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

Article abstract of DOI:10.1021/jm9500582

4-N-Subsituted and -unsubstituted 3-alkyl- and 3-(alkylamino)-4H- pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides were synthesized and tested vs diazoxide and selected 3-alkyl- and 3-(alkylamino)-7-chloro-4H-1,2,4- benzothiadiazine 1,1-dioxides as potassium channel openers on pancreatic and vascular tissues. Several 4-N-unsubstituted 3-(alkylamino)pyridothiadiazines and some 3-(alkylamino)-7-chlorobenzothiadiazines were found to be more potent than diazoxide for the inhibition of the insulin-releasing process. Moreover, the 3-(alkylamino)pyridothiadiazines appeared to be more selective for the pancreatic than for the vascular tissue. By means of the pharmacological results obtained on pancreatic B-cells, structure-activity relationships were deduced and a pharmacophoric model for the interaction of these drugs with their receptor site associated to the pancreatic K(ATP) channel was proposed. According to their selectivity for the B-cell (endocrine tissue) vs the vascular (smooth muscle tissue) ionic channel, selected 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides may serve as pharmacological tools in studying the K(ATP) channels ('pancreatic- like' K(ATP) channels) in other tissues.

Full text of DOI:10.1021/jm9500582

J . Med. Chem. 1996, 39, 937-948
937
3
- a n d 4-Su bstitu ted 4H-P yr id o[4,3-e]-1,2,4-th ia d ia zin e 1,1-Dioxid es a s P ota ssiu m
Ch a n n el Op en er s: Syn th esis, P h a r m a cologica l Eva lu a tion , a n d
Str u ctu r e-Activity Rela tion sh ip s
,
†
†
‡
§
|
‡
Pascal de Tullio,* Bernard Pirotte, Philippe Lebrun, J eanine Fontaine, L e´ on Dupont, Marie-H e´ l e` ne Antoine,
‡
†
‡,§
†
†
†
Raogo Ouedraogo, Smail Khelili, Carine Maggetto, Bernard Masereel, Ousmane Diouf, Tchao Podona, and
†
J acques Delarge
Laboratoire de Chimie Pharmaceutique, Universit e´ de Li e` ge, 3, rue Fusch, B-4000, Li e` ge, Belgium, Laboratoire de
Pharmacodynamie et de Th e´ rapeutique, Universit e´ Libre de Bruxelles, 808, Route de Lennik, B-1070, Bruxelles, Belgium,
Laboratoire de Pharmacologie, Universit e´ Libre de Bruxelles, Campus Plaine, Boulevard du Triomphe, B-1050, Bruxelles,
Belgium, Laboratoire de Cristallographie, Universit e´ de Li e` ge, Sart Tilman, B-4000, Li e` ge, Belgium
X
Received J anuary 30, 1995
4
-N-Subsituted and -unsubstituted 3-alkyl- and 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadi-
azine 1,1-dioxides were synthesized and tested vs diazoxide and selected 3-alkyl- and
-(alkylamino)-7-chloro-4H-1,2,4-benzothiadiazine 1,1-dioxides as potassium channel openers
3
on pancreatic and vascular tissues. Several 4-N-unsubstituted 3-(alkylamino)pyridothiadiazines
and some 3-(alkylamino)-7-chlorobenzothiadiazines were found to be more potent than diazoxide
for the inhibition of the insulin-releasing process. Moreover, the 3-(alkylamino)pyridothiadi-
azines appeared to be more selective for the pancreatic than for the vascular tissue. By means
of the pharmacological results obtained on pancreatic B-cells, structure-activity relationships
were deduced and a pharmacophoric model for the interaction of these drugs with their receptor
site associated to the pancreatic KATP channel was proposed. According to their selectivity for
the B-cell (endocrine tissue) vs the vascular (smooth muscle tissue) ionic channel, selected
3
-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides may serve as pharmacological
tools in studying the KATP channels (“pancreatic-like” KATP channels) in other tissues.
In tr od u ction
Potassium channels (K channels) play a crucial role
+
1
,2
in controlling the cellular membrane potential. Among
those, the ATP-sensitive potassium channels (or KATP
channels) have been characterized in numerous cell
types such as cardiac cells, pancreatic B-cells, skeletal
and smooth muscle cells, and central neurons.3 These
channels appear to be involved in important physiologi-
cal processes.4 In pancreatic B-cells, these channels
have been shown to mediate the glucose-induced insulin
,4
5
,6
secretion.
In the vascular tissue, the KATP channels
play an important role in controlling muscle tone and
7
contractility. Hypoglycemic sulfonylureas such as tol-
butamide and glibenclamide are generally reported as
selective blockers of the KATP channels. Conversely,
F igu r e 1. Chemical structure of different potassium channel
openers (1-3) and of AO44 (4).
8
several compounds such as cromakalim, minoxidil sul-
fate, nicorandil, diazoxide (1, Figure 1) and pinacidil (2)
channels but without important tissue selectivity. In
contrast, pinacidil and cromakalim show a stronger
activity on vascular smooth muscle cells than on pan-
creatic B-cells and exhibit a clear selectivity for the
are typical examples of different chemical classes of
potassium channel openers (PCOs).9
,10
PCOs exert their
+
biological effects by increasing the K channel perme-
ability, a probable result of their opening properties on
1
2,14,15
smooth muscle vs the endocrine tissue.
3
,11
KATP channels.
Recently, we synthesized novel 4H-pyrido[4,3-e]-1,2,4-
thiadiazine 1,1-dioxides (3, Figure 1), bearing different
The ability of PCOs to activate KATP channels may
vary considerably according to the tissue localization of
the channel. The antihypertensive agent diazoxide
seems to exert a comparable efficacy in relaxing vascu-
lar smooth muscle and in inhibiting insulin secre-
16
aminoalkyl side chains in the 3-position. It was found
that some of these compounds were the most powerful
16-19
inhibitors of insulin secretion reported to date.
Further pharmacological investigations have shown
that the biological target of BPDZ 44 (3, R ) CH(CH3)-
CH(CH3)2) on pancreatic B-cells, like diazoxide and
1
2,13
tion.
These effects of diazoxide result from its ability
to activate both the vascular and the pancreatic KATP
1
7,18
pinacidil, was the KATP channel.
The activity of
†
Laboratoire de Chimie Pharmaceutique, Universit e´ de Li e` ge.
Laboratoire de Pharmacodynamie et de Th e´ rapeutique, Universit e´
these new PCOs on other cell types, in particular on
vascular smooth muscle cells, remained to be explored.
‡
Libre de Bruxelles.
§
Laboratoire de Pharmacologie, Universit e´ Libre de Bruxelles.
Laboratoire de Cristallographie, Universit e´ de Li e` ge.
Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
|
The present work constitutes an expanded version of
X
16
our previous Communication to the Editor giving a
0
022-2623/96/1839-0937$12.00/0 © 1996 American Chemical Society

9
38 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
de Tullio et al.
Sch em e 1a
Sch em e 2a
a
(i) Acetic anhydride.
larger chemical and pharmacological exploration of the
pyrido- and benzothiadiazinedioxide class of PCOs.
Thus, pyridothiadiazinedioxides bearing a variety of 3-
or 4-alkyl and 3-aminoalkyl side chains, and 7-chlo-
robenzothiadiazine dioxides bearing selected 3-alkyl and
3
-aminoalkyl side chains were synthesized and evalu-
ated as PCOs on pancreatic B-cells and on isolated rat
aorta. The most interesting pyrido- and benzothiadi-
azinedioxides from different series of compounds were
also investigated on guinea pig ileum and on rat heart
ventricle. Particular attention was paid to the possible
improved selectivity of these pyridothiadiazines and
7
-chlorobenzothiadiazines for one of these tissues.
The pharmacological results obtained with the new
compounds allowed us to deduce structure-activity
relationships that could explain the biological activity
of pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides on pan-
creatic B-cells. Assuming that their activity on insulin
secretion results from their K channel-opening proper-
ties, a pharmacophoric model for their interaction with
the pancreatic KATP channel was proposed.
a
(i) R′-COOCOR′; (ii) trifluoroacetic anhydride; (iii) OPCl3, ∆;
iv) cyclopentanecarboxylic acid, OPCl3; (v) NaOH, H2O; (vi) ∆.
(
+
Sch em e 3a
Ch em istr y
The 4-substituted-3-methyl-4H-pyrido[4,3-e]-1,2,4-
thiadiazine 1,1-dioxides 11-16 were obtained by cy-
clization of the corresponding (4N-substituted-4-ami-
nopyrid-3-yl)sulfonamides20 5-10 with acetic anhydride
(Scheme 1). The synthesis of compounds 19-23 was
achieved by action of the appropriate anhydride on (4-
aminopyrid-3-yl)sulfonamide 17 (Scheme 2). Starting
from the same compound 17, treatment with trifluoro-
acetic anhydride and cyclization of the monoacylated
intermediate 24 by means of OPCl3 led to the pyrido-
thiadiazine 25. Compound 28 was obtained in three
steps as described in Scheme 2 from 17 via a diacylated
a
(i) RNH ; (ii) R′-NHR′′.
2
2
2
6 and a monoacylated 27 intermediate. Hydrolysis of
6 in mild alkaline conditions resulted in a selective
mediate 47 compared to the non-4-methyl compound 29.
This higher reactivity of 47 may be explained by the
higher electrophilicity of the carbon atom in the 3-posi-
tion compared to that of compound 29, since no depro-
tonation can occur in the 4-position in the presence of
the amine.
deacylation in the 4-position. Such selectivity was
explained by the reduction of the electrophilic character
of the acylsulfonamide fonction due to its deprotonation
in the alkaline medium. The synthesis of the com-
pounds 32, 39, 42, and 44 was achieved by following
the experimental conditions previously described for the
compounds 31, 33-38, 40, 41, and 43,16 starting from
the key intermediate 29, and treating them with an
excess of the appropriate amine (Scheme 3). For the
Compound AO44 was prepared from the reaction of
diazoxide and methyl iodide in alcoholic alcaline solution
2
1
as reported in the litterature.
Compound 56 was obtained in four steps (Scheme 5)
starting from the reaction of p-chloroaniline 52 with
chlorosulfonyl isocyanate according to a previously
described process leading to the corresponding 3-ox-
3
-(alkylamino)-4-methyl-4H-pyrido[4,3-e]-1,2,4-thiadiaz-
ine 1,1-dioxides 48-51, the intermediate 47, obtained
from 5 in three steps (Scheme 4), was treated with an
excess of the appropriate amine as described above for
the 3-aminoalkyl compounds 32-43. However, milder
conditions were required for these reactions, probably
due to an increasing reactivity of the 4-methyl inter-
2
2
obenzothiadiazine dioxide 53. Hydrolysis of 53 gave
the corresponding aminobenzenesulfonamide 5422 which
was acylated in the presence of cyclopentanecarboxylic
acid chloride to give the monoacylated intermediate 55.

4
H-Pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 939
Sch em e 4a
Sch em e 6a
a
(i) RNH2.
pancreatic islets incubated in the presence of an insuli-
notropic glucose concentration (16.7 mM). A 90%
inhibition (10% residual insulin secretion) may be
considered as a full effect relative to the glucose-
insensitive basal insulin release. The same compounds
were also investigated for their vasorelaxant activity on
+
K -depolarized rat aorta without endothelium.
As observed in Table 1, 3-alkyl- and 4-alkyl-3-methyl-
4
H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides were less
active than diazoxide and 7-chloro-3-cyclopentyl-4H-
,2,4-benzothiadiazine 1,1-dioxide (56) on pancreatic
1
B-cells and on rat aorta. Compounds 14, 23, and 28
exhibited a poor vasorelaxant activity with ED50 values
a
(
i) NH2CONH2, ∆; (ii) P2S5, pyridine; (iii) NaHCO3, CH3I; (iv)
(
5
∼250 µM) higher than that of diazoxide (21 µM) and
RNH2.
6 (2.8 µM). Interestingly, compound 56 (BPDZ 84),
Sch em e 5a
which is the 3-cyclopentyl analog of diazoxide and is also
the chlorobenzenic analog of 28, was found to be of
comparable potency than the reference compound dia-
zoxide on pancreatic B-cells but was clearly more potent
than the former on vascular tissue. The present in vitro
results on isolated rat aorta corroborate previous ob-
servations reported with several diazoxide analogs. In
a series of 3-alkyl-4H(2H)-1,2,4-benzothiadiazine 1,1-
dioxides, an increasing blocking activity on the norepi-
nephrine-induced contractile response of the rat aorta
was observed when the size of the alkyl substituent in
2
3
the 3-position was increased.
The pharmacological results obtained with 3-(alkyl-
amino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides
and 3-(alkylamino)-7-chloro-4H-1,2,4-benzothiadiazine
1
,1-dioxides on the pancreatic and vascular tissues are
shown in Table 2. Some results on the B-cell secretory
1
6
process have been previously reported. Compound 31,
which is devoided of an alkyl substituent on the exocy-
clic amino group, and the morpholinyl derivative 42
were found to be essentially inactive on the two tissues.
However, the pyridinic and the chlorobenzenic monoalky-
lamine derivatives (32-41 and 58-60) showed a strong
inhibitory activity on the insulin secretion. The pyri-
dinic but not the chlorobenzenic compounds clearly
exhibited a lower vasorelaxant activity than that of
diazoxide.
a
(
i) ClSO2NCO, CH3NO2; AlCl3; (ii) H2SO4 50%; ∆; (iii)
C5H9COCl, C5H5N; (iv) NaOH 1%, ∆.
Cromakalim, which is a strong vasorelaxant agent,
was reported to be ineffective as a PCO in exerting an
agonistic activity on the pancreatic KATP channel.24 On
the other hand, the antihypertensive agent pinacidil (2)
is known to exert a stronger activity on the vascular
than on the pancreatic tissue onto which this drug was
found to be 10 times less active than diazoxide in
Cyclization of the former into the benzothiadiazine
dioxide 56 was achieved in aqueous alkaline medium.
Compounds 58, 59,16 and 60 were obtained from the
reaction of the 3-(methylthio)benzothiadiazine interme-
diate 57 with an excess of the appropriate amine
1
6
according to a previously described process (Scheme 6).
1
2,14
inhibiting insulin release.
Interestingly, the pyrido
Resu lts a n d Discu ssion
compounds 36, 38, and 39, bearing a short branched
alkyl chain closely related (36, 38) to or identical (39)
to that of pinacidil, were the most efficacious pyrido-
The different compounds reported in Tables 1-3 were
tested as inhibitors of the insulin release from rat

9
40 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
de Tullio et al.
Ta ble 1. Percentage of Residual Insulin Secretion and Contractile Activity of 4-Alkyl-3-methyl- and 3-Alkyl-4H-pyrido-[4,3-c]-
,2,4-thiadiazine 1,1-Dioxides Compared to Diazoxide and 7-Chloro-3-cyclopentyl-4H-1,2,4-benzothiadiazine 1,1-Dioxide 56
1
%
residual insulin
% residual contractile
activity ( SEM (n)
a
b
secretion ( SEM (n)
compd
R1
R2
50 µM
10 µM
500 µMc
ED50 (µM)d
1
1
1
1
1
1
1
1
2
2
2
2
2
2
1c
2
3
4
5
CH3
CH(CH3)2
C6H11 (cyclohexyl)
C8H15 (cyclooctyl)
NC4H8O (N-morpholinyl)
NC7H12 (azabicyclooctyl)
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH2CH3
CH2CH2CH3
CH(CH3)2
C(CH3)3
108.8 ( 4.7 (8)
68.1 ( 8.3 (8)
87.9 ( 3.8 (14)
90.6 ( 6.9 (8)
93.5 ( 6.7 (8)
71.9 ( 4.7 (8)
99.5 ( 6.2 (8)
100.4 ( 6.8 (16)
90.2 ( 5.9 (16)
85.1 ( 6.2 (16)
79.7 ( 6.4 (16)
81.1 ( 5.0 (13)
90.4 ( 6.3 (23)
99.3 ( 4.6 (32)
28.8 ( 2.5 (22)
14.1 ( 0.9 (15)
94 ( 1 (3)
86 ( 3 (3)
52 ( 4 (5)
+++f
89 ( 9 (3)
53 ( 15 (4)
94 ( 6 (3)
92 ( 2 (5)
74 ( 8 (6)
77 ( 4 (4)
71 ( 7 (6)
+++
248 ( 57 (5)
6
8
9
e
e
0
1
2
3
5
8
258 ( 78 (6)
CF3
96 ( 4 (3)
+++
+++
+++
C5H9 (cyclopentyl)
257 ( 44 (5)
21 ( 7 (6)
2.8 ( 0.7 (5)
diazoxide
5
70.0 ( 3.6 (22)
85.7 ( 2.8 (16)
6
a
b
Percentage of residual insulin release from rat pancreatic islets incubated in presence of 16.7 mM glucose. Activity measured on
+
c
d
K -depolarized rat aorta. Percentage of residual contractile response to 30 mM KCl in isolated rat aorta. ED50: drug concentration
giving 50% relaxation of the KCl-induced contraction. For the synthesis see ref 25. g +++ indicates that a maximal vasorelaxant activity
was observed at 500 µM.
e
Ta ble 2. Percentage of Residual Insulin Secretion and Contractile Activity of 3-(Alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine
1
,1-Dioxides and 3-(Alkylamino)-7-chloro-4H-1,2,4-benzothiadiazine 1,1-Dioxides
%
residual insulin secretion ( SEM (n)a
% residual contractile activity ( SEM (n)b
compd
R
50 µM
10 µM
1 µM
500 µMc
ED50 (µM)d
3
1c
2
H
104.1 ( 6.5 (14)
59.2 ( 3.6 (14)
77 ( 13 (3)
87 ( 2 (5)
88 ( 7 (4)
89 ( 2 (4)
74 ( 3 (4)
34 ( 10 (6)
+++f
3
3
3
3
3
3
3
3
4
4
4
5
5
6
CH2CH3
(CH2)2CH3
CH(CH3)2
(CH2)3CH3
CH(CH3)CH2CH3
CH2CH(CH3)2
CH(CH3)CH(CH3)2
CH(CH3)C(CH3)3
C5H9 (cyclopentyl)
C6H11 (cyclohexyl)
NC4H8O (N-morpholinyl)
CH(CH3)CH2CH3
CH(CH3)CH(CH3)2
CH(CH3)C(CH3)3
c
3
4
5
6
7
8
57.7 ( 3.8 (14) 87.3 ( 6.7 (15)
97.1 ( 5.9 (8)
110.3 ( 11.3 (6)
83.7 ( 6.2 (8)
97.8 ( 9.1 (8)
84.2 ( 11.0 (8)
70.5 ( 4.4 (19)
70.0 ( 3.4 (24)
82.8 ( 9.7 (8)
74.4 ( 4.5 (8)
c
42.6 ( 6.9 (8)
38.6 ( 5.6 (7)
9.4 ( 0.9 (8)
23.4 ( 3.1 (8)
8.6 ( 0.9 (23) 13.7 ( 1.2 (23)
10.3 ( 0.8 (20) 41.0 ( 2.2 (19)
31.6 ( 4.0 (7)
40.5 ( 4.3 (8)
98.8 ( 3.9 (16)
5.5 ( 0.4 (16) 19.9 ( 1.4 (14)
11.0 ( 0.8 (16) 62.7 ( 3.6 (15)
9.3 ( 1.0 (30) 46.9 ( 4.1 (16)
28.8 ( 2.5 (21) 70.0 ( 3.6 (22)
55.3 ( 8.1 (8)
82.0 ( 6.2 (8)
49.0 ( 5.4 (7)
71.8 ( 7.2 (16)
c
c
c
205 ( 53 (12)
302 ( 68 (8)
104 ( 80 (7)
c
+++
9
0
1
2
+++
c
71.1 ( 11.2 (7)
64.4 ( 9.5 (7)
60 ( 4 (4)
40 ( 12 (4)
93 ( 6 (6)
+++
c
c
8
9
85.4 ( 3.7 (15)
88.9 ( 6.0 (15)
87.7 ( 4.5 (16)
78.9 ( 3.8 (21)
25 ( 8 (11)
32 ( 15 (5)
10 ( 3 (15)
21 ( 7 (6)
c
+++
0
+++
diazoxide
+++
a
b
Percentage of residual insulin release from rat pancreatic islets incubated in presence of 16.7 mM glucose. Activity measured on
+
c
d
K -depolarized rat aorta. Percentage of the contractile response to 30 mM KCl in isolated rat aorta. ED50: drug concentration giving
e
5
0% relaxation of the KCl-induced contraction. For the synthesis see ref 16. f +++ indicates that a maximal vasorelaxant activity was
observed at 500 µM.
thiadiazines tested on B-cells. They were more active
than diazoxide at a 50 and at a 10 µM concentration.
Compound 38 (BPDZ 44) exhibited an IC50 value of 3.80
were equal or superior to diazoxide as putative PCOs
acting on the pancreatic tissue and seemed to confirm
the favorable influence of an aminoalkyl chain in the
(
0.46 µM. When introduced on the 7-chlorobenzothia-
3
-position in targeting the pancreatic receptor.
diazine structure, these three selected aminoalkyl side
chains led to the compounds 58-60, exhibiting a potent
inhibitory activity on the insulin-releasing process
which was of the same order of magnitude to that
expressed by their pyridinic counterparts. Thus, they
On isolated rat aorta, compound 39 (BPDZ 62) was
found to be the most active pyridinic derivative (ED50
) 104 µM), but remained less potent than diazoxide
(ED50 ) 21 µM) or the other 7-chloro-(3-alkyl/3-alkyl-

4
H-Pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 941
Ta ble 3. Percentage of Residual Insulin Secretion and Contractile Activity of 3-(Dialkylamino)- and 3-(Alkylamino)-4-methyl-4H-
pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides Compared to Diazoxide and AO44
%
residual insulin
% residual contractile
activity ( SEM (n)
a
b
secretion ( SEM (n)
compd
R1
R2
CH3
C4H8NCH3 (N-methylpiperazinyl)
R3
50 µM
10 µM
500 µMc
ED50 (µM)d
4
4
4
4
5
5
3
4
8
9
0
1
H
H
CH3
CH3
CH3
CH3
(CH2)3CH3
76.1 ( 4.3 (7)
90.6 ( 7.6 (15)
79.4 ( 5.9 (15)
81.0 ( 7.1 (13)
95.4 ( 5.3 (14)
88.5 ( 8.2 (12)
83.9 ( 5.4 (23)
28.8 ( 2.5 (21)
47 ( 8 (3)
+++e
84 ( 7 (3)
61 ( 15 (3)
69 ( 12 (3)
74 ( 13 (3)
125 ( 30 (11)
H
H
H
H
CH(CH3)2
CH(CH3)CH2CH3
CH(CH3)CH(CH3)2
CH(CH3)C(CH3)3
AO44
diazoxide
70.0 ( 3.6 (22)
+++
21 ( 7 (6)
a
b
Percentage of residual insulin release from rat pancreatic islets incubated in presence of 16.7 mM glucose. Activity measured on
+
c
d
K -depolarized rat aorta. Percentage of the contractile response to 30 mM KCl in isoalted rat aorta. ED50: drug concentration giving
e
5
0% relaxation of the KCl-induced contraction. +++ indicates that a maximal vasorelaxant activity was observed at 500 µM.
amino)benzothiadiazine derivatives, and markedly less
potent than pinacidil (ED50 ) 0.55 µM).15
A very interesting observation in this series of com-
pounds was that the 3-(alkylamino)-4H-pyrido[4,3-e]-
1
,2,4-thiadiazine 1,1-dioxides (i.e. 34, 36, 38, 39) were
clearly more selective for the pancreatic tissue than for
the vascular tissue. By contrast, the 3-alkyl- and
3
-(aminoalkyl)-7-chloro-4H-1,2,4-benzothiadiazine 1,1-
dioxides studied (diazoxide, 56, 58, 59, 60) were less
selective. The most potent drug on insulin secretion,
and also the most selective for the pancreatic tissue, was
the compound 38 with an ED50 (vasorelaxant activity)/
IC50 (insulin release inhibition) ratio of 302 µM/3.8 µM
)
∼80. The ED50/IC50 ratio of diazoxide (21 µM/24.8
µM), which is close to the unity, clearly indicates a poor
tissue selectivity for the reference compound.
F igu r e 2. Molecular structure of compound 34 with atom-
labeling scheme. Displacement ellipsoids are shown at 50%
probability levels. H atoms are drawn as small circles of
arbitrary units.
Some 3-N,N-disubstituted aminoalkyl compounds
were also prepared and evaluated on the pancreatic and
vascular tissue (Table 3). In particular, compound 44
is the pyridinic analog of 7-chloro-3-(N-methylpiperazi-
nyl)-4H-1,2,4-benzothiadiazine 1,1-dioxide, a diazoxide
2
5
analog reported as an antihypertensive agent.
It
appeared that the absence of a hydrogen atom on the
exocyclic nitrogen atom (compounds 43 and 44) led to a
decrease of the activity on pancreatic B-cells. However,
compound 44 exhibited a poor but measurable efficacy
+
in relaxing K -depolarized rat aorta.
Since the 4-methyl derivative of diazoxide AO44 was
reported to exhibit a stronger in vivo hyperglycemic
activity than diazoxide,20 this compound and the 3-(alkyl-
amino)-4-methyl-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-
dioxides 48-51 were synthesized and tested on pan-
creatic B-cells. These compounds were found to be less
active than their non-methyl counterparts (34, 36, 38,
and 39) at a 50 µM concentration. Moreover, at this
concentration, AO44 did not show any significant in
vitro activity, in apparent contradistinction to its previ-
ously reported in vivo effect.20
F igu r e 3. Chemical structure of selected compounds repre-
sentative of the different classes of pyrido- and benzothiadi-
azinedioxide PCOs.
Selected compounds i.e. 28, 38, 39, 56, and 59 (Figure
3
), which are representative of the different classes of
pyrido- and benzothiadiazinedioxides tested on the
pancreatic and the vascular tissues, were also examined
vs diazoxide and pinacidil on the guinea pig ileum and
on the rat ventricle.
PCOs i.e. cromakalim and pinacidil have been found
to exert interesting biological activities on a variety of
gastrointestinal tissue preparations such as inhibition
of contractions evoked by electrical field stimulation or

9
42 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
de Tullio et al.
by high K+ (10-30 mM KCl) solutions.10 Since glib-
enclamide antagonized the relaxant effects of the two
PCOs on the gastrointestinal smooth muscle cells, the
An electron-deficient hydrogen atom in the 4-position
is also expected to help the establishment of a strong
hydrogen bond interaction with the receptor site.
1
0
presence of putative KATP channels was suggested.
Because the acidic character of pyrido- and benzothia-
diazinedioxides is related to the lability of the former
4-H atom, it was of considerable interest to determine
the pKa values of some representative drugs. The
ionization constant was determined for selected 3-alkyl-
(19, pKa ) 7.65; 28, pKa ) 7.95) and 3-(alkylamino)-
4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides (32, pKa
) 8.09; 39, pKa ) 8.15). The 3-alkyl derivatives
presented a lower pKa value compared to that of the
3-aminoalkyl ones, the latter drugs having also a lower
The inhibitory effect of diazoxide, pinacidil, and the
selected drugs 28, 38, 39, 56, and 59 on the electrically
induced contractions (coaxial stimulation) of the guinea
pig ileum was measured. Pinacidil was found to be the
most potent drug with an ED50 of 18.0 ( 6.6 µM (n ) 5)
-
4
(
∼75% inhibition at a 1 × 10 M concentration of drug).
None of the other drugs, including diazoxide, were found
to exert a strong inhibitory activity on the ileum
-
4
contractions (<20% inhibition at 1 × 10 M; n ) 5)
2
6
except compound 59 which gave ∼40% inhibition at 1
pKa value than that of diazoxide (pKa ) 8.62 ). In
accordance with their pKa values, diazoxide as well as
compounds 32 and 39 are mainly present in solution
as nonionic species at physiological pH. Thus, it is
concluded that the neutral form of the drug, which
warrants the hydrogen atom in the 4-position, is re-
quired for optimal interaction with the binding site.
-
4
×
10 M (n ) 5). Thus the most active compound on
vascular tissue (compound 56) was found to be clearly
less active than the compound 59, indicating different
possible responses of PCOs when tested on different
smooth muscle cell types.
In addition to shortening the cardiac action potential
duration, PCOs such as cromakalim have displayed
negative inotropic activity in isolated cardiac tissue.
However, the effects of PCOs on this tissue were mainly
observed at considerably higher concentrations than
those required for effective vasorelaxation.10
Assuming that crystallographic informations on
3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-
dioxides could also help our knowledge of the structural
requirements responsible for optimal activity on the
pancreatic tissue, we tried to determine the X-ray
structure of the most active and most representative
compound 38. However, due to some difficulties in
obtaining suitable crystals of 38, its isopropyl homolog
The contractile activity of right ventricular strips from
rat heart was measured in the absence and the presence
of the selected pyrido- and benzothiadiazines and of the
two reference compounds diazoxide and pinacidil. The
negative inotropic effect of the two references and the
3
4 was ultimately and successfully selected for this
purpose.
It was found that the N(2)-C(3) length (1.315 Å) of
-
4
five selected compounds at a 1 × 10 M concentration
of drug (n ) 4) never exceeded 15% decrease in the
systolic tension of rat ventricles.
compound 34 was shorter than its C(3)-N(4) length
(1.366 Å), supporting the view that the CdN double
bond of the thiadiazine ring is preferentially located in
the 2,3-positions corresponding for 34 to the adoption
of a 4H-tautomeric form. Such a preferred conformation
These results on two other muscle cell types seem to
indicate that the selected pyrido- and benzothiadiaz-
inedioxide PCOs examined in the present work are not
expected to exert a significant activity on these tissues
at concentrations for which a biological response was
observed on the vascular and/or the pancreatic endo-
crine tissues.
From pharmacological results reported on pancreatic
B-cells, structure-activity relationships for the inhibi-
tory activity of the new drugs can be deduced. 3-(Alkyl-
amino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides
2
7
has already been observed with diazoxide and with
previously reported 3-alkylpyridothiadiazines2
the solid state.
6,28,29
in
Interestingly, the displacement-ellipsoid representa-
tion of compound 34 (Figure 2) has clearly shown that
the two N-H hydrogen atoms located either on the
exocyclic or on the 4-N nitrogen atom adopt a spatial
orientation which is quite similar to that taken by the
H atoms of the two cyanoguanidine N-H groups of
(
1
compounds 32-41) and 3-(alkylamino)-7-chloro-4H-
3
0
pinacidil in the solid state.
(See Figure 1 for the
,2,4-benzothiadiazine 1,1-dioxides (compounds 58-60)
crystal conformation of pinacidil.) It is tempting to
speculate that such a similar conformation of the
were active drugs on insulin secretion but the pyridinic
derivatives are the most selective for the pancreatic
tissue. The activity of the pyridothiadiazines increased
with the enhancement of the size and the ramification
of the alkylamino chain in the 3-position. A limit of
steric hindrance for the alkylamino substituent in this
position should correspond to the side chain of com-
pounds 38 and 39 since a decreasing activity was
observed with more bulky substituents such as cy-
cloalkylamino side chains (see compounds 40 and 41).
Interestingly, one of the best alkylamino substituents
for the pyrido- and benzothiadiazines is the (1,2,2-
trimethylpropyl)amino chain also encountered in the
chemical structure of pinacidil.
“guanidinic” moiety will be that preferentially adopted
by both molecules in solution and maybe during their
binding site interaction with their pancreatic B-cell
receptors.
From X-ray data of diazoxide27 and compound 34,
their isopotential map was performed using SYBIL 6.1
software package (atomic charge distribution calculated
with MOPAC AM1). The comparison between these two
stereoelectronical representations did not show marked
differences (Figure 4). The present result confirms, in
this particular case, the excellent analogy between a
chlorobenzenic and a pyridinic moiety for which the
heterocyclic nitrogen atom is located at the same posi-
tion as that of the halogeno-substituted carbon atom.
The presence of an hydrogen atom on the 4-N nitrogen
atom and on the exocyclic nitrogen atom seemed to be
required to maintain a strong inhibitory activity on
insulin secretion. Indeed, methylation of these two
positions considerably reduced the biological activity.
By means of the physicochemical and pharmacological
results discussed, structure-activity relationships were
deduced and a pharmacophoric model for the interaction

4
H-Pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 943
F igu r e 4. Electrostatic isopotential maps of diazoxide and of compound 34. Negative fields (-1 kcal/mol) are presented in bold
black (inferior contour), neutral fields (0 kcal/mol) in light gray, and positive fields (+1 kcal/mol) in dark gray (superior contour).
acter of the hydrogen atom in the 4-position, a possible
favorable requirement for an optimal activity.
Finally, in the series of 3-aminoalkyl derivatives, an
aromatic ring with an halogen atom at the 7-position,
or better, a 7-aza pyridinic ring isostere, is favorable to
an optimal binding site interaction. More examples of
compounds remains to be synthesized and tested in
order to define the exact nature of the substituent in
the 7-position.
Interestingly, the structure-activity relationships
found for an agonistic activity on the pancreatic KATP
channel ressembles, at least in part, those suggested
for benzopyrans (i.e. cromakalim) on the smooth muscle
3
1
KATP channel.
Indeed, the presence of either an
aromatic ring with adequate substituents at the 6-posi-
tion or (6-N)-pyridinic isosteres, a strong electronegative
moiety as hydrogen bond acceptor site (i.e. the pyroli-
dinone carboxamide group) and a hydrophobic (di)alkyl
group (i.e. the dimethyl group) are all structural re-
quirements that could be satisfactorily superimposed to
those suggested for the pyrido- and benzothiadiazine
dioxide PCOs. However, the present pharmacophore is
concerned with an endocrine type of KATP channels
(pancreatic KATP channels) onto which benzopyran
PCOs such as cromakalim are devoid of strong agonistic
activity.
In summary, we have synthesized pyridothiadiazines
bearing a variety of 3- or 4-alkyl and 3-aminoalkyl side
chains. The synthesis of the 3-cyclopentyl analog of
diazoxide and of selected 3-(alkylamino)-7-chloro-4H-
1,2,4-benzothiadiazine 1,1-dioxides was also made and
these benzothiadiazines as well as the pyridothiadiaz-
ines were tested vs diazoxide as inhibitors of insulin
release on pancreatic B-cells and as vasorelaxants on
isolated rat aorta. Three 3-(alkylamino)-4H-pyrido[4,3-
e]-1,2,4-thiadiazine 1,1-dioxides (compounds 36, 38, and
39) appeared to be powerful inhibitors of insulin release
and are clearly more selective than their chlorobenzenic
homologs (compounds 58-60) or than diazoxide for the
pancreatic tissue vs the vascular tissue. The pharma-
cophoric model proposed for their interaction with the
pancreatic KATP channel could help the design of new
therapeutic agents acting on specific tissues. Moreover,
pyridothiadiazine PCOs may serve as interesting phar-
F igu r e 5. Pharmacophoric model for an agonistic activity on
the pancreatic KATP channel.
of the active compounds with the pancreatic KATP
channel was proposed (Figure 5).
Critical structural requirements influence the ago-
nistic activity on the B-cell KATP channel. Firstly, a
short branched lipophilic chain on the nitrogen atom
in the 3-position should be required for an optimal
interaction with an hydrophobic pocket of a limited size
at the receptor site. Up to now, the side chain of
compound 38 constitutes the best choice of size and
ramification. But since the compound is a racemate,
the stereochemistry of the best alkyl chain remains to
be determined.
Secondly, strong hydrogen bond interactions are
probably established between the receptor site and one
or better two N-H groups. Indeed, methylation of these
two former positions decreased the activity. The pres-
ence of a sulfonyl group should be required in order to
impose a particular tautomeric form to the molecules
that adequately orientates the two “guanidinic” N-H
groups. A strong electron-withdrawing functional group
at the 1-position is also able to establish strong hydro-
gen bond interactions with the receptor site, but such a
mechanism remains to be proved i.e. by comparing the
biological response of compounds devoid of a sulfonyl
group in this position. Nevertheless, the presence of the
former group could explain the electron-deficient char-

9
44 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
de Tullio et al.
mide (17,26 1.0 g, 5.8 mmol) and propionic anhydride (10 mL)
was heated at 150 °C for 8 h. After cooling, an equal volume
of diethyl ether was added to the solution. The white
precipitate of crude 20 was collected by filtration, washed with
diethyl ether, dried, and recrystallized from hot water (0.67
g, 50%): mp 220-223 °C; IR (KBr) 3488 (NsH), 1637, 1614,
macological tools in studying the KATP channel (“pan-
creatic-like” KATP channels) in numerous tissues.
Lastly, potent inhibitors of the insulin secretion with
possible lower effects than those of diazoxide on blood
pressure can be expected to become excellent substitutes
for diazoxide in the treatment of pancreatic disorders
associated to an excess of insulin release.
-
1
1
1577, 1510 (CdN, CdC, NsH), 1295, 1167 (SdO) cm ; H
NMR (DMSO-d , 80 MHz) δ 1.15 (t, 3H, 3-CH CH ), 2.55 (q,
), 7.15 (d, 1H, 5-H), 8.6 (d, 1H, 6-H), 8.9 (s, 1H,
-H), 12.0 (bs, 1H, NsH). Anal. (C S‚H O) C, H, N,
6
2
3
2
8
2 3
H, 3-CH CH
H
9
N
3
O
2
2
Exp er im en ta l Section
8
S.
Ch em istr y. Melting points were determined on a B u¨ chi-
Tottoli capillary apparatus and are uncorrected. IR spectra
were recorded as KBr pellets on a Perkin-Elmer 1750 FT
3
-P r op yl-4H-p yr id o[4,3-e]-1,2,4-th ia d ia zin e 1,1-Dioxid e
Mon oh yd r a te (21). A mixture of 4-aminopyridine-3-sulfona-
mide (17, 1.0 g, 5.8 mmol) and butyric anhydride (10 mL) was
heated at 180 °C for 18 h. After cooling, the precipitate of
crude 21 was collected by filtration, washed with butyric
anhydride and diethyl ether, dried, and recrystallized from hot
water (0.78 g, 55%): mp 210-212 °C; IR (KBr) 3511 (NsH),
1
spectrophotometer. The H NMR spectra were taken on a
6
Brucker AW-80 (80 MHz) instrument in DMSO-d with HMDS
as an internal standard; chemical shifts are reported in δ
values (ppm) relative to internal HMDS. The abbreviation s
)
singlet, d ) doublet, t ) triplet, m ) multiplet, and b )
1
cm
CH
CH
648, 1614, 1578, 1509 (CdN, CdC, NsH), 1295, 1167 (SdO)
broad are used throughout. Elemental analyses (C, H, N, S)
were realized on a Carlo-Erba EA 1108-elemental analyser and
were within (0.4% of the theoretical values. All reactions
were routinely checked by TLC on silica gel Merck 60F 254.
-1
1
;
H NMR (DMSO-d
3
6
, 80 MHz) δ 0.85 (t, 3H, 3-CH
2
-
-
2
3
CH ), 1.6 (m, 2H, 3-CH
2
CH CH ), 2.5 (t, 2H, 3-CH CH
2
3
2
2
), 7.15 (d, 1H, 5-H), 8.6 (d, 1H, 6-H), 8.9 (s, 1H, 8-H), 12.2
S‚H O) C, H, N, S.
-Isop r op yl-4H-p yr id o[4,3-e]-1,2,4-th ia d ia zin e 1,1-Di-
oxid e (22). A mixture of 4-aminopyridine-3-sulfonamide (17,
.0 g, 5.8 mmol) and isobutyric anhydride (10 mL) was heated
(bs, 1H, NsH). Anal. (C
9
H
11
N
3
O
2
2
4
-Isop r op yl-3-m et h yl-4H -p yr id o[4,3-e]-1,2,4-t h ia d ia z-
3
in e 1,1-Dioxid e (12). A mixture of 4-(isopropylamino)-
2
0
pyridine-3-sulfonamide (6, 1.0 g, 4.65 mmol) and acetic
anhydride (10 mL) was heated under reflux for 4 h. After
cooling, an equal volume of diethyl ether was added under
stirring. The resulting precipitate was collected by filtration,
washed with diethyl ether, and dried. Recrystallization from
methanol and water gave 12 as a white crystalline compound
1
at 170 °C for 72 h. After cooling, the precipitate of crude 22
was collected by filtration, washed with diethyl ether, dried,
and dissolved in hot methanol. This solution was discolored
with charcoal, and the filtrate was supplemented with 3
volumes of water. After one night at 4 °C, the crystalline 22
was collected, washed with water, and dried (0.59 g, 45%): mp
(
1
d
(
0.78 g, 80%): mp 196-197 °C; IR (KBr) 1657, 1596, 1568,
535 (CdN, CdC), 1315, 1163 (SdO) cm-1; 1H NMR (DMSO-
, 80 MHz) δ 1.55 (d, 6H, 4-CH(CH ), 2.5 (s, 3H, 3-CH ), 4.8
), 7.65 (d, 1H, 5-H), 8.65 (d, 1H, 6-H), 8.9
s, 1H, 8-H). Anal. (C10 S) C, H, N, S.
-Cycloh exyl-3-m eth yl-4H-p yr id o[4,3-e]-1,2,4-th ia d ia z-
in e 1,1-Dioxid e (13). The title compound was obtained from
-(cyclohexylamino)pyridine-3-sulfonamide (7, 1.0 g, 3.9 mmol)
2
48-250 °C; IR (KBr) 3291 (NsH), 1613, 1573, 1510 (CdN,
6
3
)
2
3
CdC, NsH), 1288, 1154 (SdO) cm ; H NMR (DMSO-d
MHz) δ 1.15 (d, 6H, 3-CH(CH ), 2.8 (m, 1H, 3-CH(CH ), 7.2
d, 1H, 5-H), 8.7 (d, 1H, 6-H), 8.9 (s, 1H, 8-H), 12.1 (bs, 1H,
NsH). Anal. (C H N O S) C, H, N, S.
-1 1
6
, 80
3 2
m, 1H, 4-CH(CH )
3
)
2
3 2
)
(
13 3 2
H N O
(
4
9
11
3
2
3
-ter t-Bu tyl-4H-p yr id o[4,3-e]-1,2,4-th ia d ia zin e 1,1-Di-
4
oxid e (23). The title compound was obtained as described
for compound 22, by starting from 4-aminopyridine-3-sulfona-
mide (17, 1.0 g, 5.8 mmol) and trimethylacetic anhydride (10
mL) (0.63 g, 45%): mp 290-293 °C; IR (KBr) 3303 (NsH),
by following the experimental conditions described for 12 (0.71
g, 65%), mp 224-226 °C; IR (KBr) 2943, 2860 (CsH), 1596,
1
1
566, 1535 (CdN, CdC), 1312, 1174 (SdO) cm-1
;
H NMR
(
DMSO-d
H, 3-CH
6
, 80 MHz) δ 1.3-2.2 (bm, 10H, 4-CHC
5
H
10), 2.5 (s,
-1
1
614, 1569, 1500 (CdN, CdC, NsH), 1291, 1164 (SdO) cm ;
3
3
), 4.2 (m, 1H, 4-CHC
5
H
10), 7.75 (d, 1H, 5-H), 8.7 (d,
S) C, H, N, S.
-Cyclooctyl-3-m eth yl-4H-p yr id o[4,3-e]-1,2,4-th ia d ia z-
in e 1,1-Dioxid e (14). The title compound was obtained from
-(cyclooctylamino)pyridine-3-sulfonamide (8, 1.0 g, 3.53 mmol)
1
H NMR (DMSO-d
H, 5-H), 8.65 (d, 1H, 6-H), 8.9 (s, 1H, 8-H), 11.4 (bs, 1H,
NsH). Anal. (C H N O S) C, H, N, S.
6
, 80 MHz) δ 1.2 (s, 9H, 3-C(CH
3 3
) ), 7.5 (d,
1
H, 6-H), 8.9 (s, 1H, 8-H). Anal. (C13
4
17 3 2
H N O
1
1
0
13
3
2
N-[(4-Am in opyr id-3-yl)su lfon yl]tr iflu or oacetam ide (24).
A mixture of 4-aminopyridine-3-sulfonamide (17, 1.0 g, 5.8
mmol) and trifluoroacetic anhydride (7.5 mL) was heated
under reflux for 4 h. After cooling, the precipitate was
collected by filtration, washed with diethyl ether, dried, and
recrystallized from hot methanol (1.33 g, 85%): mp 262-266
4
by following the experimental conditions described for 12 (0.70
g, 65%): mp 207-210 °C; IR (KBr) 2924, 2854 (CsH), 1601,
1
1
567, 1542 (CdN, CdC), 1313, 1172 (SdO) cm-1
;
H NMR
(
3
DMSO-d
H, 3-CH
6
, 80 MHz) δ 1.3-2.2 (bm, 14H, 4-CHC
7
H
14), 2.5 (s,
3
), 4.5 (m, 1H, 4-CHC
7
H
14), 7.45 (d, 1H, 5-H), 8.65
S) C, H, N,
°C; IR (KBr) 3390, 3312, 3219 (NsH), 2688 (N
form), 1647, 1520 (CdN, CdC, NsH), 1311, 1194 (SdO) cm-1
;
+
sH zwitterionic
(d, 1H, 6-H), 8.9 (s, 1H, 8-H). Anal. (C15
H
21
N
3
O
2
S.
1
H NMR (DMSO-d , 80 MHz) δ 6.9 (d, 1H, 5-H), 7.4 (bs, 2H,
NH ), 8.0 (d, 1H, 6-H), 8.45 (s, 1H, 2-H), 13.0 (bs, 1H,
6
4
-N-Mor p h olin yl-3-m eth yl-4H-p yr id o[4,3-e]-1,2,4-th ia -
2
d ia zin e 1,1-Dioxid e (15). The title compound was obtained
from 4-(N-morpholinylamino)pyridine-3-sulfonamide (9, 1.0 g,
SO NHCO). Anal. (C H N O SF ) C, H, N, S.
2
7
6
3
3
3
3-(Tr iflu or om et h yl)-4H -p yr id o[4,3-e]-1,2,4-t h ia d ia z-
in e 1,1-Dioxid e Mon oh yd r a te (25). N-[(4-Aminopyrid-3-yl)-
sulfonyl]trifluoroacetamide (24, 1.0 g, 3.7 mmol) was heated
under reflux in phosphorus oxychloride (20 mL) for 18 h. Most
of the excess reactant was eliminated by distillation under
reduced pressure, and the oily residue obtained was triturated
with cold water (5 mL). The precipitate was collected by
filtration, washed with water, and dried. Recrystallization
from methanol, diethyl ether, and petroleum ether (40-60 °C)
1:1:3 gave 25 (0.61 g, 65%): mp 236-240 °C; IR (KBr) 2623,
3
.87 mmol) by following the experimental conditions described
for 12 (0.76 g, 70%): mp 285-288 °C; IR (KBr) 2966, 2927,
893, 2869 (CsH), 1609, 1576, 1553 (CdN, CdC), 1304, 1165
2
-1 1
(SdO) cm ; H NMR (DMSO-d
6
, 80 MHz) δ 2.5 (s, 3H, 3-CH
.1-3.9 (bm, 8H, 4-N-morpholinyl), 7.65 (d, 1H, 5-H), 8.7 (d,
H, 6-H), 8.9 (s, 1H, 8-H). Anal. (C11 S) C, H, N, S.
-N-Aza bicyclo[3.3.0]octyl-3-m eth yl-4H-p yr id o[4,3-e]-
,2,4-th ia d ia zin e 1,1-Dioxid e (16). The title compound was
3
),
3
1
14 4 3
H N O
4
1
obtained from 4-(N-azabicyclo[3.3.0]octylamino)pyridine-3-sul-
fonamide (10, 1.0 g, 3.5 mmol) by following the experimental
conditions described for 12 (0.64 g, 60%): mp 219-222 °C; IR
+
2144 (N sH zwitterionic form), 1646, 1617, 1535, 1503 (CdN,
-
1 1
CdC), 1302, 1168 (SdO), 1317, 1183, 1143 (CF
3
) cm ; H NMR
O + NH), 7.45 (d, 1H, 5-H),
(
KBr) 2953, 2867 (CsH), 1610, 1575, 1553 (CdN, CdC), 1303,
(DMSO-d , 80 MHz) δ 6.9 (brs, H
6
2
-1 1
1167 (SdO) cm ; H NMR (DMSO-d
6H, 3′,4′,5′-CH azabicyclo), 2.5 (s, 3H, 3-CH
6H, azabicyclo), 7.25 (d, 1H, 5-H), 8.7 (d, 1H, 6-H), 8.9 (s, 1H,
8-H). Anal. (C14 S) C, H, N, S.
-Eth yl-4H-p yr id o[4,3-e]-1,2,4-th ia d ia zin e 1,1-d ioxid e
m on oh yd r a te (20). A mixture of 4-aminopyridine-3-sulfona-
6
, 80 MHz) δ 1.2-1.9 (bm,
8.5 (d, 1H, 6-H), 9.2 (s, 1H, 8-H). Anal. (C
C, H, N, S.
7
H
4
N
3
O
2
SF
3
2
‚H O)
2
3
), 2.8-3.5 (bm,
N-[4-(Cyclop en ta n eca r boxa m id op yr id -3-yl)su lfon yl]-
cyclop en ta n eca r boxa m id e (26). A mixture of 4-aminopyri-
dine-3-sulfonamide (17, 1.0 g, 5.8 mmol), cyclopentanecar-
boxylic acid (2 mL), and phosphorus oxychloride (8 mL) was
18 4 2
H N O
3

4
H-Pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 945
heated under reflux for 10 min. Most of the excess reactant
was eliminated by distillation under reduced pressure, and the
resulting oily residue was triturated with cold water (100 mL),
giving a white precipitate of crude 26. The product was
collected by filtration, washed with water, and dried. Recrys-
tallization from methanol and water 1:2 gave 26 (1.8 g, 85%):
1H, 3-NHCH
1H, NsH). Anal. (C
2
CH
3
), 8.4 (d, 1H, 6-H), 8.6 (s, 1H, 8-H), 12.1 (bs,
S) C, H, N, S.
8
10 4 2
H N O
3-[(1′,2′,2′-Tr im et h ylp r op yl)a m in o]-4H -p yr id o[4,3-e]-
1,2,4-th ia d ia zin e 1,1-Dioxid e Mon oh yd r a te (39). 1,2,2-
Trimethylpropylamine was obtained from the hydrochloride
30b (5.0 g) after dissolution of the former in the minimum
amount of water (3 mL) and addition of a solution of NaOH
(2.2 g) in water (3 mL). The sparingly water-soluble amine
which separates was decanted, filtered, and rectified by
distillation (boiling point 85 °C at 760 mmHg). A solution of
3-(methylthio)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide
monohydrate (29, 0.5 g, 2.02 mmol) in 1,2,2-trimethylpropy-
lamine (5 mL) was heated under reflux during 2 days. The
solution was concentrated under reduced pressure, and the
residue was dissolved in aqueous NaOH (0.1 N). The solution
was treated with charcoal and filtered. The filtrate was
adjusted to pH 5 with formic acid, and the precipitate of crude
mp 219-222 °C; IR (KBr) 3370 (NsH amide), 2958, 2870
+
(
CsH aliphatic), 2738, 2602 (N sH zwitterionic form), 1708
(
CdO, amide), 1583, 1499 (CdN, CdC), 1320, 1154 (SdO)
-
1
1
cm
;
)
H NMR (DMSO-d
, cyclopentyl), 2.75 (bs, 2H, 2 × COCHC
-H), 8.7 (d, 1H, 6-H), 8.95 (s, 1H, 2-H), 9.7 (s, 1H, CNHCO-
cyclopentyl). Anal. (C17 S) C, H, N, S.
6
, 80 MHz) δ 1.2-2 (m, 16H, 2 ×
(
5
CH
2
4
4 8
H
), 8.4 (d, 1H,
23 3 4
H N O
N-[(4-Am in op yr id -3-yl)su lfon yl]cyclop en ta n eca r box-
a m id e (27). The solution of N-[(4-cyclopentanecarboxami-
dopyrid-3-yl)sulfonyl]cyclopentanecarboxamide (26, 1.0 g, 2.8
mmol) in an aqueous solution of NaOH (0.22 g of NaOH in 20
mL water) was refluxed for 2 h. After treatment with charcoal
and cooling, the solution was adjusted to pH 2 with 0.1 N HCl.
The resulting precipitate was collected by filtration, washed
with water, and dried (0.64 g, 85%): mp 218-220 °C; IR (KBr)
3
9 was collected by filtration, washed with water, and dried.
Recrystallization from methanol and water 1:2 gave 39 (0.4
g, 66%): mp 257-260 °C, IR (KBr) 3297 (NsH), 1631, 1581
-1 1
(
CdN, CdC, NsH), 1281, 1162 (SdO) cm ; H NMR (DMSO-
d
6
, 80 MHz) δ 0.8 (s, 9H, NCH(CH
3 3 3
)C(CH ) ), 1.0 (d, 3H, NCH-
3
2
388, 3310, 3212 (NsH), 2958, 2939, 2867 (CsH aliphatic),
+
(CH )C(CH ) ), 3.8 (m, 1H, NCH(CH )C(CH ) ), 7.0 (d, 1H,
656, 2047 (N sH zwitterionic form), 1653 (CdO), 1605, 1516
3
3
3
3
3 3
-
1
1
3
5-H), 7.25 (bs, 1H, 3-NHCH(CH )), 8.45 (d, 1H, 6-H), 8.7 (s,
(
CdN, CdC), 1287, 1154 (SdO) cm ; H NMR (DMSO-d
MHz) δ 1.5 (bs, 8H, CO-CHC ), 2.5 (bs, DMSO + COCHC
.75 (d, 1H, 5-H), 7.15 (bs, 2H, NH
H, 2-H). Anal. (C11 S) C, H, N, S.
-Cyclop en t yl-4H-p yr id o[4,3-e]-1,2,4-t h ia d ia zin e 1,1-
6
, 80
1
H, 8-H), 12.1 (bs, 1H, NsH). Anal. (C12
H, N, S.
-(Meth ylbu tyla m in o)-4H-p yr id o[4,3-e]-1,2,4-th ia d ia z-
H
18
N
4
O
2
S‚H
2
O) C,
4
H
8
4
H
8
),
6
1
2
), 8.05 (d, 1H, 6-H), 8.4 (s,
3
15 3 3
H N O
in e 1,1-Dioxid e (43). 3-(Methylthio)-4H-pyrido[4,3-e]-1,2,4-
thiadiazine 1,1-dioxide monohydrate (29, 0.5 g, 2.02 mmol) was
heated under reflux with N-methylbutylamine (5 mL) for 24
h. Most of the excess of N-methylbutylamine was removed
by distillation under reduced pressure. The residue was
dissolved in water (10 mL), and the solution was adjusted to
pH 5 with formic acid. The precipitate of the crude 43 was
collected by filtration, washed with water, dried, and recrystal-
lized from methanol and water 1:1 (0.39 g, 66%): mp 201-
3
Dioxid e (28). N-[(4-Aminopyrid-3-yl)sulfonyl]cyclopentan-
ecarboxamide (27, 0.7 g, 2.6 mmol) was heated at 220 °C
(fusion) for 10 min. The residue was dissolved in hot methanol,
discolored with charcoal and cooled. The crude 28 which
precipitates was collected by filtration (0.47 g, 72%): mp 298-
3
1
02 °C; IR (KBr) 3288 (NsH), 2952, 2870 (CsH aliphatic),
618, 1571, 1506 (CdN, CdC, NsH), 1293, 1155 (SdO) cm-1
;
1
H NMR (DMSO-d
6
, 80 MHz) δ 1.55 (bs, 8H, CHC
), 7.2 (d, 1H, 5-H), 8.65 (d, 1H, 6-H), 8.9 (s, 1H,
-H), 12.1 (bs, 1H, NsH). Anal. (C11 S) C, H, N, S.
,2,2-Tr im eth ylp r op a n a l Oxim e (30a ). An aqueous solu-
tion of Na CO (2.6 g in 10 mL of water) was added dropwise
4 8
H ), 3.0 (bs,
2
04 °C; IR (KBr) 3287, 3202 (NsH), 1613, 1598, 1580 (CdN,
1
8
4 8
H, CHC H
-
1 1
CdC, NsH), 1283, 1154 (SdO) cm ; H NMR (DMSO-d
MHz) δ 0.9 (t, 3H, 3-N(CH CH ), 1-1.8 (m, 4H, 3-NCH (CH
CH ), 3.05 (s, 3H, 3-NCH ), 3.5 (t, 2H, 3-NCH (CH CH ) 7.35
d, 1H, 5-H), 8.5 (d, 1H, 6-H), 8.7 (s, 1H, 8-H), 10.6 (bs, 1H,
NsH). Anal. (C11 S) C, H, N, S.
-(4′-Meth yl-1′-p ip er a zin yl)-4H-p yr id o[4,3-e]-1,2,4-th ia -
d ia zin e 1,1-Dioxid e (44). 3-(Methylthio)-4H-pyrido[4,3-e]-
,2,4-thiadiazine 1,1-dioxide monohydrate (29, 0.6 g, 2.43
6
, 80
13 3 2
H N O
2
)
3
3
2
2 2
) -
1
3
3
2
2
)
2
3
2
3
(
with vigorous stirring to a suspension of pinacolone (5.0 g, 50
mmol) in an aqueous solution of hydroxylamine hydrochloride
16 4 2
H N O
3
(3.5 g/50 mmol in 7 mL of water). After 3 h, the crystalline
oxime 30a was collected by filtration, washed with water, and
dried (4.72 g, 82%): mp 73-75 °C; IR (KBr) 3284 (OsH), 1663
1
mmol) was heated under reflux with N-methylpiperazine (3
mL) for 30 min. After cooling, diethyl ether (60 mL) was added
to the mixture, and an oily product was isolated by decanta-
tion. The oil was dissolved in a small volume of methanol,
after which diethyl ether was added until precipitation of the
crude 44 occurred. After cooling, the precipitate was collected
by filtration, washed with diethyl ether, and dried. Recrys-
tallization from methanol and diethyl ether 1:4 gave 44 (0.55
g, 80%): mp 257-260 °C; IR (KBr) 3250, 3162 (NsH), 1607,
-1 1
(
1
CdN) cm ; H NMR (DMSO-d
.65 (s, 3H, 1-CH ), 10.1 (s, 1H, NOH).
,2,2-Tr im eth ylp r op yla m in e Hyd r och lor id e (30b). So-
dium metal (3.0 g, 0.13 mol) was added slowly to a solution of
,2,2-trimethylpropanal oxime (30a , 2.0 g, 17.4 mmol) in
6
, 80 MHz) δ 1.0 (s, 9H, (CH
3 3
) ),
3
1
1
absolute ethanol (10 mL). After complete addition, the mixture
was refluxed until total dissolution of sodium was achieved.
The solution was cooled in an ice bath, mixed with brine (50
mL), and extracted with diethyl ether (100 mL). The organic
layer was washed with water (20 mL) and extracted with 6 N
HCl (30 mL). The aqueous solution was concentrated to
dryness under reduced pressure. The crude hydrochloride was
dissolved in the minimum ethanol, filtered, and mixed with 5
-1
1
1
570, 1521 (CdN, CdC, NsH), 1290, 1158 (SdO) cm ; H
NMR (DMSO-d , 80 MHz) δ 2.25 (s, 3H, 4′-NCH ), 2.4 (t, 4H,
6
3
3′,5′-CH ), 3.6 (t, 4H, 2′,6′-CH ), 7.05 (d, 1H, 5-H), 8.3 (d, 1H,
2
2
6-H), 8.6 (s, 1H, 8-H), 12.1 (bs, 1H, NH). Anal. (C H N O S)
C, H, N, S.
1
1
15
5
2
volumes of diethyl ether to give the title compound (1.2 g,
4
-Meth yl-3-oxo-2,3-d ih yd r o-4H-p yr id o[4,3-e]-1,2,4-th ia -
1
6
9
8%): mp 289-292 °C; H NMR (DMSO-d
6
, 80 MHz) δ 0.9 (s,
d ia zin e 1,1-Dioxid e (45). A mixture of 4-(methylamino)-
pyridine-3-sulfonamide (5, 9.4 g, 50.2 mmol) and urea (6.1 g,
H, (CH
3
)
3
), 1.1 (d, 3H, CHCH
16NCl) C, H, N.
-(Eth yla m in o)-4H-p yr id o[4,3-e]-1,2,4-th ia d ia zin e 1,1-
3
), 2.9 (m, 1H, CHCH ). Anal.
3
(C
6
H
1
00.4 mmol) was heated at 200 °C (fusion). After cooling, the
3
residue was dissolved in 1 N NaOH and the solution was
adjusted to pH 5 with formic acid. The precipitate of 45 was
collected by filtration, washed with water, and dried (6.75 g,
63%): mp 273-275 °C; IR (KBr) 3363 (NsH), 1637, 1568, 1511
Dioxid e (32). A solution of 3-(methylthio)-4H-pyrido[4,3-e]-
,2,4-thiadiazine 1,1-dioxide monohydrate (29, 0.5 g, 2.02
1
mmol) in a 70% w/v aqueous solution of ethylamine (10 mL)
was heated in a hermetically closed autoclave at 120 °C during
-
1
1
(CdN, CdC, NsH, CdO), 1291, 1174 (SdO) cm ; H NMR
(DMSO-d , 80 MHz) δ 3.35 (s, 3H, 4-N-CH ), 6.1 (bs, H O +
NH), 7.5 (d, 1H, 5-H), 8.55 (d, 1H, 6-H), 8.9 (s, 1H, 8-H). Anal.
(C S) C, H, N, S.
4-Met h yl-3-t h ioxo-2,3-d ih yd r o-4H -p yr id o[4,3-e]-1,2,4-
6
h. The excess of ethylamine was removed under reduced
6
3
2
pressure and the residue was dissolved in water. The solution
was adjusted to pH 6 with 1 N HCl and the title product which
precipitates was collected by filtration, washed with water, and
dried (0.33 g, 72%): mp 246-248 °C; IR (KBr) 3383, 3288, 3166
7
7 3 3
H N O
th ia d ia zin e 1,1-Dioxid e (46). The mixture of 4-methyl-3-
oxo-2,3-dihydro-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide
(45, 6.0 g, 28.1 mmol) and phosphorus pentasulfide (12.0 g,
27 mmol) in anhydrous pyridine was heated under reflux for
(
(
NsH), 1651, 1604, 1582, 1551 (CdN, CdC, NsH), 1278, 1173
-
1
1
SdO) cm
CH
;
H NMR (DMSO-d
6
, 80 MHz) δ 1.0 (t, 3H,
N-CH
2
3
), 3.2 (q, 2H, NCH CH ), 7.05 (d, 1H, 5-H), 7.25 (bs,
2
3

9
46 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
de Tullio et al.
2
days. The resulting suspension was concentrated under
1H, 5-H), 8.65 (d, 1H, 6-H), 8.75 (s, 1H, 8-H). Anal.
reduced pressure, and the residue was dissolved in the
minimum 2 N NaOH. This solution was treated with charcoal
and filtered, and the filtrate was adjusted to pH 2 with 1 N
HCl. The yellow precipitate was collected by filtration, washed
with water, and dissolved in a saturated aqueous solution of
(C13
H
20
N
4
O
2
S) C, H, N, S.
5
-C h l o r o -2 -c y c l o p e n t a n e c a r b o x a m i d o b e n z e n e -
su lfon a m id e (55). A solution of 2-amino-5-chlorobenzene-
sulfonamide (54, 1.0 g, 4.8 mmol), pyridine (0.5 mL), and
23
cyclopentanecarboxylic acid chloride (0.7 g, 5.3 mmol) in
dioxane (15 mL) was stirred at room temperature for 1 h. The
solvent was removed by distillation under reduced pressure,
and the residue was triturated with water (50 mL). The
resulting insoluble material was collected by filtration, washed
with water and recrystallized from methanol/water 1:1 (1.26
g, 86%): mp 165-168 °C; IR (KBr) 3377, 3236 (NsH), 2955,
3
NaHCO (150 mL). The insoluble material was filtered off,
and the filtrate was adjusted again to pH 2 with 1 N HCl.
The crystalline 46 was collected by filtration, washed with
water, and dried (4.0 g, 62%): mp 243-245 °C; IR (KBr) 3495
(
NsH), 2581 (SsH tautomeric form) 1633, 1539, 1490 (CdN,
-
1 1
CdC, NsH), 1289, 1166 (SdO) cm ; H NMR (DMSO-d
MHz) δ 4.0 (s, 3H, 4-N-CH O), 7.55
d, 1H, 5-H), 8.65 (d, 1H, 6-H), 8.95 (s, 1H, 8-H). Anal.
6
, 80
3
), 5.95 (bs, 4H, NH + H
2
2
1
2
868 (CsH aliphatic), 1636 (CdO), 1564, 1521 (CdN, CdC),
344, 1157 (SdO) cm ; H NMR (DMSO-d
.0 (bm, 8H, COCHC ), 2.7 (m, 1H, COCHC H
(
(
-1 1
6
, 80 MHz) δ 1.2-
C
7
H
7
N
3
O
2
S
2
) C, H, N, S.
4
H
8
4 8
), 7.55 (d,
4
-Meth yl-3-(m eth ylth io)-4H-p yr id o[4,3-e]-1,2,4-th ia d i-
1H, 4-H), 7.8 (bs, 3H, 6-H + SO NH ), 8.2 (d, 1H, 3-H), 9.3 (s,
1H, CONH). Anal. (C H N O SCl) C, H, N, S.
12 15 2 3
2
2
a zin e 1,1-Dioxid e (47). 4-Methyl-3-thioxo-2,3-dihydro-4H-
pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (46, 3.2 g, 14 mmol)
was dissolved in a solution of NaHCO3 (2.4 g) in water (106
mL). Under stirring, methanol (80 mL) and methyl iodide
7
-Ch lor o-3-cyclop en tyl-4H-1,2,4-ben zoth ia d ia zin e 1,1-
Dioxid e (56). The solution of 2-cyclopentanecarboxamido-5-
chlorobenzenesulfonamide (55, 0.64 g, 2.12 mmol) in NaOH
(10.2 mL, 164 mmol) were successively added to this solution.
1
% w/v (32 mL) was refluxed for 30 min. The solution was
After 30 min under agitation at room temperature, the mixture
was adjusted to pH 3 with 1N HCl, and the methanol was
removed under reduced pressure. After cooling, the title
product which precipitated, was collected by filtration, washed
adjusted to pH 7 with 1 N HCl, and the resulting precipitate
was collected by filtration, washed with water, and dried.
Recrystallization from acetone/petroleum ether 1:3 gave the
title compound (0.38 g, 63%): mp 278-280 °C; IR (KBr) 3285
with water, and dried (2.65 g, 78%): mp 208-210 °C; IR (KBr)
(NsH), 2957, 2870 (CsH aliphatic), 1618, 1578, 1525, 1482
1
1
591, 1564, 1520 (CdN, CdC), 1306, 1173 (SdO) cm-1
;
H
-1 1
(
CdN, CdC), 1288, 1157 (SdO) cm ; H NMR (DMSO-d
), 2.9 (m, 1H, COCHC
.30 (d, 1H, 5-H), 7.65 (d, 1H, 6-H), 7.7 (s, 1H, 8-H). Anal.
SCl) C, H, N, S.
-Ch lor o-3-[(1′-m eth ylpr opyl)am in o]-4H-1,2,4-ben zoth i-
a d ia zin e 1,1-Dioxid e (58). The title compound was obtained
6
, 80
NMR (DMSO-d
-NCH ), 7.45 (d, 1H, 5-H), 8.7 (d, 1H, 6-H), 8.9 (s, 1H, 8-H).
Anal. (C ) C, H, N, S.
-(Isop r op yla m in o)-4-m et h yl-4H -p yr id o[4,3-e]-1,2,4-
th ia d ia zin e 1,1-Dioxid e (48). A solution of 4-methyl-3-
methylthio)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (47,
.5 g, 2.05 mmol) in isopropylamine (5 mL) was heated under
6
, 80 MHz) δ 2.5 (s, 3H, 3-SCH
3
), 3.65 (s, 3H,
MHz) δ 1.2-2.0 (bm, 8H, COCHC
4
H
8
4
H
8
),
4
3
7
8
9 3 2 2
H N O S
12 13 2 2
(C H N O
3
7
(
0
1
6
as previously described for the compound 59 by starting from
the intermediate 5716 (0.5 g, 1.90 mmol) and sec-butylamine
reflux for 1 h. The excess of amine was removed under reduced
pressure, and the residue was triturated with water. The title
compound was collected by filtration, washed with water, and
recrystallized from methanol and water 1:3 (0.35 g, 69%): mp
(
(
5 mL) (0.49 g, 89%): mp 240-242 °C; IR (KBr) 3296, 3182
NsH), 2970, 2934 (CsH aliphatic), 1630, 1583, 1480 (CdN,
-
1 1
CdC, NsH), 1250, 1157 (SdO) cm ; H NMR (DMSO-d
MHz) δ 0.8 (t, 3H, 3-NCH(CH )CH CH ), 1.05 (d, 3H,
-NCH(CH )CH CH ), 1.4 (m, 2H, 3-NCH(CH )CH CH ), 3.7
m, 1H, 3-NCH(CH )CH CH ), 6.95 (bd, 1H, 3-NH), 7.10 (d,
H, 5-H), 7.5 (d, 1H, 6-H), 7.55 (s, 1H, 8-H), 10.3 (s, 1H, 4-NH).
SCl) C, H, N, S.
-Ch lor o-3-[(1′,2′,2′-t r im et h ylp r op yl)a m in o]-4H -1,2,4-
ben zoth ia d ia zin e 1,1-Dioxid e (60). The title compound was
6
, 80
3
2
3
2
72-274 °C; IR (KBr) 3299 (NsH), 1598, 1554 (CdN, CdC,
3
(
1
3
2
3
3
2
3
-
1 1
NsH), 1285, 1158 (SdO) cm ; H NMR (DMSO-d
δ 1.1 (d, 6H, 3-NCH(CH ), 3.4 (s, 3H, 4-NCH ), 4.05 (m, 1H,
), 7.45 (d, 1H, 5-H), 7.65 (bs, 1H, NsH), 8.65 (d,
H, 6-H), 8.8 (s, 1H, 8-H). Anal. (C10 S) C, H, N, S.
-Meth yl-3-[(1′-m eth ylp r op yl)a m in o]-4H-p yr id o[4,3-e]-
,2,4-th ia d ia zin e 1,1-Dioxid e (49). The title compound was
6
, 80 MHz)
3
2
3
3
)
2
3
3
1
3 2
-NCH(CH )
Anal. (C11
14 3 2
H N O
14 4 2
H N O
7
4
1
1
6
obtained as described for the compound 59 by starting from
the intermediate 5716 (0.5 g, 1.90 mmol) and 1,2,2-trimethyl-
propylamine (5 mL) (0.44 g, 73%): mp 288-290 °C; IR (KBr)
3301, 3184 (NsH), 2967 (CsH aliphatic), 1630, 1582, 1480
obtained as described for the compound 48, by starting from
the intermediate 47 (0.5 g, 2.05 mmol) and sec-butylamine (5
mL) (0.36 g, 67%): mp 215-217 °C; IR (KBr) 3312 (NsH),
599, 1554 (CdN, CdC, NsH), 1285, 1156 (SdO) cm-1; H
1
1
-
1 1
(CdN, CdC, NsH), 1250, 1161 (SdO) cm ; H NMR (DMSO-
NMR (DMSO-d
6
, 80 MHz) δ 0.75 (t, 3H, 3-N-CH(CH
)CH CH ), 1.5 (m, 2H,
), 3.4 (s, 3H, 4-NCH ), 3.75 (m, 1H,
), 7.35 (d, 1H, 5-H), 7.45 (bs, 1H, NsH),
S) C,
3
)-
d
3
6
, 80 MHz) δ 0.85 (s, 9H, 3-NCH(CH
3
)C(CH
3
)
3
), 1.0 (d, 3H,
CH CH ), 1.1 (d, 3H, 3-NCH(CH
2
3
3
2
3
-NCH(CH )C(CH ), 3.7 (m, 1H, 3-NCH(CH
3
3
)
3
3
)C(CH ), 6.8
3 3
)
3
3
8
-NCH(CH
-NCH(CH
3
3
)CH
)CH
2
2
CH
CH
3
3
3
(bd, 1H, 3-NH), 7.10 (d, 1H, 5-H), 7.5 (d, 1H, 6-H), 7.55 (s, 1H,
8
-H), 10.2 (s, 1H, 4-NH). Anal. (C13
Ion iza tion Con sta n ts. The pK
9, 28, 32, and 39 was determined spectroscopically by means
H
18
N
3
O
2
SCl) C, H, N, S.
.65 (d, 1H, 6-H), 8.75 (s, 1H, 8-H). Anal. (C11
16 4 2
H N O
H, N, S.
-[(1′,2′-Dim et h ylp r op yl)a m in o]-4-m et h yl-4H -p yr id o-
4,3-e]-1,2,4-th ia d ia zin e 1,1-Dioxid e (50). The title com-
a
value of the compounds
1
3
of 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 pK values were calculated
a
[
pound was obtained as described for 48 starting from 47 (0.5
g, 2.05 mmol) and 1,2-dimethylpropylamine (5 mL) (0.48 g,
by the Debye-H u¨ kkel equation at the maximum basic form
8
5%): mp 160-162 °C; IR (KBr) 3339 (NsH), 1595, 1549
absorbance.32
-1 1
(CdN, CdC, NsH), 1289, 1158 (SdO) cm ; H NMR (DMSO-
d
3
3
6
, 80 MHz) δ 0.85 (d, 6H, 3-NCH(CH
-NCH(CH )CH(CH ), 1.75 (m, 1H, 3-NCH(CH
3 2
.42 (s, 3H, 4-NCH ), 3.75 (m, 1H, 3-NCH(CH )CH(CH )
3
)CH(CH
3
)
2
), 1.1 (d, 3H,
)CH(CH ),
), 7.35
Ca lcu la tion s a n d Molecu la r Gr a p h ics. The displace-
ment-ellipsoid view of compound 34 was undertaken with the
program ORTEP-II.33 The molecular design and the isopoten-
3
3
)
2
3
3 2
)
3
3
3
4
(
8
d, 1H, 5-H), 7.5 (bs, 1H, NH), 8.65 (d, 1H, 6-H), 8.75 (s, 1H,
-H). Anal. (C12 S) C, H, N, S.
-Meth yl-3-[(1′,2′,2′-tr im eth ylpr opyl)am in o]-4H-pyr ido-
4,3-e]-1,2,4-th ia d ia zin e 1,1-Dioxid e (51). The title com-
tial map were performed using SYBIL 6.1 software package
H
18
N
4
O
2
on a Silicon Graphics Personal IRIS Indigo Elan workstation.
The geometries of diazoxide27 and of the compound 34 were
taken from the X-ray crystallographic structure. The crystal-
lographic models were submitted to AM1 calculations (MOPAC
version 6.0)35 to determine the atomic charge distributions.
4
[
pound was obtained as described for the compound 48 starting
from the compound 47 (0.5 g, 2.05 mmol) and 1,2,2-trimeth-
ylpropylamine (5 mL) (0.32 g, 54%): mp 253-256 °C; IR (KBr)
X-r a y Cr ysta llogr a p h y. A crystal of compound 34 was
grown by slow evaporation from a methanol-water solution.
Diffraction data were measured at room temperature using a
Stoe-Siemens AED four-circle diffractometer. Final cell di-
mensions of crystal 34 were obtained by a least-squares fit to
3
325 (NsH), 1597, 1555 (CdN, CdC, NsH), 1303, 1163 (SdO)
-
1 1
cm ; H NMR (DMSO, 80 MHz) δ 0.9 (s, 9H, 3-NCH(CH
CH ), 1.1 (d, 3H, 3-NCH(CH )C(CH ), 3.6 (s, 3H, 4-NCH
.1 (m, 1H, 3-NCH(CH )C(CH ), 7.2 (bs, 1H, NH), 7.45 (d,
3
)C-
(
4
3
)
3
3
3
)
3
3
),
3
3 3
)

4
H-Pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 947
the automatically centered setting for 22 reflexions (70.51 <
θ < 78.61°). Two reference reflexions monitored during each
Tissues were incubated at 30 °C in oxygenated (95% O
CO ) Tyrode’s solution (composition (in mM): NaCl 137; KCl
6; CaCl 1.8; MgCl 1; NaH PO 0.42; NaHCO 11.5; glucose
5.5) under an initial tension of 0.5 g and were stimulated with
rectangular 10 ms pulses at a frequency of 1 Hz and 1-10 V.
Drugs were added after an initial equilibration period of 90
min and systolic tension was followed for 35-40 min after their
addition. Tension in the presence of drugs was expressed as
percentage of initial value.
2
-5%
2
2
experiment showed no significant variation. Intensity data
were corrected for Lorentz polarization effects and for absorp-
tion (semiempirical method, ψ scan). Space group assignments
were suggested by cell geometry and average values of
normalized structure factors; choices were confirmed by suc-
cessful refinement.
2
2
2
4
3
3
6
The structure was solved by direct methods (SHELXS 86 ).
3
7
The least-square refinement (SHELXL 93 ) included inde-
pendent position parameters and anisotropic displacement
coefficients for all non-hydrogen atoms, and two global isotro-
pic thermal parameters for hydrogen atoms (one for non-
methyl and one for methyl atoms). During refinement, H
atoms have been included as riding atoms at calculated
positions, except H4(N4) and H(water) in 34, whose positions
obtained from Fourier-difference map were kept fixed. For
both experiments, the final difference Fourier map had no
significant features. Atomic scattering factors were taken from
ref 38.
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. Lebrun and B. Pirotte
are Senior Research associates and from which L.
Dupont has received financial support for the info-
graphic materials. The assistance of Mrs. P. Neven, D.
Dewalque, M. L. Pirard, M. Vermeire, M. Hermann and
F. Maranca is gratefully acknowledged.
(
Biologica l Assa ys. Mea su r em en ts of In su lin Relea se
fr om In cu ba t ed Islet s. 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
Su p p or tin g In for m a tion Ava ila ble: Crystal data for
compound 34 (7 pages); observed and calculated structure
factors for 34 (4 pages). Ordering information is given on any
current masthead page.
1
15, KCl 5, CaCl
with 2.8 mM glucose, 0.5% (w:v) dialyzed albumin (Sigma) and
equilibrated against a mixture of O (95%) and CO (5%). The
2 2 3
2.56, MgCl 1, NaHCO 24] supplemented
2
2
Refer en ces
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,
either diazoxide, AO44, or the pyridothiadiazine derivatives.
The release of insulin was measured radioimmunologically
using rat insulin as a standard. IC50 values were graphically
assessed.
(
1) Cook, N. S. The Pharmacology of Potassium Channels and their
Therapeutic Potential. Trends Pharmacol. Sci. 1988, 9, 21-27.
(2) Potassium Channels, Structure, Classification, Function and
Therapeutic Potential; Cook, N. S., Ed.; Ellis Horwood Ltd.:
Chichester, 1990.
3
9
(3) Edwards, G.; Weston, A. H. The Pharmacology of ATP-Sensitive
Potassium Channels. Annu. Rev. Pharmacol. Toxicol. 1993, 33,
Mea su r em en ts of Ten sion in Ra t Aor ta : All experi-
ments were performed in aortae removed from fed Wistar rats
5
97-637.
(
4) Lazdunski, M. ATP-Sensitive Potassium Channels: An Over-
(
250-300 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
view. J . Cardiovasc. Pharmacol. 1994, 24, S1-S5.
(5) Ashcroft, F. M. Adenosine 5′-Triphosphate-sensitive Potassium
Channels. Annu. Rev. Neurosci. 1988, 11, 97-118.
(
(
6) Petersen, O. H.; Dunne, M. J . Regulation of K+ Channels Plays
the intimal surface with forceps. The segments were sus-
pended under 1 g tension by means of steel hooks in an organ
bath containing 20 mL of a Krebs-bicarbonate-buffered solu-
tion of the following composition (in mM): NaCl 118, KCl 4.7,
a Crucial Role in the Control of Insulin Secretion. Pfl u¨ gers Arch.
1
989, 414, S115-120.
(7) Kolb, H. A. Potassium Channels in Excitable and Non-Excitable
Cells. Rev. Physiol. Biochem. Pharmacol. 1990, 15, 51-79.
CaCl
physiological solutions were maintained at 36 ( 1 °C and
bubbled continuously with a mixture of O (95%) and CO (5%).
2
2.5, NaHCO
3
25, KH
2
PO
4
1.2, MgSO
4
1.2, glucose 5. The
(8) Malaisse, W. J .; Lebrun, P. Mechanisms of Sulfonylurea-Induced
Insulin Release. Diabetes Care 1990, 13, suppl 3, 9-17.
(
9) Robertson, D. W.; Steinberg, M. I. Potassium Channel Modula-
tors: Scientific Applications and Therapeutic Promise. J . Med.
Chem. 1990, 33, 1529-1541.
2
2
The isometric contractions of the aortic rings were measured
with a Grass force-displacement transducer. After 60 min of
equilibration, the rings were exposed to 30 mM KCl. When
the tension had stabilized (plateau tension between 1 and 2
g), the agonist was added to the bath at increasing concentra-
tions until maximal relaxation (or until 1 mM). The relaxation
response was expressed as the percentage of the contractile
response to KCl. The ED50 value was graphically assessed for
each dose response curve as the concentration evoking 50%
inhibition of the plateau induced by KCl. Results were
expressed as the mean (( sem) of 3-12 experiments.
(10) Longman, S. D.; Hamilton, T. C. Potassium Channel Activator
Drugs: Mechanism of Action, Pharmacological Properties, and
Therapeutic Potential. Med. Res. Rev. 1992, 12, 73-148.
(
11) Weston, A. H.; Edwards, G. Recent Progress in Potassium
Channel Opener Pharmacology. Biochem. Pharmacol. 1992, 43,
4
7-54.
(
12) Lebrun, P.; Devreux, V.; Hermann, M.; Herchuelz, A. Similarities
Between the Effects of Pinacidil and Diazoxide on Ionic and
Secretory Events in Rat Pancreatic Islets. J . Pharmacol. Exp.
Ther. 1989, 250, 1011-1018.
13) Antoine, M.-H.; Berkenboom, G.; Fang, Z.-Y.; Fontaine, J .;
Herchuelz, A.; Lebrun, P. Mechanical and Ionic Response of Rat
Aorta to Diazoxide. Eur. J . Pharmacol. 1992, 216, 299-306.
14) Lebrun, P.; Devreux, V.; Hermann, M.; Herchuelz, A. Pinacidil
Inhibits Insulin Release by Increasing K+ Outflow from Pan-
creatic B-Cells. Eur. J . Pharmacol. 1988, 156, 283-286.
15) Lebrun, P.; Fang, Z.-Y.; Antoine, M.-H.; Herchuelz, A.; Hermann,
M.; Berkenboom, G.; Fontaine, J . Hypoglycemic Sulfonylureas
(
(
(
Exp er im en ts on Gu in ea P ig Ileu m . Adult guinea pigs
of either sex (body weight: 300-400 g) were stunned and bled.
Segments of the ileum (4 cm long) were removed at least 10
cm from the cecum. They were set up under an initial load of
1
g in a Krebs-bicarbonate-buffered solution (composition (in
mM): NaCl 118, KCl 4.7, CaCl 2.5, NaHCO 25, KH PO 1.2,
MgSO 1.2, glucose 5), maintained at 37 °C and gassed with a
mixture of 5% CO and 95% O Coaxial stimulation was
carried out as described by Paton with rectangular pulses of
2
3
2
4
8
6
Antagonize the Effects of Cromakalim and Pinacidil on Rb
Fluxes and Contractile Activity in the Rat Aorta. Pharmacology
1990, 41, 36-48.
4
2
2
.
4
0
(16) 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.
0
.5 ms duration, 0.1 Hz, 5-25 V delivered and measured by a
Grass stimulator. Muscle contractions were recorded iso-
metrically by a transducer.
The inhibitory effects of drugs were assessed on electrically
induced contractions by adding increasing concentrations to
the bath until maximal effect. Results were expressed as
percentages of control responses (measured during 5 min
before adding the drug).
(17) Pirotte, B.; Antoine, M.-H.; de Tullio, P.; Hermann, M.; Her-
chuelz, A.; Delarge, J .; Lebrun, P. A Pyridothiadiazine (BPDZ
4
4) as a New and Potent Activator of ATP-Sensitive K+ Chan-
nels. Biochem. Pharmacol. 1994, 47, 1381-1386.
18) 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.
(
Stu d ies on Ra t Ven tr icle. The contractile activity of right
ventricular strips (10 mm length) from adult male Wistar rats
(
200-250 g) was recorded as described by Finet et al.41

9
48 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
de Tullio et al.
(
19) Lebrun, P.; Antoine, M.-H.; Ouedraogo, R.; Herchuelz, A.; de
Tullio, P.; Delarge, J .; Pirotte, B. Pyridothiadiazines as Potent
Inhibitors of Glucose-Induced Insulin Release. Adv. Exp. Med.
Biol. 1995, in press.
(30) Pirotte, B.; Dupont, L.; de Tullio, P.; Masereel, B.; Schynts, M.;
Delarge, J . Structural Study of Pinacidil, a Potassium Channel
Opener Belonging to the Chemical Class of N-alkyl-N′′-cyano-
N′-pyridylguanidines. Helv. Chim. Acta 1993, 76, 1311-1318.
(
20) Delarge, J .; Lapi e` re, C. Synthesis and properties of some
substituted anilinopyridines. J . Pharm. Belg. 1973, 3, 283-290.
21) Lopez, N.; Basade, J .; Grant, A.; Krees, S.; Terry, M.; Viktora,
J .; Wolff, F. AO44: an Improved Hyperglycemic Agent. Metab.
Clin. Exp. 1971, 20, 373-383.
(
31) Atwal, K. S. Pharmacology and Structure-Activity Relationships
for KATP Modulators: Tissue-Selective KATP Openers. J . Cardio-
vasc. Pharmacol. 1994, 24, S12-S17.
(
(32) Albert, A.; Serjeant, E. P. In Determination of Ionization
Constants; Chapman & Hall, London, 1971.
(33) J onhson, C. K. ORTEP-II, a Fortran Thermal-Ellipsoid Plot
Program for Crystal Structure Illustrations; Report ORNL-5318;
Oak Ridge National Laboratory, TN, 1976.
(
(
(
(
(
22) 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-
1
047.
23) Topliss, J . G.; Yudis, M. D. Correlation of Antihypertensive
Activity with Structure in a Series of 2H-1,2,4-Benzothiadiazine
(34) Tripos Associates, Inc., 1699 S. Harley Rd., Suite 303, St. Louis,
MO.
1
,1-dioxides Using the Substituent Constant Approach. J . Med.
(35) Stewart, J . J . P. MOPAC, a Semiempirical Molecular Orbital
Program. J . Comput.-Aided Mol. Des. 1990, 4, 1-105.
Chem. 1972, 15, 394-400.
24) Lebrun, P.; Antoine, M.-H.; Devreux, V.; Hermann, M.; Her-
8
6
chuelz, A. Paradoxical Inhibitory Effect of Cromakalim on Rb
(36) Sheldrick, G. M. SHELXS 86, a Program for the Solution of
Crystal Structures. University of G o¨ ttingen, Germany, 1986.
Outflow from Pancreatic Islets Cells. J . Pharmacol. Exp. Ther.
1
990, 255, 948-953.
(
37) Sheldrick, G. M. SHELXL 93, a Program for the Refinement of
Crystal Structures from Diffraction Data. University of G o¨ ttin-
gen, Germany, 1993.
25) Wollweber, H.; Horstmann, H.; Stoepel, K.; Garthoff, B.; Puls,
W.; Krause, H. P.; Thomas, G. 3-Amino-2H-benzothiadiazine-
1
,1-dioxide with Antihypertensive and Possibly Diabetogenic
(
38) International Tables for X-Ray Crystallography; Hahn, Th., Ed.;
Reidel Publishing Company: Dordrecht, The Netherlands, 1992;
Vol. C, Tables 4.2.6.8 and 6.1.1.4.
Activity. Arzneim.-Forsch./ Drug. Res. 1981, 31, 279-288.
26) 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:
(39) Leclercq-Meyer, V.; Marchand, J .; Rebolledo, O.; Malaisse, W.
J .; Leclercq, R. A. Combined Radioimmunoassay for Glucagon
and Insulin. Diabetologia 1975, 11, 419-425.
1
,2,4-Pyridothiadiazine 1,1-Dioxides, Pyridyl Analogues of 1,2,4-
Benzothiadiazine 1,1-Dioxides. Tetrahedron 1995, 51, 3221-
234.
3
(
40) Paton, W. D. M. The Response of the Guinea-Pig Ileum to
Electrical Stimulation by Coaxial Electrodes. J . Physiol. (Lon-
don) 1955, 127, 40P-41P.
(
(
(
27) Bandoli, G.; Nicolini, M. Crystal and Molecular structure of
Diazoxide, an Antihypertensive Agent. J . Cryst. Mol. Struct.
1
977, 7, 229-240.
28) Dupont, L.; Pirotte, B.; de Tullio, P.; Masereel, B.; Delarge, J .
-Methyl-4H-pyrido[3,2-e][1,2,4]thiadiazine 1,1-dioxide. Actra
(
41) Finet, M.; Godfraind, T.; Khoury, G. The Positive Inotropic
Action of the Nifedipine Analog K 8644 in Guinea-Pig and Rat
Isolated Cardiac Preparations. Br. J . Pharmacol. 1985, 86,
27-32.
3
Crystallogr. 1995, C51, 944-946.
29) Dupont, L.; de Tullio, P.; Pirotte, B.; Masereel, B.; Delarge, J .
3
-Methyl-4H-pyrido[4,3-e][1,2,4]thiadiazine 1,1-dioxide Mono-
hydrate. Actra Crystallogr. 1995, C51, 946-948.
J M9500582