-d) with phosphorus pentasulfide in
pyridine led to 7-substituted 3-thioxo-3,4-dihydro-2H-
1,2,4-benzothiadiazine 1,1-dioxides (8a-d). In the next
step, the synthesis of 7-substituted 3-(methylsulfanyl)-
4H-1,2,4-benzothiadiazine 1,1-dioxides (9a-d)28 was
achieved, with an excellent yield, by reaction of the
thioxo derivatives (8a-d) with methyl iodide in the
presence of sodium hydrogenocarbonate. Finally, the
different 7-substituted 3-alkylamino-4H-1,2,4-benzothi-
adiazine 1,1-dioxides (10a-d, 11a-d, 12a-c, 13a-c)
were obtained by heating the methylsulfanyl intermedi-
ates (9a-d) in a sealed vessel with an excess of the
appropriate alkylamine.
2-Amino-5-trifluoromethylbenzenesulfonamide (18a)
(Scheme 2) was prepared from commercially available
2-chloro-5-trifluoromethylbenzenesulfonyl chloride (14).
The latter intermediate (14), by reaction with a diluted
solution of ammonia, gave access to 2-chloro-5-trifluoro-
methylbenzenesulfonamide (15). Nucleophilic substitu-
tion of the chlorine atom was achieved by reaction of
15 with benzylamine to form the intermediate 16.
Removal of the benzyl group by catalytic hydrogenolysis
led to the desired key intermediate 2-amino-5-trifluo-
romethylbenzenesulfonamide (18a). The 4-amino-3-sul-
famoylbenzoic acid synthesis (Scheme 2) started from
7-methyl-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxide (7a) which was oxidized by means of potassium
permanganate to the corresponding acid (17). Subse-
quent hydrolysis of 17 under acidic conditions led to the
desired compound 18b.
The ring closure reaction between 5-substituted 2-ami-
nobenzenesulfonamides and 1,1′-thiocarbonyldiimida-
zole may give access, according to the starting materials
and the experimental conditions used, either to 7-sub-
stituted 3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxides or to 3-(1H-imidazol-1-yl)-4H-1,2,4-ben-
zothiadiazine 1,1-dioxides (19a-c).29 In the case of
2-aminobenzenesulfonamides (18a-c), reaction with an
excess of 1,1′-thiocarbonyldiimidazole in dioxane gave
access to the 3-(1H-imidazol-1-yl)-substituted interme-
diates. The latter were converted, with good yields, to
the corresponding 7-substituted 3-alkylamino-4H-1,2,4-
benzothiadiazine 1,1-dioxides (20a-c, 21a-c, 22a-e)
after reaction with the appropriate alkylamine.
a Reagents and conditions: (i) 1: ClSO2NCO, CH3NO2; 2: AlCl3;
(ii) P2S5, pyridine, ∆; (iii) NaHCO3, CH3I, CH3OH/H2O; (iv) R2NH2,
∆.
found in smooth muscle cells, is activated both by
pinacidil and diazoxide.19-22 Thus, and due to the
ubiquitous distribution of KATP channels, the develop-
ment of novel PCOs should be linked to the expression
of a high selectivity for a single channel subtype located
on a single target tissue.
Previous works performed in our laboratories led to
the development of original 3-alkylamino-4H-pyrido[4,3-
e]-1,2,4-thiadiazine 1,1-dioxides, among which BPDZ 44
(4) was reported to be a more potent and more selective
pancreatic B-cell KATP channel opener than diazoxide
(Figure 1).23,24 To improve activity and selectivity for
the pancreatic B-cell KATP channel, we also prepared the
7-halobenzenic counterparts of pyridothiadiazines. From
those original compounds, BPDZ 73 (5) (7-chloro-3-
isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide,
Figure 1) appeared to be one of the most potent
pancreatic KATP channel activator reported to date.12,25,26
In the present study, we examined different series of
4H-1,2,4-benzothiadiazine 1,1-dioxides structurally re-
lated to BPDZ 73 (5). Particular attention was paid to
the influence of the nature of the substituent in the
7-position (steric, lipophilic, electronic impact) and the
size as well as the branching of the alkylamino side
chain in the 3-position. The new compounds were
examined as putative potassium channel openers on rat
pancreatic islets and on rat aorta rings. The structure-
activity relationships of these putative PCOs will also
be discussed.
As described in Scheme 3a, 3-alkylamino-7-cyano-4H-
1,2,4-benzothiadiazine 1,1-dioxides (24a,b) were ob-
tained from the substitution of the iodine atom of the
corresponding 3-alkylamino-7-iodo-4H-1,2,4-benzothia-
26
diazine 1,1-dioxides (23a,b) with cyanide ion as the
Chemistry
cuprous salt in hot DMF. Compound 25 was obtained
by reaction of 21b with MeOH in the presence of H2-
SO4 whereas compound 26 resulted from the reaction
of 21b with carbonyldiimidazole in DMF followed by
reaction of the activated intermediate with ammonia
(Scheme 3b).
The synthetic pathways used to prepare the different
3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides re-
ported here are illustrated in Schemes 1-4.
The strategy described in Scheme 1 was based on
previously reported synthetic procedures.26 3-Alkylami-
no-4H-1,2,4-benzothiadiazine 1,1-dioxides bearing a
methyl (10a-d), a pentyl (11a-d), a methoxy (12a-
c), and an ethoxy group (13a-c) in the 7-position were
obtained in a four-step reaction starting from the
appropriate para-substituted aniline (6a-d). First of
all, the anilines were transformed after reaction with
chlorosulfonyl isocyanate into the corresponding 7-sub-
stituted 3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxides (7a-d).27 Subsequent thionation of the oxo
3-Alkylamino-7-methylsulfonyl-4H-1,2,4-benzothiadi-
azine 1,1-dioxides (31a-c) were not obtained from the
ring closure reaction of 2-amino-5-methylsulfonylben-
zenesulfonamide (29) with 1,1′-thiocarbonyldiimidazole
and subsequent reaction with an appropriate alky-
lamine due to low yields of the imidazolyl intermediate
obtained in that case. An alternative route in a four-
step reaction, starting from 4-chloro-1-methylsulfonyl-
benzene (29), was used to gave access to the desired

3494 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Boverie et al.
Scheme 2a
a Reagents and conditions: (i) NH4OH; (ii) benzylamine, ∆; (iii) Pd/C 5%, H2 5 bars, MeOH; (iv) KMnO4; (v) H2SO4 50%, ∆; (vi) 1,1′-
thiocarbonyldiimazole, dioxane, DMF, ∆; (vii) R2NH2, ∆.
Scheme 3a
(27) with chlorosulfonic acid, followed by the reaction
of the sulfonyl chloride intermediate with aqueous
ammonia. Treatment of 28 with a saturated aqueous
solution of ammonia in a sealed vessel gave 2-amino-
5-methylsulfonylbenzenesulfonamide (29). The latter
was converted into the N-alkyl-N′-(2-amino-5-methyl-
sulfonylbenzenesulfonyl)-thioureas (30a-c) after reac-
tion with the appropriate alkylisothiocyanates. The
sulfonylthioureas were converted by ring closure into
the corresponding 3-alkylamino-7-methylsulfonyl-4H-
1,2,4-benzothiadiazine 1,1-dioxides (31a-c) by treat-
ment with a toluenic solution of phosgene in the
presence of triethylamine at 0 °C.30
Results and Discussion
The ability of the newly synthesized compounds to
inhibit the glucose-induced insulin secretion was evalu-
ated on isolated rat pancreatic islets. Tables 1-4 report
the in vitro results expressed as the percentage of
residual insulin release at different drug concentrations.
The data revealed that several drugs markedly inhibited
the insulin releasing process. Moreover, some newly
synthesized derivatives (10a-d, 12a-d, 20b, and 22a-
e) were found to be more potent than diazoxide used as
a reference PCO.23
a Reagents and conditions: (i) MeOH, H2SO4; (ii) 1: carbonyl-
diimidazole; 2: NH4OH.
Scheme 4a
The nature of the substituent in the 7-position and
in the 3-position strongly affected the activity on insulin
secreting cells. 7-COOH-substituted compounds (21a-
c) appeared to be completely inactive (Table 3). It is
expected that the carboxylic group in the 7-position will
be deprotonated at physiological pH (7.4), giving an
anionic site in this position. Such an event could maybe
explain the lack of biological activity. However, even
when the carboxylic group was converted into the
nonionic methyl ester group (25), the activity on B-cells
remained to be negligible. Surprisingly, a carboxamido
group (26) conferred a better activity. 7-Pentyl- (11a-
c) (Table 1), 7-ethoxy- (13a,c) (Table 2), 7-cyano- (24a,b)
(Table 3), 7-methylsulfonyl- (31a-c) (Table 3), 7-nitro-
(32a,c-e)31 (Table 4), 7-amino- (33)31 (Table 4) and
7-acetamido- (34)31 (Table 4) substituted 4H-1,2,4-
benzothiadiazine 1,1-dioxide were also found to express
a weak activity as inhibitors of insulin release. The
a Reagents and conditions: (i) 1: ClSO3H, 2: NH4OH; (ii) concd
NH4OH, ∆; (iii) R2NCS, K2CO3, acetone; (iv) N(C2H5)3, Cl2CO,
THF.
compounds (Scheme 4). Compound 28 was obtained
from the reaction of 4-chloro-1-methylsulfonylbenzene

4H-1,2,4-Benzothiadiazine 1,1-Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3495
Table 1. Effects of 7-Substituted 3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides on Insulin Secretion from Rat Pancreatic
Islets and Contractile Activity of K+-Depolarized Rat Aorta Rings
rat pancreatic B-cells % RISa
compd
R
50 µM
10 µM
1 µM
rat aorta rings ED50b (µM)
22a
22b
22c
22d
22e
10a
10b
10c
10d
11a
11b
11c
CH2CH3
N.D.
N.D.
25.8 ( 2.0 (12)
54.2 ( 3.2 (21)
34.9 ( 2.0 (16)
42.7 ( 1.5 (16)
29.8 ( 1.6 (14)
8.6 ( 0.9 (12)
8.5 ( 0.7 (14)
50.7 ( 3.1 (23)
19.7 ( 1.3 (24)
104.6 ( 5.2 (16)
106.0 ( 5.3 (16)
95.3 ( 6.1 (15)
73.9 ( 4.4 (16)c
13.7 ( 1.2 (23)
4.9 ( 0.4 (32)c
77.4 ( 3.8 (23)
93.8 ( 4.6 (16)
73.7 ( 5.2 (15)
90.4 ( 5.0 (16)
79.9 ( 4.5 (16)
83.8 ( 4.2 (23)
71.3 ( 3.5 (15)
93.6 ( 5.9 (22)
99.5 ( 4.7 (23)
N.D.
>300 (4)
>300 (4)
CH2CH2CH3
CH(CH3)2
N.D.
>300 (4)
CH(CH3)CH2CH3
N.D.
precipitate
precipitate
d
CH(CH2)3
N.D.
CH2CH3
N.D.
210.2 ( 32.2 (6)
200.0 ( 47.6 (3)
15.5 ( 3.2 (10)
58.4 ( 7.0 (4)
6.6 ( 0.3 (4)
20.1 ( 4.6 (4)
>30 (4)
CH(CH3)2
3.5 ( 0.3 (13)
N.D.
CH(CH3)CH2CH3
d
CH(CH2)3
3.7 ( 0.3 (11)
N.D.
CH2CH3
CH(CH3)2
N.D.
N.D.
N.D.
d
CH(CH2)3
N.D.
diazoxide
BPDZ 44
BPDZ 73
26.7 ( 1.6 (16)c
8.6 ( 0.9 (23)
5.7 ( 0.5 (35)c
87.5 ( 5.0 (15)c
70.5 ( 4.4 (19)
36.2 ( 2.4 (31)c
22.4 ( 2.1 (11)c
154.4 ( 14.5 (8)c
36.3 ( 2.2 (6)c
a RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ( SEM
(n)). b ED50: drug concentration giving 50% relaxation of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n)).
c Published results (ref 26). d Cyclobutyl.
Table 2. Effects of 7-Substituted 3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides on Insulin Secretion from Rat Pancreatic
Islets and Contractile Activity of K+-Depolarized Rat Aorta Rings
rat pancreatic B-cells, % RISa
compd
R
50 µM
10 µM
1 µM
rat aorta rings ED50b (µM)
12a
12b
12c
12d
13a
13b
13c
20a
20b
20c
CH2CH2CH3
6.3 ( 0.6 (12)
4.4 ( 0.7 (12)
8.5 ( 1.4 (9)
4.0 ( 0.2 (12)
N.D.
47.4 ( 3.6 (14)
8.5 ( 0.9 (24)
23.4 ( 1.7 (15)
14.1 ( 0.9 (15)
75.0 ( 4.0 (24)
65.9 ( 4.4 (14)
84.9 ( 4.0 (16)
77.9 ( 4.4 (22)
7.4 ( 0.6 (13)
76.1 ( 4.2 (22)
73.9 ( 4.4 (16)c
13.7 ( 1.2 (23)
4.9 ( 0.4 (32)c
101.7 ( 4.7 (16)
67.6 ( 4.3 (20)
92.4 ( 5.8 (15)
71.3 ( 3.8 (14)
N.D.
>300 (4)
c
CH(CH3)2
274.0 ( 19.0 (5)
17.5 ( 3.6 (4)
157.3 ( 7.4 (6)
162.5 ( 12.8 (4)
233.2 ( 29.2 (4)
>300 (4)
CH(CH3)CH2CH3
c,d
CH(CH2)3
CH2CH3
CH(CH3)2
CH(CH2)3
N.D.
N.D.
d
N.D.
N.D.
N.D.
CH2CH2CH3
CH(CH3)2
CH(CH3)CH2CH3
diazoxide
BPDZ 44
BPDZ 73
52.0 ( 2.9 (14)
7.1 ( 1.0 (13)
28.6 ( 1.8 (15)
26.7 ( 1.6 (16)c
8.6 ( 0.9 (23)
5.7 ( 0.5 (35)c
2.9 ( 0.5 (4)
71.5 ( 4.3 (14)
N.D.
13.1 ( 3.4 (6)
8.9 ( 2.6 (4)
87.5 ( 5.0 (15)c
70.5 ( 4.4 (19)
36.2 ( 2.4 (31)c
22.4 ( 2.1 (11)c
154.4 ( 14.5 (8)c
36.3 ( 2.2 (6)c
a RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ( SEM
(n)). b ED50: drug concentration giving 50% relaxation of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n)).
c Published results or previously reported compounds (refs 26, 35, 39). d Cyclobutyl.
reduced B-cell activity observed with 3-alkylamino-7-
pentyl- and 3-alkylamino-7-ethoxy-4H-1,2,4-benzothia-
diazine 1,1-dioxides compared to that observed with
7-methyl- and 7-methoxy-substituted compounds (com-
pare 11a, 11b, and 11c with 10a, 10b, and 10d,
respectively; compare 13b and 13c with 12b and 12d,
respectively), could be explained at least in part by the
increase of the size of the hydrocarbon chain in the
7-position (steric effect).
The rank order of potency, as inhibitors of insulin
release, for compounds bearing an isopropylamino side
chain in the 3-position appeared to be: 7-COOH com-
pound (21b) ) 7-COOMe compound (25) ) 7-C5H11
compound (12b) e 7-SO2CH3 compound (31b) ) 7-NH2
compound (33) ) 7-NHCOCH3 compound (34) < 7-CN
compound (24a) ) 7-CONH2 compound (26) e 7-C2H5O
compound (13b) ) 7-NO2 compound (32b) < 7-H com-
pound (22c) < 7-CF3 compound (20b) ) 7-CH3 com-
pound (10b) ) 7-CH3O compound (12b) < 7-chloro
compound (BPDZ 73) (Tables 1-4).
The effect of the introduction of an electron-with-
drawing group in the 7-position remained, however,
unclear (i.e. CN 24a and SO2CH3 31b compared to CF3
20b). Indeed, the presence of a CN or a SO2CH3 group
provoked a drastic decrease of activity on insulin-
secreting B-cells. Such an effect has already been
described in a previous work with 7-NO2-substituted
compounds.31 Surprisingly, the CF3 group did not induce

3496 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Boverie et al.
Table 3. Effects of 7-Substituted 3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides on Insulin Secretion from Rat Pancreatic
Islets and Contractile Activity of K+-Depolarized Rat Aorta Rings
rat pancreatic B-cells % RISa
compd
R
50 µM
10 µM
1 µM
rat aorta rings ED50b (µM)
21a
21b
21c
24a
24b
25
CH2CH2CH3
CH(CH3)2
95.3 ( 3.3 (15)
98.6 ( 3.5 (16)
91.2 ( 4.8 (16)
23.5 ( 1.2 (21)
54.3 ( 4.3 (21)
94.3 ( 6.2 (20)
N.D.
97.6 ( 6.8 (15)
96.6 ( 5.0 (23)
93.0 ( 6.1 (23)
82.2 ( 5.0 (15)
102.6 ( 8.3 (15)
95.7 ( 4.9 (22)
76.6 ( 3.7 (23)
91.6 ( 3.4 (16)
90.5 ( 4.8 (15)
97.2 (3.9 (16)
73.9 ( 4.4 (16)c
13.7 ( 1.2 (23)
4.9 ( 0.4 (32)c
N.D.
N.D.
>300 (4)
>300 (4)
CH(CH3)CH2CH3
CH(CH3)2
N.D.
>300 (4)
N.D.
42.7 ( 2.9 (4)
57.9 ( 6.6 (4)
>200 (8)
175.2 ( 35.2 (4)
>300 (4)
127.2 ( 17.9 (4)
126.1 ( 19.5 (4)
22.4 ( 2.1 (11)c
154.4 ( 14.5 (8)c
36.3 ( 2.2 (6)c
d
CH(CH2)3
N.D.
CH(CH3)2
CH(CH3)2
CH2CH3
N.D.
26
N.D.
31a
31b
31c
N.D.
N.D.
CH(CH3)2
CH(CH3) CH2CH3
Diazoxide
BPDZ 44
N.D.
N.D.
N.D.
N.D.
26.7 ( 1.6 (16)c
8.6 ( 0.9 (23)
5.7 ( 0.5 (35)c
87.5 ( 5.0 (15)c
70.5 ( 4.4 (19)
36.2 ( 2.4 (31)c
BPDZ 73
a RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ( SEM
(n)). b ED50: drug concentration giving 50% relaxation of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n)).
c Published results (ref 26). d Cyclobutyl.
Table 4. Effects of 7-Substituted 3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides on Insulin Secretion from Rat Pancreatic
Islets and Contractile Activity of K+-Depolarized Rat Aorta Rings
rat pancreatic B-cells % RISa
compd
R
50 µM
10 µM
1 µM
rat aorta rings ED50b (µM)
32a
32b
32c
32d
32e
33
CH2CH2CH3
88.9 ( 4.6 (16)c
4.8 ( 0.6 (14)c
63.3 ( 3.8 (18)c
90.4 (5.2 (16)c
59.6 ( 4.9 (13)c
85.8 ( 4.3 (15)c
89.3 ( 5.8 (16)c
26.7 ( 1.6 (16)c
8.6 ( 0.9 (23)
N.D.
64.0 ( 3.8 (37)c
N.D.
N.D.
N.D.
15.7 ( 3.0 (7)
23.1 ( 3.5 (4)
6.1 ( 0.9 (4)
2.8 ( 0.8 (4)
13.8 ( 1.6 (4)
>300 (4)
CH(CH3)2
CH(CH3)CH2CH3
CH(CH3)CH(CH3)2
N.D.
N.D.
N.D.
d
CH(CH2)3
N.D.
N.D.
CH(CH3)2
CH(CH3)2
Diazoxide
BPDZ 44
BPDZ 73
N.D.
N.D.
34
N.D.
N.D.
>300 (4)
73.9 ( 4.4 (16)c
13.7 ( 1.2 (23)
4.9 ( 0.4 (32)c
87.5 ( 5.0 (15)c
70.5 ( 4.4 (19)
36.2 ( 2.4 (31)c
22.4 ( 2.1 (11)c
154.4 ( 14.5 (8)c
36.3 ( 2.2 (6)c
5.7 ( 0.5 (35)c
a RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ( SEM
(n)). b ED50: drug concentration giving 50% relaxation of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n)).
c Published results (refs 26 and 31). d Cyclobutyl.
any marked reduction of activity on B-cells. The pro-
nounced inhibitory effect of the trifluoromethyl-substi-
tuted derivative bearing an isopropylamino side chain
in the 3-position (20b) may be compared to that of its
7-chloro-substituted counterpart BPDZ 73. Both com-
pounds bear a small size lipophilic substituent in the
7-position which exerts either a moderate (Cl) or pro-
nounced (CF3) electron-withdrawing effect.32 By con-
trast, the CN, SO2CH3, and NO2 substituents represent
electron-withdrawing groups which rather increase
hydrophilicity. The presence of an electron-donor group
such as a methyl (i.e. 10a, 10b) or a methoxy moiety
(i.e. 12b, 12d) appeared to increase activity on the
pancreatic tissue (electronic effect). Derivatives having
just an hydrogen atom in the 7-position (22a-e) were
found to exhibit, on insulin-secreting cells, an interme-
diate activity between the electron-withdrawing group
(i.e. compounds 21, 24, 31) and the electron-donor group
(i.e. compounds 10, 12).
The choice of the alkylamino side chains introduced
in the 3-position of the 4H-benzothiadiazine 1,1-dioxides
was inferred from our preliminary investigations with
3-alkylamino-7-halo-4H-1,2,4-benzothiadiazine 1,1-di-
oxides.26 As expected, the most potent inhibitors of
insulin secretion carry a short linear or branched
hydrocarbon chain such as ethyl and isopropyl (see 12b,
20b, 22a, 10a, 10b). In the same way, the activity of
the 3-cyclobutylamino-substituted derivatives (see 10d,
12d and 22e) was close to that of the 3-ethyl (10a and
22a) or 3-isopropylamino-substituted (10b, 12b and
22c) compounds in their respective series.
The myorelaxant activity of the newly synthesized
compounds as well as previously synthesized31 7-NO2,
7-NH2 and 7-NHCOCH3 compounds was evaluated on

4H-1,2,4-Benzothiadiazine 1,1-Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3497
Table 5. Tissue Selectivity Ratio: Pancreatic versus Aortic Tissue for Selected Compounds
rat pancreatic B-cells:
rat aorta rings:
c
compd
R1
R2
IC50a (µM)
ED50b (µM)
ED50/IC50
22c
10a
10b
10c
10d
12a
12b
12c
12d
20b
H
CH(CH3)2
3.8
2.5
>300 (4)
>79
84
100
1.4
10
>34
74
4.4
72
CH3
CH2CH3
210.2 ( 32.2 (6)
200.0 ( 47.6 (3)
15.5 ( 3.2 (10)
58.4 ( 7.0 (4)
>300 (4)
CH3
CH(CH3)2
2.0
CH3
CH(CH3)CH2CH3
11.2
5.6
e
CH3
CH(CH2)3
CH3O
CH3O
CH3O
CH3O
CF3
CH2CH2CH3
CH(CH3)2
8.9
3.7
274.0 ( 19.0 (5)
17.5 ( 3.5 (4)
157.3 ( 7.4 (6)
13.1 ( 3.4 (6)
22.4 ( 2.1 (11)d
36.3 ( 2.2 (6)d
154.4 ( 14.5 (8)d
CH(CH3)CH2CH3
CH(CH2)3
CH(CH3)2
4.0
e
2.2
1.7
7.7
1.0
52
diazoxide
BPDZ 73
BPDZ 44
22.6d
0.7d
4.4d
35
a IC50: drug concentration giving 50% inhibition of insulin release (estimated value). b ED50: drug concentration giving 50% relaxation
of the 30 mM KCl-induced contraction of rat aortic rings (mean ( SEM (n). c ED50/IC50: estimated selectivity ratio. d Published results
(refs 26). e Cyclobutyl.
K+-depolarized rat aorta rings (Tables 1 to 4). As
previously noted for the pancreatic model, the potency
of the drugs was affected by the nature of the hydro-
carbon chain on the exocyclic nitrogen atom in the
3-position but also by the nature of the substituent in
the 7-position. Except for 7-CF3-substituted (20a-c),
7-pentyl-substituted (11a,b), 7-NO2-substituted (32a-
e), and 3-sec-butylamino-substituted compounds (10c,
12c), most of the 4H-1,2,4-benzothiadiazine 1,1-dioxides
exhibited a weaker vasorelaxant activity on rat aorta
rings than diazoxide. As a rule, the introduction of an
electron-withdrawing group (i.e. CF3, NO2, CN) rather
than an electron-donor group (CH3, OCH3) in the
7-position appeared to improve the myorelaxant activity
(compare 10b, 12b, and 13b with 20b, 24a, and 32b).
7-COOH-substituted compounds (21a-c) were found to
be inactive, probably due at least in part to the same
reason than that mentioned for the pancreatic model
(deprotonation at physiological pH). The ester 25, which
cannot be deprotonated at physiological pH, was also
inactive, and the amide 26 only exhibited a poor
myorelaxant activity. Likewise, the absence of a sub-
stituent in the 7-position was responsible for a lack of
effect on rat aorta rings. The weak activity of 7-SO2-
CH3-substituted compounds 31a-c and of the 7-car-
boxamido compound (26) could also be due to the drastic
increase of hydrophilicity induced by the substituent
introduced in the 7-position.32
ed compounds 10b and 12b). Such a result is in
accordance with previous structure-activity relation-
ships reported for cyanoguanidines33 and pyrido-24 and
benzothiadiazine 1,1-dioxides.26 Surprisingly, in the
7-pentyl series of compounds (11a-c), the most potent
compound has an ethylamino side chain in the 3-posi-
tion. However, in this particular series of compounds
(11a-c), and supporting the view that the global steric
hindrance and the global lipophilicity of the drugs have
to be limited, the presence of a large pentyl group in
the 7-position could favor the myorelaxant activity of
drug bearing a small alkylamino side chain in the
3-position.
ED50/IC50 ratios have been calculated in order to
appreciate the apparent tissue selectivity (vascular
versus pancreatic tissue) of some selected drugs (see
Table 5). Diazoxide, which is known for its lack of tissue
selectivity was found to be nearly equipotent on both
tissues. BPDZ 73 (5), as previously described in the
literature,12,26 was more selective for the pancreatic than
the aortic tissue (selectivity ratio ) 52).
7-Methyl-substituted and 7-methoxy-substituted 4H-
1,2,4-benzothiadiazine 1,1-dioxides were found to be the
most interesting drugs in terms of potency and selectiv-
ity for the pancreatic tissue. Indeed, such compounds,
exhibiting an ethylamino (10a), an isopropylamino (10b,
12b), or a cyclobutylamino (10d, 12d) side chain in the
3-position, were potent inhibitors of the insulin releasing
process while their myorelaxant activity was very less
pronounced. The selectivity ratio for 10b was estimated
to be 105. As expected, the loss of selectivity of the 7-Me
and 7-MeO-substituted compounds for the pancreatic
versus aortic smooth muscle tissue was linked to the
increase of the size of the hydrocarbon chain in the
3-position (see 10c and 12c, Table 5).
3-Alkylamino-7-trifluoromethyl- (20a-c) and 3-alkyl-
amino-7-pentyl-4H-1,2,4-benzothiadiazine 1,1-dioxides
(11a,b) were found to express a marked myorelaxant
activity on vascular smooth muscle cells. Thus, com-
pared to 7-methyl-substituted compounds 10a, b, and
d, the introduction of a trifluoromethyl or a pentyl group
in the 7-position, which increased the lipophilicity, also
improved the myorelaxant effect of the drugs.
The presence of an hydrogen atom in the 7-position
(22c) was responsible for a lack of effect on rat aortic
ring but appeared to further pancreatic B-cells activity.
In contrast, the 7-trifluoromethyl-4H-1,2,4-benzothia-
diazine 1,1-dioxide 20b, despite of a marked pancreatic
The myorelaxant activity was usually improved with
the increase of the size and the branching of the
alkylamino chain in the 3-position (see the 3-sec-
butylamino-substituted compounds 10c and 12c com-
pared to the corresponding 3-isopropylamino-substitut-

3498 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Boverie et al.
Table 6. pKa Values of Selected
to be less acidic than diazoxide and 7-hydrogeno-
substituted compound 22c. They should be mainly
nonionized at physiological pH. In contrast, compounds
24a and BPDZ 145, two compounds bearing an electron-
withdrawing group, were more acidic than diazoxide
and compound 22c. The calculated ionized fraction at
pH 7.4 was 23.6% for the nitro derivative (BPDZ 145)
and 6.5% for the cyano derivative (24), respectively. To
some extent, the weaker activity of 7-nitro compounds
as inhibitors of insulin release, could be explained by a
pKa value lower than 8. In the same context, previously
described 3-alkyl-4H-1,2,4-pyridothiadiazine 1,1-dioxide
with a pKa value close to 7.5-7.6 were shown to be
inactive on the pancreatic tissue.24
Taken as a whole, these results confirm that the
nonionized form of 3-alkylamino-4H-1,2,4-benzothiadi-
azine 1,1-dioxides is required, but insufficient, for
optimal interaction with the receptor (hydrogen bond
interaction between the N-H group in the 4-position
and the receptor).
Radioisotopic experiments conducted with the pan-
creatic selective compound 12b indicated that the drug
(50 µM) was able to increase 86Rb (42K substitute)
outflow from prelabeled and perifused rat pancreatic
islets. Such an effect was completely abolished by the
addition of glibenclamide (10 µM) in the medium (data
not shown). Those data, as well as previously reported
results,35 confirmed that the 7-methoxy compound
expressed the classical pharmacological profile of an
ATP-sensitive potassium channel opener.
3-Isopropylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides
compd
22c
R1
pKa valuea
H
9.98
9.63b
9.51b
9.41b
9.28b
8.96
BPDZ 138
BPDZ 73
BPDZ 135
BPDZ 69
20b
F
Cl
Br
I
CF3
CN
CH3
24a
8.56
10b
10.21
10.11
7.91b
10.49b
9.80b
8.62b
12b
CH3O
NO2
NH2
BPDZ 145
BPDZ 212
BPDZ 213
diazoxide
CH3CONH
a The ionization constant was determined by UV spectroscopy
in different aqueous buffers of pH ranking from 5 to 12. b Pub-
lished results (refs 26, 31, 34).
activity, exhibited, like diazoxide, a poor tissue selectiv-
ity.
Biological results also indicated that the 7-pentyl-
substituted compound 11a exhibited a tissue selectivity
(vascular smooth muscle versus pancreatic tissue) op-
posite to that usually observed with 3-(ethylamino)-
substituted compounds (i.e. 22a and 10a, Table 1). This
finding suggests that a minor structural modification
can reverse tissue selectivity.
Conclusions
As previously reported for diazoxide and 3-alkylami-
no-4H-1,2,4-pyridothiadiazine 1,1-dioxides, the acidity
of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides
is correlated with the presence of a labile proton in the
4-position of the thiadiazine ring.24,26,34 The presence
of this hydrogen atom, and thus the nonionization of
the 4-position, appeared to be required for biological
activity on the pancreatic tissue.24,26,34 Using the UV
spectrophotometry method, we have determined and
compared with previously published values 26,31 the pKa
values (Table 6) of a selection of 4H-1,2,4-benzothiadi-
azine dioxides diversely substituted in the 7-position.
When comparing diazoxide (8.69) 34 to BPDZ 73 (9.51),
two 7-chloro-substituted benzothiadiazine 1,1-dioxides,
it appeared that the introduction of an alkylamino side
chain in the 3-position increased the pKa value (Table
6). This was probably due to the lone pair delocalization
of the exocyclic nitrogen atom into the guanidinic
system. The pKa values of previously synthesized halo-
substituted compounds (BPDZ 69, BPDZ 73, BPDZ 135,
BPDZ 138) showed that the increase of the acidic
character (7-F: 9.63, 7-Cl: 9.51, 7-Br: 9.41, and 7-I:
9.28) can be satisfactorily correlated with the increase
of the Hammett σp constant of the substituent (0.15,
0.24, 0.26, 0.28, respectively; compare to 7-hydrogeno-
substituted compound 22c with σp ) 0 and pKa value
of 9.98).26 As expected, the halo-substituted compounds
should exist in more than 99% in the nonionized state
at the physiological pH (7.4). Table 6 also reports
derivatives bearing electron-withdrawing groups (20b,
24a, BPDZ 145, BPDZ 213) or electron-donating groups
(10b, 12b, BPDZ 212) in the 7-position. Derivatives
substituted with an electron-donating group were shown
The presence of an electron-donating group such as
a methyl or a methoxy moiety or the absence of
substituent in the 7-position of 3-alkylamino-4H-1,2,4-
benzthiadiazine 1,1-dioxides appeared to be required for
increasing activity and selectivity for the pancreatic
tissue. The higher selectivity for the pancreatic endo-
crine tissue was obtained with derivatives bearing an
isopropylamino, an ethylamino, or a cyclobutylamino
side chain in the 3-position (10a, 10b, 10d, 12b, 12d,
22c).
In contrast, two series of compounds bearing an
electron-withdrawing group such as CF3 (20a-c) and
NO2 (32a-e) or a larger lipophilic hydrocarbon chain
in the 7-position (11a,b) were found to express a marked
myorelaxant activity on vascular smooth muscle cells.
Compound 11a even exhibited a tissue selectivity
(vascular smooth muscle vs pancreatic tissue) opposite
to that usually observed with compounds bearing small
groups such as an ethylamino side chain in the 3-posi-
tion.
Taken as a whole, these results indicated that the
modulation of the nature of the substituent in the
7-position can profoundly affect biological activity and
tissue selectivity. The present work further indicated
that parameters such as lipophilicity, electron-donating
or -withdrawing properties and steric effect of the
substituent in the 7-position notably influenced the
pharmacological profile of the drugs.
Last, radioisotopic experiments conducted on rat
pancreatic islets with a typical example of pancreatic
B-cell selective drug (12b) confirmed that the target of
such compounds was the KATP channel.

4H-1,2,4-Benzothiadiazine 1,1-Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3499
described for 8a starting from 7c (22 g, 95.15 mmol). The reflux
was maintained during 3 h. (19.78 g, 66%); mp 219-222 °C;
IR (KBr); 1H NMR (DMSO-d6, 500 MHz). Anal. (C8H8N2O3S2)
C, H, N, S.
7-Ethoxy-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (8d). The title compound was obtained as
described for 8a starting from 7d (28 g, 115.03 mmol). The
reflux was maintained during 3 h. (22.87 g, 77%); mp 223-
225 °C; IR (KBr); 1H NMR (DMSO-d6, 500 MHz). Anal.
(C9H10N2O3S2) C, H, N, S.
7-Methyl-3-methylsulfanyl-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide Monohydrate (9a). 7-Methyl-3-thioxo-3,4-
dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (8a) (12.0 g,
52.63 mmol) was dissolved in an aqueous solution of NaHCO3
(7 g/280 mL). Under stirring, methanol (300 mL) and methyl
iodide (12 mL, 192.34 mmol) were successively added to this
solution. After 30 min under stirring at room temperature, the
mixture was adjusted to pH 3 with 1 N HCl and methanol
was removed under reduced pressure. After cooling, the title
product was collected by filtration, washed with water and
dried (10.93 g, 85%); mp 275-278 °C; IR (KBr); 1H NMR
(DMSO-d6, 500 MHz). Anal. (C9H10N2O2S2‚H2O) C, H, N, S.
3-Methylsulfanyl-7-pentyl-4H-1,2,4-benzothiadiazine
1,1-Dioxide (9b). The title compound was obtained as de-
scribed for 9a starting from 8b (12.0 g, 42.19 mmol) (10.70 g,
85%); mp 237-241 °C; IR (KBr); 1H NMR (DMSO-d6, 500
MHz). Anal. (C13H18N2O2S2) C, H, O, S.
The structure-activity relationships deduced from
this study will permit to further improve the pharma-
cophoric model initially proposed for drugs activating
the pancreatic KATP channel.25,26
Experimental Section
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. The 1H NMR spectra were taken on a
Bruker AW-80 (80 MHz) instrument in DMSO-d6 with HMD-
SO as internal standard or on a Bruker Avance 500 (500 MHz)
instrument in DMSO-d6 with TMS as internal standard;
chemical shifts are reported in δ values (ppm) relative to
internal HMDSO or TMS. All reactions were routinely checked
by TLC on silica gel Merck 60F 254.
7-Methyl-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (7a). Chlorosulfonyl isocyanate (16.8 mL,
193.24 mmol) and nitromethane (160 mL) were mixed together
in a closed dried vessel. The mixture was cooled at -5 °C (ice-
salt bath) and protected from moisture during the slow
addition, under vigorous stirring, of 4-toluidine (17.8 g, 166.12
mmol) dissolved in nitromethane (50 mL). When the addition
was completed, anhydrous AlCl3 (28.0 g, 210.1 mmol) was
added to the resulting suspension and the mixture was
refluxed for 30 min. The hot solution was poured onto ice (800
g) and, after stirring and the complete melting of ice, the
resulting precipitate was collected by filtration and washed
with water (75 mL). The insoluble crude material was sus-
pended in an aqueous solution of NaHCO3 (10 g/200 mL) and
heated until most of the precipitate was solubilized. The
suspension was treated with charcoal, filtered, and the filtrate
was adjusted to pH 1 with 6 N HCl. The resulting white
product was collected by filtration, washed with water, and
dried. (19.38 g, 55%); mp 298-302 °C (lit.: 316-318 °C 27).
7-Pentyl-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (7b). The title compound was obtained from
4-pentylaniline (27.01 g, 166.00 mmol) by following the
experimental conditions described for 7a, except that the crude
material was dissolved in a hydromethanolic 1:1 solution of
NaHCO3 (30 g/600 mL). (23.16 g, 55%); mp > 300 °C; IR (KBr);
1H NMR (DMSO-d6, 500 MHz). Anal. (C12H16N2O3S) C, H, N,
S.
7-Methoxy-3-methylsulfanyl-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide Monohydrate (9c). The title compound was
obtained as described for 9a starting from 8c (12.0 g, 49.12
mmol) (11.80 g, 93%); mp 270-276 °C, IR (KBr); 1H NMR
(DMSO-d6, 500 MHz). Anal. (C9H10N2O3S2‚H2O) C, H, N, S.
7-Ethoxy-3-methylsulfanyl-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (9d). The title compound was obtained as
described for 9a starting from 8d (12.0 g, 46.45 mmol) (9.94
1
g, 78%); mp 243-248 °C; IR (KBr); H NMR (DMSO-d6, 500
MHz). Anal. (C10H12N2O3S2) C, H, N, S.
2-Chloro-5-trifluoromethylbenzenesulfonamide (15). A
suspension of 2-chloro-5-trifluoromethylbenzenesulfonyl chlo-
ride (14, 10.0 g, 38.4 mmol) in an aqueous solution of ammonia
(10% w/v, 150 mL) was stirred at room temperature. After 1
h, the solution was treated with charcoal, the filtrate was
concentrated by an half and adjusted to pH 1 with 6 N HCl.
The precipitate was collected by filtration, washed with water
and dried (8.97 g, 90%); mp 153-155 °C (Lit.: 158.5-160 °C36).
2-Benzylamino-5-trifluoromethylbenzenesulfon-
amide (16). A solution of 2-chloro-5-trifluoromethylbenzene-
sulfonamide (15, 3 g, 11 mmol) in benzylamine (30 mL) was
heated at 100 °C. Most of the amine was removed by distil-
lation under reduced pressure and the residue was suspended
in water (30 mL). An aqueous (5% w/v) solution of NaOH was
added (30 mL). The alkaline solution was treated with charcoal
and filtered, and the filtrate was adjusted to pH 2 with 6 N
HCl. The precipitate was collected by filtration, washed with
water and dried (3.05 g, 80%); mp 125-127 °C; IR (KBr);1H
NMR (DMSO-d6, 500 MHz). Anal. (C14H13F3N2O2S) C, H, N,
S.
3-Oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-carbox-
ylic acid 1,1-Dioxide Monohydrate (17). 7-Methyl-3-oxo-
3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (7a, 5 g,
23.56 mmol) was suspended in water (500 mL) and dissolved
by addition of 2.5 N NaOH. KMnO4 (12 g, 75.95 mmol) was
added portionwise, and the resulting mixture was stirred at
70 °C for 3 h. The insoluble material was filtered off, and
NaHSO3 was added to the filtrate until the pink coloration
disappeared. The solution was treated with charcoal and
filtered. The filtrate was adjusted to pH 1 with 12 N HCl. The
precipitate was collected by filtration, washed with water, and
dried (3.76 g, 66%); mp > 300 °C; IR (KBr); 1H NMR (DMSO-
d6, 500 MHz). Anal. (C8H6N2O5S‚H2O) C, H, N, S.
7-Methoxy-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (7c). The title compound was obtained from
4-methoxyaniline (20.44 g, 166.00 mmol) by following the
experimental conditions described for 7a (17.05 g, 45%); mp
275-277 °C (lit.: 300 °C27).
7-Ethoxy-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (7d). The title compound was obtained from
4-ethoxyaniline (22.77 g, 166.00 mmol) by following the
experimental conditions described for 7a (20.11 g, 50%); mp
1
243-244 °C; IR (KBr); H NMR (DMSO-d6, 500 MHz). Anal.
(C9H10N2O4S) C, H, N, S.
7-Methyl-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (8a). The mixture of 7-methyl-3-oxo-3,4-
dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (7a) (20.3 g,
95.65 mmol) and phosphorus pentasulfide (40 g, 179.95 mmol)
in anhydrous pyridine (250 mL) was heated under reflux for
5 h. The resulting suspension was concentrated under reduced
pressure and the residue was dissolved in the minimum of 2
N NaOH. This solution was treated with charcoal and filtered,
and the filtrate was adjusted to pH 1 with 6 N HCl. The
crystalline solid was collected by filtration, washed with water,
and dried (14.75 g, 65%); mp 220-224 °C; IR (KBr); 1H NMR
(DMSO-d6, 500 MHz). Anal. (C8H8N2O2S2) C, H, N, O, S.
7-Pentyl-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (8b). The title compound was obtained as
described for 8a starting from 7b (25.7 g, 95.65 mmol) (16.32
1
g, 60%); mp 198-202 °C; IR (KBr); H NMR (DMSO-d6, 500
2-Amino-5-trifluoromethylbenzenesulfonamide (18a).
A solution of 2-benzylamino-5-trifluoromethylbenzenesulfon-
amide (16, 2 g, 6 mmol) in methanol (20 mL) was supple-
mented with Pd/C (0.2 g, 5%). The mixture was poured in a
MHz). Anal. (C12H16N2O2S2) C, H, N, S.
7-Methoxy-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadi-
azine 1,1-Dioxide (8c). The title compound was obtained as

3500 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Boverie et al.
hermetically closed autoclave under hydrogen atmosphere (5
bar). The insoluble material was filtered off and the solvent
removed under reduced pressure. The residue was dissolved
in a minimum aqueous NaOH solution (10% w/v), treated with
charcoal and filtered, and the filtrate was adjusted to pH 1
with 6 N HCl. The precipitate was collected by filtration,
washed with water and dried (1.27 g, 85%); mp 142-142 °C;
IR (KBr); 1H NMR (DMSO-d6, 500 MHz). Anal. (C7H7F3N2O2S)
C, H, N, S.
1.25 g, 5.0 mmol) in dry acetone (20 mL) under stirring at room
temperature. Ethyl isothiocyanate (0.58 mL, 6.6 mmol) was
added dropwise, and the resulting mixture was stirred at
gentle reflux for 4 h. The cooled mixture was evaporated to
dryness under reduced pressure. The residue was dissolved
in water (25 mL). The pH was adjusted to 6 with 6 N HCl,
and the solution was stirred at 0 °C until the product solidified.
The precipitate was collected by filtration, washed with water,
1
and dried (1.01 g, 60%); mp 148-150 °C; IR (KBr); H NMR
(DMSO-d6, 500 MHz). Anal. (C10H15N3O4S3) C, H, N, S.
N-(2-Amino-5-methylsulfonylbenzenesulfonyl)-N′-iso-
propylthiourea (30b). The title compound was obtained as
described for 30a using isopropyl isothiocyanate (0.62 g, 6.1
mmol) (1.13 g, 60%); mp 149-151 °C; IR (KBr); 1H NMR
(DMSO-d6, 500 MHz). Anal. (C11H17N3O4S3) C, H, N, S.
R/S-N-(2-Amino-5-methylsulfonylbenzenesulfonyl)-N′-
2-butylthiourea (30c). The title compound was obtained as
described for 30a using R/S-2-butyl isothiocyanate (0.66 g,
4-Amino-3-sulfamoylbenzoic Acid (18b). 3-Oxo-3,4-di-
hydro-2H-1,2,4-benzothiadiazine-7-carboxylic acid 1,1-dioxide
(17, 5 g, 20.64 mmol) was suspended in an aqueous solution
of sulfuric acid (50% w/v) and stirred under reflux. When the
insoluble has disappeared, the solution was cooled to 0 °C and
supplemented with an aqueous solution of NaOH (30% w/v)
until pH 2. The precipitate was collected by filtration, washed
with water, and dried (4.06 g, 91%); mp 214-219 °C; IR (KBr);
1H NMR (DMSO-d6, 500 MHz). Anal. (C7H8N2O4S) C, H, N, S.
3-(1H-Imidazol-1-yl)-7-trifluoromethyl-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (19a). A solution of 2-amino-5-
trifluoromethylbenzenesulfonamide (18a, 5.5 g, 22 mmol) and
N,N′-thiocarbonyldiimidazole (14.0 g, 78,22 mmol) in dioxane
(55 mL) was refluxed for 4 h. The solvent was removed by
distillation under reduced pressure, and the residue was
suspended in water (150 mL). A solution of NaOH in water
(2.5 g/20 mL) was added, and the solution was stirred at room
temperature for 10 min. The alkaline solution was treated with
charcoal and filtered. The filtrate was adjusted to pH 2 with
6 N HCl. The precipitate was collected by filtration, washed
with water,, and dried. (4.87 g, 70%); mp 211-214 °C; IR
(KBr); 1H NMR (DMSO-d6, 500 MHz). Anal. (C11H7 F3N4O2S)
C, H, N, S.
3-(1H-Imidazol-1-yl)-4H-1,2,4-benzothiadiazine-7-car-
boxylic Acid 1,1-Dioxide (19b). The title compound was
obtained as described for 19a starting from 18b (5.5 g, 25.44
mmol) (5.1 g, 68%); mp > 300 °C; IR (KBr); 1H NMR (DMSO-
d6, 500 MHz). Anal. (C11H8N4O4S) C, H, N, S.
3-(1H-Imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (19c). The title compound was obtained as described
for 19a starting from 2-aminobenzenesulfonamide (5.5 g, 31.94
mmol) (6.26 g, 79%); mp 246-248 °C (Lit.: 246-248 °C 37);
IR (KBr); 1H NMR (DMSO-d6, 500 MHz). Anal. (C10H8N4O2S)
C, H, N, S.
2-Chloro-5-methylsulfonylbenzenesulfonamide (28).
Chlorosulfonic acid (25 mL) was cooled on an ice-salt bath and
then carefully supplemented with 4-chloro-1-methylsulfonyl-
benzene (27, 5 g, 26.22 mmol). The mixture was heated under
reflux for 1 h and thionyl chloride (1.5 mL) was added. After
2 h reflux, the reaction mixture was cooled and poured on ice
(50 g) under stirring. The resulting precipitate was collected
by filtration and washed with cold water. The solid was
suspended under stirring in an aqueous solution of ammonia
(150 mL, 10 w/v). After 30 min, the alkaline suspension was
concentrated under reduced pressure up to a volume of 75 mL.
The precipitate was collected by filtration, washed with water,
and dissolved in an aqueous solution of NaOH (50 mL, 10 w/v).
The alkaline solution was treated with charcoal and filtered,
and the filtrate was adjusted to pH 5 with 6 N HCl. The
crystalline precipitate was collected by filtration washed with
water, and dried. (3.54 g, 50%); mp 225-227 °C (Lit.: 227-
22939); Anal. (C7H8ClNO4S2) C, H, N, S.
2-Amino-5-methylsulfonylbenzenesulfonamide (29). The
mixture of 2-chloro-5-methylsulfonylbenzenesulfonamide (28,
5 g, 18.54 mmol) in a saturated aqueous solution of ammonia
(50 mL) was heated in a sealed vessel at 140 °C for 2 h. After
cooling, the solution was concentrated to a small volume (20
mL), and the resulting mixture was adjusted to pH 5 with 6
N HCl. The crystalline precipitate was collected by filtration,
washed with water, and dried. (4.17 g, 90%); mp 209-210 °C;
IR (KBr); 1H NMR (DMSO-d6, 500 MHz). Anal. (C7H10N2O4S2)
C, H, N, S.
1
6.1 mmol) (1.28 g, 68%); mp 144-146 °C; IR (KBr); H NMR
(DMSO-d6, 500 MHz). Anal. (C12H19N3O4S3) C, H, N, S.
General Procedure for the Synthesis of the 7-Substi-
tuted 3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Diox-
ides 10a-d, 11a-c, 12a-d, and 13a-c. The mixture of the
appropriate 7-substituted 3-methylsulfanyl-4H-1,2,4-benzothi-
adiazine 1,1-dioxide intermediate (9a-d) (0.5 g) and the
appropriate alkylamine was heated in a sealed vessel for 4 h
at 140 °C. The excess of amine was eliminated by distillation
under reduced pressure, and the residue was suspended in
water (20 mL). An aqueous solution of NaOH (5% w/v) was
added dropwise until dissolution of the residue. The alkaline
solution was treated with charcoal and filtered. 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 the appropriate solvent.
3-Ethylamino-7-methyl-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (10a). The title compound was obtained from 9a (0.5
g) following the general procedure using an aqueous solution
of ethylamine 70% (10 mL). Yield: 0.35 g (70%); mp 250-252
°C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz). Anal. (C10H13N3O2S)
C, H, N, S.
3-Isopropylamino-7-methyl-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (10b). The title compound was obtained from
9a (0.5 g) following the general procedure using isopropy-
lamine (5 mL). Yield: 0.40 g (76%); mp 266-268 °C IR (KBr);
1H NMR (DMSO-d6, 80 MHz). Anal. (C11H15N3O2S) C, H, N,
S.
R/S-3-(2′-Butyl)amino-7-methyl-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (10c). The title compound was obtained
from 9a (0.5 g) following the general procedure using R/S-2-
butylamine (5 mL). Yield: 0.35 g (63%); mp 219-220 °C; IR
1
(KBr); H NMR (DMSO-d6, 80 MHz). Anal. (C12H17N3O2S) C,
H, N, S.
3-Cyclobutylamino-7-methyl-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (10d). The title compound was obtained from
9a (0.5 g) following the general procedure using a solution of
cyclobutylamine (1 mL) in dioxane (5 mL). Yield: 0.43 g (79%);
mp 289-290 °C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz). Anal.
(C12H15N3O2S) C, H, N, S.
3-Ethylamino-7-pentyl-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (11a). The title compound was obtained from 9b (0.5
g) following the general procedure using an aqueous solution
of ethylamine 70% (10 mL).Yield: 0.35 g, (70%); mp 202-205
°C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz). Anal. (C14H21N3O2S)
C, H, N, S.
3-Isopropylamino-7-pentyl-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (11b). The title compound was obtained from
9b (0.5 g) following the general procedure using isopropy-
lamine (5 mL). Yield: 0.34 g (65%); mp 198-202 °C; IR (KBr);
1H NMR (DMSO-d6, 80 MHz). Anal. (C15H23N3O2S) C, H, N,
S.
3-Cyclobutylamino-7-pentyl-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (11c). The title compound was obtained from
9b (0.5 g) following the general procedure using a solution of
cyclobutylamine (1 mL) in dioxane (5 mL). Yield: 0.35 g (66%);
N-(2-Amino-5-methylsulfonylbenzenesulfonyl)-N′-eth-
ylthiourea (30a). K2CO3 (0.85 g, 6.2 mmol) was added to a
solution of 2-amino-5-methylsulfonylbenzenesulfonamide (29,

4H-1,2,4-Benzothiadiazine 1,1-Dioxides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3501
1
mp 239-241 °C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz). Anal.
(C16H23N3O2S) C, H, N, S.
°C; IR (KBr); H NMR (DMSO-d6, 80 MHz). Anal. (C12H14F3-
N3O2S) C, H, N, S.
7-Methoxy-3-propylamino-4H-1,2,4-benzothiadiazine
1,1-Dioxide Monohydrate (12a). The title compound was
obtained from 9c (0.5 g) following the general procedure using
propylamine (5 mL). Yield: 0.42 g (78%); mp 194-199 °C; IR
(KBr); 1H NMR (DMSO-d6, 80 MHz). Anal. (C11H15N3O3S‚H2O)
C, H, N, S.
3-Propylamino-4H-1,2,4-benzothiadiazine-7-carboxy-
lic Acid 1,1-Dioxide (21a). The title compound was obtained
from 19b (0.5 g) following the general procedure using pro-
1
pylamine (5 mL). Yield: 0.34 (71%); mp 300 °C; IR (KBr); H
NMR (DMSO-d6, 80 MHz). Anal. (C11H13N3O4S) C, H, N, S.
3-isopropylamino-4H-1,2,4-benzothiadiazine-7-carbox-
ylic Acid 1,1-Dioxide (21b). The title compound was from
19b (0.5 g) following the general procedure using isopropy-
lamine (5 mL). Yield: 0.32 g (66%); mp >300 °C; IR (KBr); 1H
NMR (DMSO-d6, 80 MHz). Anal. (C11H13N3O4S) C, H, N, S.
R/S-3-(2′-butylamino)-4H-1,2,4-benzothiadiazine-7-car-
boxylic Acid 1,1-Dioxide (21c). The title compound was
obtained from 19b (0.5 g) following the general procedure using
(R/S)-2-butylamine (5 mL). Yield: 0.34 g (68%); mp >300 °C;
3-Isopropylamino-7-methoxy-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (12b). The title compound was obtained from
9c following the general procedure using a solution of isopro-
pylamine (5 mL). Yield: 0.39 g (76%); mp 227-233 °C; IR
1
(KBr); H NMR (DMSO-d6, 80 MHz). Anal. (C11H15N3O3S) C,
H, N, S.
R/S-3-(2′-Butylamino)-7-methoxy-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (12c). The title compound was obtained
from 9c (0.5 g) following the general procedure using R/S-2-
butylamine (5 mL). Yield: 0.38 g (70%); mp 197-202 °C; IR
1
IR (KBr); H NMR (DMSO-d6, 80 MHz). Anal. (C12H15N3O4S)
C, H, N, S.
1
(KBr); H NMR (DMSO-d6, 80 MHz). Anal. (C12H17N3O3S) C,
3-Ethylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxide
(22a). The title compound was obtained from 19c (0.5 g)
following the general procedure using an aqueous solution of
ethylamine 70% (10 mL). Yield: 0.27 g (60%); mp 227-232
°C (lit.:232-233 °C 38); IR (KBr); 1H NMR (DMSO-d6, 80 MHz).
Anal. (C9H11N3O2S) C, H, N, S.
H, N, S.
3-Cyclobutylamino-7-methoxy-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (12d). The title compound was obtained from
9c (0.5 g) following the general procedure using a solution of
cyclobutylamine (1 mL) in dioxane (5 mL). Yield: 0.32 g (60%);
mp 249-252 °C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz). Anal.
(C12H15N3O3S) C, H, N, S.
7-Ethoxy-3-(ethylamino)-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (13a). The title compound was obtained from 9d (0.5
g) following the general procedure using an aqueous solution
of ethylamine 70% (10 mL). Yield: 0.38 g (78%); mp 243-248
°C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz).Anal. (C11H15N3O3S)
C, H, N, S.
3-Propylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxide
Monohydrate (22b). The title compound was obtained from
19c (0.5 g) following the general procedure using propylamine
(5 mL). Yield: 0.36 g (74%); mp 190-196 °C (lit.: 195 °C 38);
1
IR (KBr); H NMR (DMSO-d6, 80 MHz). Anal. (C10H13N3O2S‚
H2O) C, H, N, S.
3-Isopropylamino-4H-1,2,4-benzothiadiazine 1,1-Diox-
ide (22c). The title compound was obtained from 19c (0.5 g)
following the general procedure using isopropylamine (5 mL).
Yield: 0.36 g (74%); mp 213-218 °C (lit.: 222-223 °C 38); IR
7-Ethoxy-3-isopropylamino-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (13b). The title compound was obtained from
9d (0.5 g) following the general procedure using isopropy-
lamine (5 mL). Yield: 0.32 g (62%); mp 211-215 °C; IR (KBr);
1H NMR (DMSO-d6, 80 MHz).Anal. (C12H17N3O3S) C, H, N, S.
3-Cyclobutylamino-7-ethoxy-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (13c). The title compound was obtained from
9d (0.5 g) following the general procedure using a solution of
cyclobutylamine (1 mL) in dioxane (5 mL). Yield: 0.31 g (58%);
mp 246-251 °C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz).Anal.
(C13H17N3O3S) C, H, N, S.
General Procedure for the Synthesis of the 7-Substi-
tuted 3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Diox-
ides 20a-c, 21a-c, and 22a-e. The mixture of the appro-
priate 7-substituted 3-(1H-imidazol-1-yl)-4H-1,2,4-benzothia-
diazine 1,1-dioxide intermediate (19a-c, 0.5 g) and the ap-
propriate alkylamine (5 mL) was heated in a hermetically
closed autoclave at 140 °C for 5 h. After cooling, the excess of
the amine was removed by distillation under reduced pressure,
and the residue was suspended in water (20 mL). An aqueous
solution of NaOH (5% w/v) was added dropwise until dissolu-
tion of the residue. The alkaline solution was treated with
charcoal and filtered, and the filtrate was adjusted to pH 4-5
(pH 2 for 21a-c) with 6 N HCl. The precipitate was collected
by filtration, washed with water, and dried. The compound
was purified by crystallization in an appropriate solvent.
3-Propylamino-7-trifluoromethyl-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (20a). The title compound was obtained
from 19a (0.5 g) following the general procedure using propy-
lamine (5 mL). Yield: 0.26 g, (54%); mp 241-243 °C; IR (KBr);
1H NMR (DMSO-d6, 80 MHz). Anal. (C11H12F3N3O2S) C, H,
N, S.
1
(KBr); H NMR (DMSO-d6, 80 MHz). Anal. (C10H13N3O2S) C,
H, N, S.
R/S-3-(2-Butylamino)-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (22d). The title compound was obtained from 19c
(0.5 g) following the general procedure using (R/S)-2-buty-
lamine (5 mL). Yield: 0.35 g (68%); mp 179-181 °C; IR (KBr);
1H NMR (DMSO-d6, 80 MHz). Anal. (C11H15N3O2S) C, H, N,
S.
3-Cyclobutylamino-4H-1,2,4-benzothiadiazine 1,1-Di-
oxide (22e). The title compound was obtained from 19c (0.5
g) following the general procedure using a solution of cyclobu-
tylamine (1 mL) in dioxane (5 mL). Yield: 0.30 g (60%); mp
239-242 °C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz). Anal.
(C11H13N3O2S) C, H, N, S.39
7-Cyano-3-isopropylamino-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (24a). 7-Iodo-3-isopropylamino-4H-1,2,4-ben-
zothiadiazine 1,1-dioxide26 (23a, 1.2 g, 3.28 mmol) and CuCN
(0.6 g, 6.76 mmol) were suspended in anhydrous DMF (10 mL)
under nitrogen, and the mixture was heated under reflux for
5 h. The mixture was supplemented with water (30 mL). The
resulting suspension was adjusted to pH 12 by means of an
aqueous solution of NaOH (10% w/v), and the alkaline solution
was treated with charcoal and filtered. The filtrate was
adjusted to pH 1 with 6 N HCl. The precipitate was collected
by filtration, washed with water, dried, and recrystallized from
hot methanol. Yield: 0.44 g (51%); mp > 300 °C; IR (KBr); 1H
NMR (DMSO-d6, 80 MHz). Anal. (C11H12N4O2S) C, H, N, S.
7-Cyano-3-cyclobutylamino-4H-1,2,4-benzothiadiaz-
ine 1,1-Dioxide (24b). The title compound was obtained as
described for 24a starting from 3-cyclopropylamino-7-iodo-4H-
1,2,4-benzothiadiazine 1,1-dioxide26 (23b, 1.2 g, 3.18 mmol).
Yield: 0.48 g (55%); mp 299-303 °C; IR (KBr); 1H NMR
(DMSO-d6, 80 MHz). Anal. (C12H12N4O2S) C, H, N, S.
Methyl 3-isopropylamino-4H-1,2,4-benzothiadiazine-
7-carboxylate 1,1-Dioxide (25). 3-Isopropylamino-4H-1,2,4-
benzothiadiazine-7-carboxylic acid 1,1-dioxide (21b, 0.35 g,
1.23 mmol) was suspended in a solution of H2SO4 (0.28 mL)
and methanol (3.5 mL), and the mixture was heated under
reflux for 4 h. The solvent was removed under reduced
pressure. The residue was poured into water (20 mL). The
3-Isopropylamino-7-trifluoromethyl-4H-1,2,4-benzothi-
adiazine 1,1-Dioxide (20b). The title compound was from
19a (0.5 g) following the general procedure using isopropy-
lamine (5 mL). Yield: 0.32 g (66%); mp 287-289 °C; IR (KBr);
1H NMR (DMSO-d6, 80 MHz). Anal. (C11H12F3N3O2S) C, H,
N, S.
R/S-3-(2′-Butylamino)-7-trifluoromethyl-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (20c). The title compound was
obtained from 19a (0.5 g) following the general procedure using
R/S-2-butylamine (5 mL). Yield: 0.34 g (68%); mp 234-236

3502 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Boverie et al.
resulting suspension was adjusted to pH 8 by means of an
aqueous solution of NaOH (10% w/v). The precipitate was
collected by filtration, washed with water, dried, and recrystal-
lized from hot methanol. Yield: 0.14 g (41%); mp 230-236 °C;
IR (KBr); 1H NMR (DMSO-d6, 500 MHz). Anal. (C12H15N3O4S)
C, H, N, S.
3-Isopropylamino-4H-1,2,4-benzothiadiazine-7-carbox-
amide 1,1-Dioxide Monohydrate (26). 3-Isopropylamino-
4H-1,2,4-benzothiadiazine-7-carboxylic acid 1,1-dioxide (21b,
0.35 g, 1.23 mmol) and carbonyldiimidazole (0.22 g, 1.35 mmol)
were solubilized in DMF (4 mL), and the mixture was heated
for 2 h at 60 °C. The mixture was added under stirring to an
aqueous solution of ammonia (5 mL, 10% w/v). After 30 min.,
the suspension was adjusted to pH 5 with 6 N HCl. The
crystalline precipitate was collected by filtration washed with
water, and dried. Yield: 0.17 g (48%), mp > 280 °C; IR (KBr);
1H NMR (DMSO-d6, 500 MHz). Anal. (C11H14N4O3S‚H2O) C,
H, N, S.
3-Ethylamino-7-methylsulfonyl-4H-1,2,4-benzothiadi-
azine 1,1-Dioxide (31a). A solution of phosgene (0.52 mL)
in toluene (20%) was added dropwise to a solution of N-(2-
amino-5-methylsulfonylbenzenesulfonyl)-N′-ethylthiourea (30a,
1 g, 2.9 mmol) and triethylamine (1 mL, 7.1 mmol) in dry THF
(30 mL). The solution was stirred at 0 °C for 1 h. The solvent
was removed under reduced pressure. The residue was poured
into water (20 mL) and stirred for 40 min. The crude product
was isolated by filtration, washed with water, dried, and
recrystallized from MeOH/water. Yield: 0.54 g (60%); mp 264-
270 °C; IR (KBr); 1H NMR (DMSO-d6, 80 MHz). Anal.
(C10H13N3O4S2) C, H, N, S.
3-Isopropylamino-7-methylsulfonyl-4H-1,2,4-benzothi-
adiazine 1,1-Dioxide (31b). The title compound was obtained
as described for 31a starting from N-(2-Amino-5-methylsul-
fonylbenzenesulfonyl)-N′-isopropylthiourea (30b, 1 g, 2.8 mmol).
Yield: 0.58 g (65%); mp 272-276 °C IR (KBr); 1H NMR
(DMSO-d6, 80 MHz). Anal. (C11H15N3O4S2) C, H, N, S.
R/S-3-(2-Butylamino)-7-methylsulfonyl-4H-1,2,4-ben-
zothiadiazine 1,1-Dioxide (31c). The title compound was
obtained as described for 31a starting from N-(R/S)-(2-
Amino-5-methylsulfonylbenzenesulfonyl)-N′-2-butylthiourea
(30c, 1 g, 2.7 mmol). Yield: 0.60 g (66%); mp 244-245 °C; IR
(KBr); 1H NMR (DMSO-d6, 80 MHz). Anal. (C12H17N3O4S2) C,
H, N, S.
Ionization Constants. The pKa values of the compounds
were determined by means of UV spectrophotometry using a
Perkin-Elmer UV/Vis 554 spectrophotometer at 25 °C. UV
spectra of compounds were taken in different aqueous buffers
of pH ranking from 5 to 12. The pKa values were calculated
by the Debye-Hu¨ckel equation at the maximum basic form
absorbance.40
Biological Assays. Measurements of Insulin Release
from Incubated Rat Pancreatic Islets. Pancreatic islets
were isolated by 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, 0.5% (w/v) dialyzed albumin (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, in
addition, the reference compound or the benzothiadiazine
derivative. The release of insulin was measured radioimmu-
nologically using rat insulin as a standard.41 Residual insulin
release was expressed as a percentage of the value recorded
in control experiment (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 aorta removed from fed
Wistar rats (180-200 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 value
(drug concentration evoking 50% inhibition of the plateau
phase induced by KCl) were assessed from dose curves using
Datanalyst software (EMKA Technologies, France).
Measurement of 86Rb Outflow from Rat Pancreatic
Islets. The method used for measuring 86Rb (42K substitute)
outflow from prelabeled and perifused rat pancreatic islets was
previously described in detail.12,23
Supporting Information Available: Elemental analyses
of the newly synthesized compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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