Hydroxylated analogues of ATP-sensitive potassium channel openers belonging to the group of 6- and/or 7-substituted 3-Isopropylamino-4H-1,2,4- benzothiadiazine 1,1-dioxides: Toward an improvement in sulfonylurea receptor 1 selectivity and metabolism stabi

Article abstract of DOI:10.1021/jm200786z

Diversely substituted 3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides are known to be potent K_ATP channel openers, with several drugs being selective for the SUR1/Kir6.2 channel subtype. This work examined the biological activity, tissu

Full text of DOI:10.1021/jm200786z

Article
pubs.acs.org/jmc
Hydroxylated Analogues of ATP-Sensitive Potassium Channel
Openers Belonging to the Group of 6- and/or 7-Substituted 3-
Isopropylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides: Toward an
Improvement in Sulfonylurea Receptor 1 Selectivity and Metabolism
Stability
,
†
‡
‡
§
†
Pascal de Tullio,* Anne-Catherine Servais, Marianne Fillet, Florian Gillotin, Fabian Somers,
§
∥
†
Patrice Chiap, Philippe Lebrun, and Bernard Pirotte
†Drug Research Center, Laboratoire de Chimie Pharmaceutique, Universite
Belgium
́
de Lieg
̀
e, 1 Avenue de l’Ho
e, 1 Avenue de l’Ho
e (CHU), 1 Avenue de l’Ho
̂
̀
e,
‡
Drug Research Center, Laboratoire d’Analyse des Med
́
icaments, Universite
́
de Lieg
̀
e,
Belgium
§
Advanced Technology Corporation (ATC s.a.), Centre Hospitalier Universitaire de Lieg
Liege, Belgium
́
Laboratoire de Pharmacodynamie et de Therapeutique, Universite Libre de Bruxelles, 808 route de Lennik, 1070 Bruxelles, Belgium
̀
̀
∥
́
*
S Supporting Information
ABSTRACT: Diversely substituted 3-isopropylamino-4H-
,2,4-benzothiadiazine 1,1-dioxides are known to be potent
1
KATP channel openers, with several drugs being selective for the
SUR1/Kir6.2 channel subtype. This work examined the
biological activity, tissue selectivity, and in vitro metabolic
stability of hydroxylated analogues of 3-isopropylaminobenzo-
thiadiazine dioxides. Because of the presence of a chiral center,
the R and S isomers were prepared separately and characterized.
R isomers were systematically found to be more potent and more selective than S isomers on pancreatic tissue (compared to
vascular smooth muscle tissue), leading to compounds with an improved sulfonylurea receptor 1 (SUR1) selectivity. An in vitro
metabolic study revealed that 7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (1a) was rapidly biotransformed
and led in part to a mixture of the corresponding (R)- and (S)-3-(1-hydroxy-2-propyl)amino-substituted derivatives.
Radioisotopic experiments characterized one of the most potent and SUR1-selective enantiomers, (R)-7-chloro-3-(1-hydroxy-2-
propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide 13a, as being a KATP channel opener. Moreover, 13a exhibited an enhanced
metabolic stability. Such a compound can be considered as a new lead candidate displaying improved physicochemical
(
hydrosolubility) and pharmacological (tissue selectivity) properties as well as improved metabolic stability compared to its
nonhydroxylated counterpart, 1a.
INTRODUCTION
brain; the SUR2A/Kir6.2 KATP channel subtype is expressed in
the cardiac and skeletal muscles, while the SUR2B/Kir6.1 and
SUR2B/Kir6.2 combinations are mainly found in smooth
■
ATP-sensitive potassium channels (KATP channels) are trans-
membrane structures that allow the passive flux of potassium
ions through the cell membrane. Regulated by changes in
9
muscles. A mitochondrial KATP channel has also been
1−4
described, but the molecular structure of this channel is yet
intracellular adenosine triphosphate (ATP) concentrations,
10−12
to be clearly established.
Activation of KATP channels
these channels have been described as complex octameric
structures composed of two different subunits: Kir6.x (inwardly
rectifying potassium channel) and SURx (sulfonylurea
receptor), the latter containing the regulatory sites for most
induces an increase in the outflow of potassium ions through
the cytoplasmic membrane and hyperpolarizes the cell
membrane. The physiological impact of this hyperpolarization
is highly dependent on the tissue localization of the channel.
Thus, potassium channel openers (PCOs) can interfere with
several physiological processes, such as the release of insulin
5
KATP channel modulators. Several isoforms of Kir6.x (Kir6.1
and Kir6.2) and SURx (SUR1, SUR2A, and SUR2B) have been
6
,7
reported. The combination of these different subunits leads
to specific KATP channel subtypes diversely distributed
8
throughout the different tissues. The SUR1/Kir6.2 KATP
Received: June 17, 2011
channel subtype is found in the endocrine pancreas and the
Published: November 14, 2011
©
2011 American Chemical Society
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Figure 1. Chemical structure of diversely substituted 3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides (1−6) reported as SUR1/Kir6.2-type
potassium channel activators. Compound 7 is a metabolite of 1a, resulting from N-dealkylation. General formula 8 illustrates the newly synthesized
hydroxylated analogues of compounds 1−6. The asterisk refers to the presence of a chiral carbon atom.
from pancreatic β-cells and contractile activity in smooth
Furthermore, two studies of the metabolism of compound 1a
and its hydroxylated derivative, 13a, were conducted to identify
the major hydroxylated stereoisomer generated during
biotransformation of 1a by hepatic microsomes and to define
whether the introduction of a hydroxy group on the alkyl chain
at position 3 could improve its metabolic stability.
1
3,14
muscle cells.
For several years, selective activation of
pancreatic KATP channels has been known to be of therapeutic
value for the treatment of critical metabolic disorders such as
1
5,16
diabetes, obesity, and hyperinsulinemia.
Taking into
account these potential therapeutic benefits, we have developed
Lastly, radioisotopic experiments were conducted with 13a to
confirm that the compound behaved as a specific KATP channel
opener on pancreatic β-cells (SUR1/Kir6.2-type KATP channel
opener).
a series of new compounds belonging to 3-isopropylamino-4H-
1
,2,4-benzothiadiazine 1,1-dioxides over the past decade.
Among these drugs, compounds 1−6 (Figure 1) have been
1
7−20
described as potent and selective pancreatic PCOs.
Structure−activity relationships, assessed from previous studies,
indicated that the presence of a small branched alkylamino
chain at position 3 (i.e., an isopropylamino chain) as well as the
presence of one or two halogen atoms (preferably Cl or F) at
position(s) 6 and/or 7 and/or the presence of a small electron-
donating group (i.e., OMe) at position 7 were favorable to both
CHEMISTRY
■
The synthetic pathways giving access to the 6- and/or 7-
substituted 3-hydroxyalkylamino-4H-1,2,4-benzothiadiazine
,1-dioxides 8, 13, and 14 are described in Scheme 1. The
1
key intermediates for the synthesis of the 6- and 7-substituted
compounds were the previously reported or newly synthesized
3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxides
17−21
in vitro activity and selectivity for pancreatic tissue.
However, a recent in vitro metabolic study demonstrated the
rapid biotransformation of compound 1a, by hydroxylation, to
compound 8a (X = H, and Y = Cl) or, by dealkylation of the
1
7,19,20
12.
Such intermediates were obtained from the
corresponding anilines 9 in three steps (Scheme 1). The first
step is the well-known Girard reaction, which allows ring
closure through Friedel−Craft conditions. The cyclic sulfony-
lureas (10) obtained were then transformed into their
sulfonylthiourea analogues (11) by the action of phosphorus
pentasulfide in pyridine. In the next step, the 3-thioxo-4H-1,2,4-
benzothiadiazine 1,1-dioxides 11 were alkylated with methyl
iodide to give the corresponding 3-methylsulfanyl-substituted
key intermediates 12. These compounds were then heated for
several hours with the appropriate racemic or optically pure 1-
hydroxy-2-propylamine at 150 °C, leading to the expected
racemic mixtures 8, R stereoisomers 13, and S stereoisomers
14.
2
2
alkylamino side chain, to compound 7 (Figure 1).
Therefore, within the framework of drug discovery and lead
optimization processes of this series of compounds, we decided
to explore the impact of the introduction of a hydroxy group
onto the 3-alkylamino side chain on biological activity, tissue
selectivity, and biotransformation. A series of new compounds
(see general structure 8 in Figure 1) bearing a hydroxylated
alkyl chain at position 3, one or two halogen atoms, and/or a
methoxy group at position 6 or 7 were synthesized and
evaluated as putative KATP channel openers on two in vitro
pharmacological models. Moreover, and according to the
position of the hydroxy group on the alkyl side chain, a
stereogenic center was introduced onto the molecule leading to
two possible stereoisomers, the R and S enantiomers. Both
enantiomers were then prepared to confirm the influence of
stereochemistry on biological activity.
The key intermediate for the synthesis of the 6,7-dichloro-
substituted compounds was the previously described 6,7-
dichloro-3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-
18
dioxide 11c. This intermediate was obtained from 2-amino-
8
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a
Scheme 1.
a
(
i) (1) ClSO NCO, CH NO ; (2) AlCl ; (ii) P S , pyridine; (iii) CH I, NaHCO , CH OH/H O; (iv) isopropylamine, Δ; (v) 1-hydroxy-2-
2
3
2
3
2
5
3
3
3
2
propylamine, Δ; (vi) (1) ClSO H; (2) NH OH; (vii) 1,1′-thiocarbonyldiimidazole; (viii) isopropylamine or 1-hydroxy-2-propylamine, Δ.
3
4
a
Scheme 2.
a
(
i) (R,S)-1-hydroxy-2-propylamine, Δ; (ii) Ac O; (iii) (R,S)-2-hydroxypropylamine, Δ; (iv) (R,S)-1-methoxy-2-propylamine, Δ.
2
8
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Table 1. Effects of Diversely Substituted 4H-1,2,4-Benzothiadiazine 1,1-Dioxides on Secretion of Insulin from Rat Pancreatic
+
Islets and on the Contractile Activity of K -Depolarized Rat Aorta Rings
a
a
a
b
c
d
X
Y
R
RIS (10 μM)
RIS (1 μM)
RIS (0.1 μM)
IC50 (μM)
EC50 (μM)
SI
f
f
1
8
1
1
1
1
1
8
1
1
1
1
8
1
1
2
8
1
1
5
8
1
1
5
8
1
1
3
1
1
4
5a
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
CH CH(CH )OH
59.2 ± 2.6 (21) at 50 μM
nd
>50
3.03
38.9
3.58
0.62
37.5
0.48
3.78
26.5
2.05
>10
0.76
1.41
0.35
8.67
0.28
1.81
0.52
>10
0.23
1.73
1.67
>10
0.20
6.31
4.93
>10
1.75
2.98
>10
0.24
>300 (4)
nd
76
nd
nd
2
3
f
a
H
CH(CH )CH OH
9.4 ± 0.7 (22)
95.0 ± 5.1 (24)
27.0 ± 2.1 (10)
8.5 ± 0.6 (13)
77.3 ± 4.7 (24)
4.8 ± 0.4 (32)
32.4 ± 2.0 (12)
82.6 ± 4.7 (21)
12.4 ± 1.3 (12)
70.8 ± 4.6 (15)
3.7 ± 0.6 (13)
16.0 ± 1.1 (11)
5.1 ± 0.4 (15)
51.0 ± 2.5 (15)
6.3 ± 0.7 (12)
13.4 ± 1.4 (13)
5.0 ± 0,3 (13)
99.1 ± 5,9 (16)
7.5 ± 0.8 (15)
10.0 ± 0.7 (16)
9.7 ± 0.9 (15)
77.6 ± 6.7 (22)
4.1 ± 0.3 (14)
42.1 ± 4.6 (21)
32.8 ± 1,9 (16)
84.1 ± 4,9 (15)
8.5 ± 0.9 (24)
22.4 ± 1.6 (14)
88.6 ± 6.2 (20)
7.7 ± 0.7 (15)
94.0 ± 4.7 (23)
nd
nd
230 ± 27 (6)
3
2
f
f
f
f
7a
6a
3a
4a
H
CH(CH )CH OCH
3
nd
nd
3
2
f
f
f
H
CH(CH )CH OCOCH
3
85.9 ± 4.8 (24)
41.7 ± 2.8 (14)
nd
nd
3
2
H
(R)-CH(CH )CH OH
97.2 ± 4.9 (22)
f
189 ± 17 (10)
124 ± 11 (7)
31.8 ± 3.9 (4)
>300 (4)
305
3.3
66
3
2
f
H
(S)-CH(CH )CH OH
nd
nd
3
2
e
a
H
CH(CH3)2
CH(CH )CH OH
36.2 ± 2.4 (31)
81.7 ± 4.0 (23)
90.4 ± 3.5 (23)
f
b
H
nd
>79
3
2
f
f
f
f
7b
3b
4b
b
H
F
CH(CH )CH OCH
3
nd
nd
nd
nd
3
2
f
H
F
(R)-CH(CH )CH OH
71.8 ± 5.7 (16)
nd
>300 (5)
>146
3
2
f
f
f
f
H
F
(S)-CH(CH )CH OH
nd
nd
nd
nd
3
2
H
F
CH(CH3)2
CH(CH )CH OH
47.3 ± 3.7 (23)
59.8 ± 4.7 (14)
21.1 ± 1.8 (16)
99.3 ± 5.6 (22)
13.2 ± 1.0 (26)
67.0 ± 5.0 (14)
35.6 ± 2.6 (14)
96.9 ± 4.9 (16)
f
43.3 ± 10.7 (5)
17.6 ± 0.7 (5)
7.5 ± 0.2 (4)
17.4 ± 2.9 (6)
2.3 ± 0.2 (8)
>200 (5)
57
c
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
H
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
nd
12.5
21.4
2.0
3
2
3c
(R)-CH(CH )CH OH
92.5 ± 5.4 (24)
f
3
2
4c
e
(S)-CH(CH )CH OH
nd
3
2
CH(CH3)2
CH(CH )CH OH
84.9 ± 4.5 (21)
f
8.2
d
nd
>110
>385
3
2
3d
4d
(R)-CH(CH )CH OH
97.3 ± 5.6 (23)
f
>200 (6)
3
2
f
f
(S)-CH(CH )CH OH
nd
nd
>200 (3)
nd
3
2
e
d
CH(CH3)2
CH(CH )CH OH
13.2 ± 1.2 (16)
66.8 ± 3.8 (13)
65.8 ± 3.1 (21)
76.7 ± 4.3 (15)
f
53.4 ± 3.6 (4)
232
f
f
e
nd
nd
nd
3
2
f
3e
4e
e
(R)-CH(CH )CH OH
nd
241 ± 22 (6)
144
3
2
f
f
f
f
(S)-CH(CH )CH OH
nd
nd
nd
nd
3
2
CH(CH3)2
CH(CH )CH OH
18.2 ± 1.4 (14)
97.9 ± 5.0 (23)
99.3 ± 5.4 (20)
68.9 ± 3.8 (16)
f
55.7 ± 2.0 (6)
279
f
f
f
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
nd
nd
nd
3
2
f
3f
H
(R)-CH(CH )CH OH
nd
>300 (5)
>61
3
2
f
f
f
f
4f
e
H
(S)-CH(CH )CH OH
nd
nd
nd
nd
3
2
f
H
CH(CH3)2
(R)-CH(CH )CH OH
67.6 ± 4.3 (20)
81.0 ± 3.9 (21)
nd
274 ± 19 (5)
53.1 ± 2.0 (4)
157
f
3g
Cl
Cl
Cl
nd
17.8
3
2
f
f
f
f
4g
e
(S)-CH(CH )CH OH
nd
nd
nd
nd
3
2
CH(CH3)2
20.6 ± 1.9 (13)
73.8 ± 4.1 (19)
37.0 ± 2.7 (6)
154
a
RIS is the percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose [mean ± SEM (n)].
b
c
Estimated IC50 is the drug concentration giving 50% inhibition of insulin release. EC is the drug concentration giving 50% relaxation of the 30
50
d
e
mM KCl-induced contraction of rat aortic rings [mean ± SEM (n)]. SI is the selectivity index (=EC /IC ). Published compounds and results
5
0
50
17−19
f
(
refs
). Not determined.
4
,5-dichlorobenzenesulfonamide 10c by a ring closure reaction
RESULTS AND DISCUSSION
■
using 1,1′-thiocarbonyldiimidazole (Scheme 1). Intermediate
1
3
Two in vitro models were used to assess the biological activity
of the original drugs on two different tissues expressing KATP
channels. The first one determined the ability of the newly
prepared compounds to inhibit the glucose-induced secretion
of insulin from rat pancreatic islets. Data collected with this
model were expressed as the percentage of residual insulin
release recorded at different drug concentrations and, for
several compounds, as the estimated IC values, expressing the
drug concentration leading to 50% inhibition of insulin release.
The results obtained within each series of compounds were
compared to previously reported data
nonhydroxylated 3-isopropylamino-substituted counterparts 1a,
0c was synthesized by a classical chlorosulfonation reaction on
,4-dichloroaniline 9c followed by treatment with aqueous
1
8
ammonia.
Compound 15a, the position isomer of 8a, was prepared by
replacement of 1-hydroxy-2-propylamine with racemic 2-
hydroxypropylamine in the last step (Scheme 2).
Compound 16a, bearing a racemic 1-acetoxy-2-propylamino
chain at position 3, was synthesized by reacting acetic
anhydride on (R,S)-7-chloro-3-(1-hydroxy-2-propyl)amino-
5
0
17−19
obtained with the
4
H-1,2,4-benzothiadiazine 1,1-dioxide (8a) (Scheme 2).
The 3-[(1-methoxy)-2-propyl]amino-substituted compounds
7a and 17b were obtained by means of the use of 1-methoxy-
-propylamine on intermediates 12a and 12b, respectively
1
b, 2−4, 5d, and 5e (Figure 1).
In the 7-chloro series, four different alkylamino chains were
introduced at position 3: the (R,S)-(2-hydroxypropyl)amino
(compound 15a), the (R,S)-(1-hydroxy-2-propyl)amino (com-
1
2
(Scheme 2).
8
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Journal of Medicinal Chemistry
Article
pound 8a), the (R,S)-(1-methoxy-2-propyl)amino (compound
(see, for example, 8a vs 1a and 8c vs 2) or negatively affected
for the S enantiomers (see, for example, 14a vs 1a and 14c vs
2), the inversion of chirality led to compounds (R enantiomers)
expressing, in most cases, an improved selectivity toward the
endocrine tissue (see 13a−13d vs 1a, 1b, 2, and 5d,
respectively). The 6-bromo-substituted compound 13e may
constitute an exception to this rule because its analogue, 5e, a
powerful inhibitor of the insulin-releasing process (IC50 = 0.20
μM), is already the most tissue-selective nonhydroxylated drug
(SI = 279).
1
1
7a), and the (R,S)-(1-acetoxy-2-propyl)amino (compound
6a) chains. The comparison between the inhibitory effects on
insulin secretion of the latter drugs with that of compound 1a
Table 1) indicated that the presence of an (R,S)-(1-hydroxy-2-
(
propyl)amino chain, which is structurally close to the
isopropylamino chain, led to a potent inhibitor of the insulin-
releasing process (compound 8a). Alkylation and, to a lesser
extent, acylation of the hydroxy group of 8a induced a decrease
in the inhibitory activity on the pancreatic endocrine tissue (see
1
7a and 16a in Table 1). According to these results, the (R,S)-
The 7-chloro-substituted and the 6-chloro-substituted R
enantiomers 13a and 13d, respectively, appeared to be the most
promising optically pure compounds in terms of activity and
tissue selectivity. These drugs exhibited a very potent activity
on pancreatic β-cells (IC50 < 1 μM) together with a high
selectivity ratio (EC /IC > 300). By contrast, 6,7-
(1-hydroxy-2-propyl)amino chain was selected to be introduced
into the other series of compounds. This modulation led to
compounds 8b−8f that had a high efficacy but were less potent
in inhibiting insulin release than their 3-isopropylamino
counterparts (compounds 1b, 2, 5d, 5e, and 3, respectively).
As previously observed, the 6,7-dichloro substitution
appeared to be favorable for activity (8c). The rank order of
potency on pancreatic β-cells was as follows: 6,7-dichloro > 6-
chloro = 6-bromo > 7-chloro ≥ 7-fluoro > 7-methoxy.
As expected, the introduction of a hydroxy group onto the
first carbon atom of the 2-isopropylamino chain was
responsible for the formation of a stereogenic center.
Therefore, in each series, the two enantiomers were prepared
and tested to characterize the influence of stereochemistry on
biological activity. Looking at the inhibitory effect of the R and
S enantiomers on the insulin-releasing process, we noticed that,
whatever the nature of the substituent on the aromatic ring, the
R isomer was systematically found to be more potent than the S
isomer and the racemate (except for 13e vs racemate 8e)
5
0
50
1
8
disubstituted hydroxylated R enantiomers such as compound
13c did not express a marked level of tissue selectivity. Such a
feature is in accordance with structure−activity relationships
deduced from previous works on 3-alkylamino-substituted
17−21
benzothiadiazine 1,1-dioxides.
A previous metabolic study highlighted the rapid bio-
tranformation of 1a into a major and nondesired N-dealkylated
derivative 7 and into a minor metabolite found to be
22
hydroxylated derivative 8a (X = H, and Y = Cl). In this
study, the determination of the exact stereochemistry of the
formed metabolites appeared to be critical to improve
extrapolation of the in vivo activity of 1a as well as of several
related 3-alkylaminobenzothiadiazine dioxides. For this pur-
pose, the resulting metabolites obtained with 1a after rat liver
microsome biotransformation were analyzed by capillary
electrophoresis using cyclodextrin for chiral separations. The
results clearly demonstrate that there is a nonstereoselective
hydroxylation of the alkylamino side chain. Indeed, both
enantiomers of 7-chloro-3-(1-hydroxy-2-propyl)amino-4H-
1,2,4-benzothiadiazine 1,1-dioxide were formed in the same
ratio during in vitro biotransformation (Figure 2). The in vitro
(
Table 1). The three most potent R isomers exhibited
estimated IC50 values below the micromolar range (0.35 μM
for 13c, 0.52 μM for 13d, and 0.62 μM for 13a).
The myorelaxant activity of most of the newly synthesized
+
compounds was evaluated on K -depolarized (30 mM KCl) rat
aorta rings, and the results are reported as EC50 values (Table
1
). In all cases, the introduction of a (1-hydroxy-2-propyl)-
amino chain at position 3 subsequently decreased the
vasorelaxant effect of the hydroxylated derivatives, in
comparison with the effects of their nonhydroxylated 3-
isopropylamino-substituted counterparts [see, for example,
racemic compounds 8a−8d vs nonhydroxylated compounds
1
a, 1b, 2, and 5d, respectively (Table 1)]. As expected from
18
previous investigations with the nonhydroxylated drugs, the
most potent hydroxylated compounds on rat aorta rings
belonged to the 6,7-dichloro series (8c, 13c, and 14c), followed
by compound 13g, belonging to the 6-chloro-7-methoxy series.
Hydroxylated compounds with other substituents on the
benzene ring failed to express a relevant myorelaxant activity.
It is interesting to note that, in this pharmacological model, the
stereochemistry of the side chain at position 3 exerted a weak
effect [see, for example, 13a vs 14a and 13c vs 14c (Table 1)].
The selectivity indexes [SI = EC /IC (see Table 1)] were
5
0
50
calculated to compare the apparent tissue selectivity (vascular
vs pancreatic tissue) of the drugs. All nonhydroxylated drugs
Figure 2. (A) Electropherogram of a standard solution of compounds
(
1a, 1b, 3, 4, 5d, and 5e), as previously reported in the
1a (62.5 μg/mL) and 8a (62.5 μg/mL) in an ACN/H
2
O mixture
1
7−20
(50:50, v/v). (B) Electropherogram resulting from in vitro metabolism
literature
(except for 5e), were found to be more selective
of compound 1a. Peaks: 1, compound 1a; 2, R enantiomer 13a; 3, S
enantiomer 14a.
for the pancreatic than for the aortic tissue. The rank order of
selectivity was as follows: 6-Br > 6-Cl > 7-methoxy = 6-Cl and
7
-methoxy > 7-Cl = 7-F > 6,7-diCl. The presence of a hydroxy
metabolic stability of the most interesting isomer, (R)-7-chloro-
group on the alkylamino chain induced various effects on the
selectivity ratio of the drugs. Indeed, while the selectivity
appeared to be weakly affected for the racemic compounds
3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-di-
oxide (13a), was further evaluated using phenobarbital-induced
8
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Journal of Medicinal Chemistry
Article
rat liver microsomes. The use of this type of microsome is a
well-established method both for producing expected in vivo
phase I metabolites and for determining the metabolic
weakness of drugs. The residue of biotransformation of
compound 13a was analyzed using the slightly modified
hydroxylated counterpart, 1a (7-chloro-3-isopropylamino-4H-
1,2,4-benzothiadiazine 1,1-dioxide), by examining the concen-
tration of the two drugs in saturated aqueous solutions. The
fraction of the molecule in solution was determined by high-
performance liquid chromatography using the same exper-
imental conditions that were used for the biotransformation
study. The experimental results confirmed the expected
increase in water solubility due to the introduction of a
hydroxy group onto the isopropyl side chain, with solubility
values of 1.71 mg/mL for compound 13a and 0.04 mg/mL for
compound 1a (>40-fold increase).
2
2
described liquid chromatography (LC) conditions. As
shown in Figure 3, the parent product 13a was eluted at a
In the last series of experiments, radioisotopic measurements
were conducted on whole pancreatic islets to confirm the
mechanism of action of the newly synthesized compounds.
Figure 4 clearly shows that the addition of the selected
compound 13a provoked a rapid, pronounced, and sustained
Figure 4. Effect of compound 13a (10 μM) on the outflow of 86Rb
from rat pancreatic islets perifused throughout in the absence (●) or
presence (○) of glibenclamide (10 μM). Basal medium contained
2
+
glucose (5.6 mM) and extracellular Ca (2.56 mM). Mean values
±SEM) refer to four individual experiments.
Figure 3. Metabolic profiles of compounds 13a (top) and 1a (down).
Retention times of parent peaks: 13.23 min for 13a and 17.90 min for
(
1
a. Retention time of compound 7: 10.83 or 10.96 min.
increase in the rate of outflow of 86Rb ( K substitute) from
42
prelabeled and perifused rat pancreatic islets. In islets exposed
retention time of approximately 13 min. Examination of the
chromatogram of the sample that had undergone P-450-
mediated biotransformation for an incubation period of 1 h
highlighted the presence, besides the parent compound, of only
one metabolite in a negligible quantity (≈2%). Comparison of
this chromatogram with the metabolic profile of compound 1a
obtained under identical experimental conditions (Figure 3)
clearly revealed the marked enhancement in metabolic stability
due to the introduction of a hydroxy group on the alkylamino
side chain at position 3. The metabolite detected at a retention
time of 10.83 min was probably the N-dealkylated derivative [7
18,24
throughout to the KATP channel blocker glibenclamide,
the
stimulatory effect of 13a was completely abolished (Figure 4).
These findings indicate that 13a activates KATP channels in
islet cells.
+
The enhancement of membrane K permeability induced by
the activation of KATP channels should hyperpolarize the
insulin-secreting cells, restrict the activity of voltage-dependent
2
+
2+
Ca channels, decrease the rate of Ca entry, and, ultimately,
inhibit the secretory process.
Such a cascade of events is indirectly revealed by the effects
4
5
(Figure 1)].
of 13a on the outflow of Ca from rat pancreatic islets
perifused in the presence of an insulinotropic glucose
concentration (Figure 5).
An improvement in the hydrosolubility could be expected
with the introduction of a hydroxy group onto the alkyl side
chain of previously described 3-alkylamino-substituted benzo-
thiadiazine dioxides. To verify such an assumption, we have
compared the water solubility at room temperature of
compound 13a [(R)-7-chloro-3-(1-hydroxy-2-propyl)amino-
Thus, 13a inhibited the fractional rate of outflow of 45Ca
from islets exposed throughout to 16.7 mM glucose and
2
+
45
extracellular Ca but failed to affect the outflow of Ca from
islets perifused throughout in the presence of 16.7 mM glucose
2
+
4
H-1,2,4-benzothiadiazine 1,1-dioxide] with that of its non-
but in the absence of extracellular Ca (Figure 5, top panel). In
8
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dx.doi.org/10.1021/jm200786z | J. Med. Chem. 2011, 54, 8353−8361

Journal of Medicinal Chemistry
Article
conducted with 13a further indicated that the compound
affected the pancreatic endocrine tissue through the activation
of KATP channels. Altogether, these data indicate that (R)-7-
chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadia-
zine 1,1-dioxide (13a) can be considered as a very promising
lead compound belonging to the group of benzothiadiazine-
type SUR1-selective KATP channel openers.
MATERIALS AND METHODS
■
Chemistry. Melting points were determined on a Stuart SMP3
capillary apparatus and are uncorrected. IR spectra were recorded as
KBr pellets on a Perkin-Elmer 1000 FTIR spectrophotometer. The H
1
NMR spectra were recorded on a Bruker AW-80 (80 MHz) or a
Bruker Avance (500 MHz) instrument using d -DMSO as the solvent
6
with TMS as an internal standard; chemical shifts are reported as δ
values (parts per million) relative to that of the internal reference. The
abbreviations s (singlet), d (doublet), m (multiplet), and b (broad) are
used throughout. Elemental analyses (C, H, N, S) were used to
confirm the purity of all the compounds (>95%) and were conducted
with a Thermo Scientific FlashEA 1112-elemental analyzer (results
within ±0.4% of the theoretical values). All reactions were routinely
checked by TLC on silica gel Merck 60 F254
.
6
-Bromo-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-Diox-
Figure 5. Effect of compound 13a (10 μM) on the outflow of 45Ca
ide (5e). The title compound was obtained according to a method
previously described for the corresponding 6-chloro-substituted
analogue 5d starting from 3-bromoaniline instead of 3-chloroaniline.
The intermediate 6-bromo-3-methylsulfanyl-4H-1,2,4-benzothiadia-
zine 1,1-dioxide (12e) obtained in three steps reacted with
isopropylamine according to ref 19 to give the title compound: mp
52−259 °C; IR (KBr) 3309, 3115, 2971, 1625, 1576, 1465, 1388,
275, 1239, 1162, 1140, 1119, 1074, 1059 cm ; H NMR (DMSO-
d ) δ 1.16 [d, 6H, CH(CH ) ], 3.92 (m, 1H, NHCH), 7.24 (bs, 1H,
NHCH), 7.42 (m, 2H, 5-H + 7-H), 7.59 (d, 1H, 8-H), 10.35 (s, 1H,
and release of insulin from perifused rat pancreatic islets. The top
1
9
panel shows the effect of 13a on the outflow of 45Ca from pancreatic
islets perifused throughout in the presence of an insulinotropic glucose
2
+
concentration (16.7 mM). Basal medium contained extracellular Ca
(
0
2+
●; 2.56 mM) or were deprived of Ca and enriched with EGTA (○;
.5 mM). The bottom panel shows the effect of 13a on the release of
2
1
−1 1
insulin from pancreatic islets perifused throughout in the presence of
an insulinotropic glucose concentration (16.7 mM). Basal medium
6
3 2
contained extracellular Ca2+ (●; 2.56 mM). Mean values (±SEM)
NH). Anal. (C H BrN O S) C, H, N, S.
refer to four to six individual experiments.
10 12
3
2
General Synthetic Pathway to 3-Hydroxyalkyl/3-Methox-
yalkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides (8a, 8b,
islets exposed to an insulinotropic glucose concentration and
8
d−8f, 13a, 13b, 13d−13g, 14a, 14b, 14d−14g, 15a, 17a, and
2
+
45
extracellular Ca , a decrease in the rate of Ca outflow is
17b) (method A). The appropriate 3-methylsulfanyl-4H-1,2,4-
2
+
18,24
17−19
known to reflect a reduction in the rate of Ca entry.
benzothiadiazine 1,1-dioxide (12a−12g)
(0.5 g) and the
Figure 5 (bottom panel) also reveals that 13a provoked
appropriate hydroxyalkylamine/methoxyalkylamine (5 mL) were
stirred and heated at 150 °C for several hours. After cooling, the
solution was supplemented with water (20 mL), and the pH was
adjusted to 12 with an aqueous solution of NaOH (5%). After
treatment with charchoal, the filtrate was acidified with 6 N HCl and
the resulting precipitate was collected by filtration, washed with water,
and dried. The isolated solid was recrystallized in a MeOH/water
mixture (yields of 50−75%).
modifications in the insulin secretory rate displaying a time
4
5
course parallel to that of the Ca outflow response.
Together, these data indicate that the inhibitory effect of 13a
on the insulin-releasing process is mediated by a reduction in
the rate of Ca2 entry resulting from the activation of KATP
channels.
+
All details relative to compounds 8b, 8d−8f, 13b, 13d−13g, 14b,
1
4d−14g, 15a, and 17b are reported in the Supporting Information.
CONCLUSION
■
General Synthetic Pathway to 6,7-Dichloro-3-hydroxyalky-
This work reveals that the introduction of a hydroxy group
onto the alkylamino side chain at position 3 of well-known
benzothiadiazine 1,1-dioxide KATP channel openers could lead
to an improvement in their pharmacodynamic profile. The
presence of an (R)-1-hydroxy-2-propylamino chain at position
lamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides 8c, 13c, and
1
4c (method B). The synthetic pathway described above was used to
obtain compounds 8c, 13c, and 14c, except that intermediates 12a−
2g were replaced by 6,7-dichloro-3-(1H-imidazol-1-yl)-4H-1,2,4-
1
18
benzothiadiazine 1,1-dioxide 11c in the reaction with the appropriate
hydroxyalkylamine. The isolated solid was recrystallized in a MeOH/
water mixture (yields of 50−60%).
All details relative to compounds 13c and 14c are reported in the
Supporting Information.
3
of the benzothiadiazine ring increased in most cases
selectivity for the pancreatic tissue. Such a pharmacomodula-
tion leads to compounds exhibiting an activity on the endocrine
tissue below the micromolar concentration range and a
selectivity ratio (EC50 vascular/IC50 pancreatic) of >300 for
the 7-chloro- and 6-chloro-substituted derivatives 13a and 13d,
respectively. Moreover, from a physicochemical point of view,
and according to the results obtained with 13a, the presence of
a hydroxy group on the exocyclic alkyl chain of 3-
alkylaminobenzothiadiazine dioxides can be expected to lead
to an enhancement of the in vitro metabolic stability and to a
marked increase in water solubility. Radioisotopic experiments
(R,S)-7-Chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (8a). The title compound was obtained
according to method A starting from 7-chloro-3-methylsulfanyl-4H-
1
7
1
,2,4-benzothiadiazine 1,1-dioxide (12a) and (R,S)-1-hydroxy-2-
propylamine: mp 216−217 °C; IR (KBr) 3429, 3285, 3102, 1626,
−1
1
1
1
3
582, 1482, 1272, 1162, 1122, 1105 cm ; H NMR (DMSO-d ) δ
6
.05 [d, 3H, CH(CH )CH OH], 3.30 [bd, 2H, CH(CH )CH OH],
.80 [m, 1H, CH(CH )CH OH], 4.85 [bs, 1H, CH(CH )CH OH],
3
2
3
2
3
2
3
2
6.90 (bd, 1H, NHCH), 7.10 (d, 1H, 5-H), 7.50 (d, 1H, 6-H), 7.55 (s,
1H, 8-H), 10.50 (bs, 1H, NH). Anal. (C H ClN O S) C, H, N, S.
1
0
12
3
3
8
359
dx.doi.org/10.1021/jm200786z | J. Med. Chem. 2011, 54, 8353−8361

Journal of Medicinal Chemistry
Article
(
R,S)-6,7-Dichloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzo-
Capillary Electrophoresis Experiments. Instrumentation.
thiadiazine 1,1-Dioxide (8c). The title compound was obtained
according to method B starting from 6,7-dichloro-3-(1H-imidazol-1-
Capillary electrophoresis (CE) experiments were conducted on a
3
D
HP CE system (Agilent Technologies, Waldbronn, Germany)
equipped with an autosampler, an on-column diode array detector,
and a temperature control system (15−60 ± 0.1 °C). A CE
Chemstation (Hewlett-Packard, Palo Alto, CA) was used for
instrument control, data acquisition, and data handling. Fused-silica
capillaries were provided by ThermoSeparation Products (San Jose,
CA).
18
yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide 14 and (R,S)-1-hydroxy-2-
propylamine: mp 222−227 °C; IR (KBr) 3436, 1640, 1577, 1461,
372, 1276, 1149, 1077, 1048 cm ; H NMR (DMSO-d ) δ 1.13 [d,
H, CH(CH )CH OH], 3.42 [m, 2H, CH(CH )CH OH], 3.85 [m,
H, CH(CH )CH OH], 4.97 [bs, 1H, CH(CH )CH OH], 7.23 (bs,
H, NHCH), 7.47 (bs, 1H, 5-H), 7.87 (s, 1H, 8-H), 10.55 (s, 1H,
NH). Anal. (C H Cl N O S) C, H, N, S.
(
diazine 1,1-Dioxide (13a). The title compound was obtained
according to method A starting from 7-chloro-3-methylsulfanyl-4H-
,2,4-benzothiadiazine 1,1-dioxide (12a) and (R)-1-hydroxy-2-
propylamine: mp 228−230 °C; IR (KBr) 3469, 3307, 2978, 1626,
581, 1481, 1279, 1248, 1153, 1123, 1095, 1047 cm ; H NMR
DMSO-d ) δ 1.10 [d, 3H, CH(CH )CH OH], 3.30 [bd, 2H,
CH(CH )CH OH], 3.80 [m, 1H, CH(CH )CH OH], 4.90 [b, 1H,
CH(CH )CH OH], 6.90 (bd, 1H, NHCH), 7.10 (d, 1H, 5-H), 7.50
d, 1H, 6-H), 7.55 (s, 1H, 8-H), 10.50 (bs, 1H, NH). Anal.
C H ClN O S) C, H, N, S.
1
(
diazine 1,1-Dioxide (14a). The title compound was obtained
according to method A starting from 7-chloro-3-methylsulfanyl-4H-
,2,4-benzothiadiazine 1,1-dioxide (12a) and (S)-1-hydroxy-2-
propylamine: mp 224−228 °C; IR (KBr) 3462, 3308, 2978, 1626,
581, 1481, 1279, 1248, 1161, 1123, 1096, 1045 cm ; H NMR
DMSO-d ) δ 1.10 [d, 3H, CH(CH )CH OH], 3.30 [bd, 2H,
CH(CH )CH OH], 3.80 [m, 1H, CH(CH )CH OH], 4.85 [bs, 1H,
CH(CH )CH OH], 6.90 (bd, 1H, NHCH), 7.10 (d, 1H, 5-H), 7.50
−1 1
1
3
1
1
6
3
2
3
2
3
2
3
2
10
11
2
3
3
Electrophoretic Technique. Electrophoretic separations were con-
ducted with uncoated fused-silica capillaries having a 50 μm internal
diameter and a 48.5 cm length (40 cm to the detector). At the
beginning of each working day, the capillary was washed with 1 N
NaOH, water, and the background electrolyte containing the
cyclodextrin (BGE-CD) for 10 min. Before each injection, the
capillary was washed successively with 1 N NaOH for 7 min and water
for 3 min and then equilibrated with the BGE-CD for 10 min. The
applied voltage was 25 kV in the negative polarity mode, and UV
detection was set at 210 nm. Injections were made by applying a
pressure of 50 mbar for a period of 4 s, and the capillary was
thermostated at 15 °C. The BGE-CD used for electrophoretic
experiments consisted of 10 mM octakis-6-O-sulfo-γ-CD in 100 mM
phosphoric acid adjusted to pH 3 with triethanolamine. Octakis-6-O-
sulfo-γ-CD was a gift from G. Vigh (Texas A&M University, College
Station, TX).
R)-7-Chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothia-
1
7
1
−
1
1
1
(
6
3
2
3
2
3
2
3
2
(
(
0
12
3
3
S)-7-Chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothia-
1
7
1
The residue from the in vitro biological metabolization test was
redissolved in 500 μL of ACN, vigorously stirred for 5 min, and then
gently evaporated to dryness under a nitrogen flux. The residue was
−
1
1
1
(
6
3
2
3
2
3
2
finally redissolved in 100 μL of an ACN/H O mixture (50:50) and
2
3
2
vigorously stirred for 3 min. The solution was finally centrifuged at
3600 rpm for 5 min and injected into the CE system.
LC Conditions for Biotransformation and Solubility Stud-
ies. The LC separations were conducted on an Agilent 1100 series
LC system equipped with a quaternary pump, a column thermostat, an
autosampler, and a diode array detector. The analyte separations were
performed on an Alltech Hypersil BDS C18 column (15 mm × 4.6
mm, inside diameter; particle size of 3 μm) from Alltech (Breda, The
Netherlands) using mobile phase A (water) and mobile phase B
(
(
d, 1H, 6-H), 7.55 (s, 1H, 8-H), 10.50 (bs, 1H, NH). Anal.
C H ClN O S) C, H, N, S.
1
1
(
0
12
3
3
R,S)-7-Chloro-3-(1-acetoxy-2-propyl)amino-4H-1,2,4-benzothia-
diazine 1,1-Dioxide (16a). The mixture of (R,S)-7-chloro-3-(1-
hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide (8a)
0.5 g, 1,72 mmol) and acetic anhydride (3 mL) was stirred at
room temperature for 2 h. The reaction mixture was then
supplemented with water (20 mL) and stirred for 20 min. The
resulting precipitate was collected by filtration, washed with water, and
dried: mp 205−206 °C; IR (KBr) 3299, 3181, 3084, 1739, 1631, 1579,
480, 1244, 1160, 1103 cm ; H NMR (DMSO-d ) δ 1.17 [d, 3H,
CH CH(CH )OAc], 2.02 (s, 3H, COCH ), 3.99 [m, 1H, CH(CH )-
CH OH], 4.11 [m, 2H, CH(CH )CH OAc], 7.24 (bm, 2H, NHCH +
-H), 7.61 (m, 1H, 6-H), 7.67 (s, 1H, 8-H), 10.62 (bs, 1H, NH). Anal.
C H ClN O S) C, H, N, S.
1
(
thiadiazine 1,1-Dioxide (17a). The title compound was obtained
according to method A starting from 7-chloro-3-methylsulfanyl-4H-
,2,4-benzothiadiazine 1,1-dioxide (12a) and (R,S)-1-methoxy-2-
propylamine: mp 150−153 °C; IR (KBr) 3294, 3186, 3118, 3084,
983, 2932, 2880, 1631, 1582, 1480, 1250, 1162, 1105 cm ; H NMR
DMSO-d ) δ 1.05 [d, 3H, CH(CH )CH OCH ], 3.00−3.40 [m, 5H,
CH(CH )CH OCH + OCH ], 3.90 [m, 1H, CH(CH )CH OCH ],
.95 (bs, 1H, NHCH), 7.10 (d, 1H, 5-H), 7.50 (d + s, 2H, 6-H + 8-H),
0.45 (bs, 1H, NH). Anal. (C H ClN O S) C, H, N, S.
Metabolism. The in vitro biological test system selected to
metabolize the parent compounds was the phenobarbital (PB)-
(
(
ACN) with a flow rate of 0.8 mL/min and the following linear
gradient: 10% ACN at 0 min, 40% ACN at 24 min, 60% ACN at 27
min, and 10% ACN at 30 min. The column temperature was set at 40
°C.
The residue from the in vitro biological metabolization test was
redissolved in 500 μL of ACN, the solution vigorously stirred for 5
min, and then the residue gently evaporated to dryness under a
nitrogen flux. The residue was finally redissolved in 100 μL of an
−1 1
1
6
2
3
3
3
2
3
2
5
(
2
14
3
4
R,S)-7-Chloro-3-(1-methoxy-2-propyl)amino-4H-1,2,4-benzo-
ACN/H O mixture (50:50) and the solution vigorously stirred for 3
2
min. The solution was finally centrifuged at 13600 rpm for 5 min and
injected into the LC system.
17
1
Solubility Studies. A suspension of 5−10 mg of compound was
stirred in 5 mL of distilled water at room temperature for 1 h. After
decantation for 30 min, 1 mL was collected and centrifuged at 4000g
for 5 min. The supernatant was decanted and used as an injection
solution.
A calibration curve for each derivative was determined using
reference solutions at different concentrations under the same LC
conditions. These curves were used to calculate the water solubility of
the two molecules.
−
1 1
2
(
6
3
2
3
3
2
3
3
3
2
3
6
1
11
14
3
3
22
induced male rat liver microsome system. The parent compounds
were dissolved in methanol and added directly to the incubation
medium, yielding a final substrate concentration of 200 μM and a final
percentage in methanol of <1%. The incubations were performed at 37
Measurements of Release of Insulin from Incubated Rat
Pancreatic Islets. The method used to measure the release of insulin
2
3,24
from incubated rat pancreatic islets was previously described.
°
C in a water shacking bath with a final protein content of 1 mg/mL in
Measurement of the Contractile Activity in Rat Aorta. The
a total volume of 1 mL. The reactions were initiated by addition of a
NADPH regenerating system. The reactions were stopped after an
incubation time of 60 min by addition of 1 mL of methanol and 2 mL
of acetonitrile and by a subsequent vortexing step. Samples were
further centrifuged at 2000g for 5 min. The supernatant was further
decanted into a glass tube, and organic solvents were evaporated under
an inert nitrogen flux to concentrate the samples.
method used to measure the myorelaxant effect of the drugs on 30
2
3,24
mM KCl-precontracted rat aortic rings was previously described.
8
6
45
Measurements of Outflow of Rb, Outflow of Ca, and Release
of Insulin from Perifused Rat Pancreatic Islets. The methods used
8
6
42
45
for measuring outflow of Rb ( K substitute), outflow of Ca, and
release of insulin from prelabeled and perifused rat pancreatic islets
19,24
were previously described.
8
360
dx.doi.org/10.1021/jm200786z | J. Med. Chem. 2011, 54, 8353−8361

Journal of Medicinal Chemistry
Article
(
10) Szewczyk, A.; Skalska, J.; Glab, M.; Kulawiak, B.; Malinska, D.;
ASSOCIATED CONTENT
Supporting Information
■
Koszela-Piotrowska, I.; Kunz, W. S. Mitochondrial Potassium
Channels: From Pharmacology to Function. Biochim. Biophys. Acta
*
S
Synthesis of compounds 8b, 8d−8f, 13b−13g, 14b−14g, 15a,
2
(
006, 1757, 715−720.
and 17b and elemental analysis of compounds 5e, 8a−8f, 13a−
11) Ardehali, H.; O’Rourke, B. Mitochondrial KATP Channels in Cell
1
3g, 14a−14g, 15a, 16a, 17a, and 17b. This material is
Survival and Death. J. Mol. Cell. Cardiol. 2005, 39, 7−16.
available free of charge via the Internet at http://pubs.acs.org.
(12) Sandler, S.; Andersson, A. K.; Larsson, J.; Makeeva, N.; Olsen,
T.; Arkhammar, P. O.; Hansen, J. B.; Karlsson, F. A.; Welsh, N.
Possible Role of an Ischemic Preconditioning-like Response
Mechanism in KATP Channel Opener-mediated Protection Against
Streptozotocin-induced Suppression of Rat Pancreatic Islet Function.
Biochem. Pharmacol. 2008, 76, 1748−1756.
AUTHOR INFORMATION
Corresponding Author
Universite de Liege, 1 avenue de l’Ho ̂p ital, 4000 Liege,
́ ̀ ̀
Belgium. Telephone: +32 4 366 43 73. Fax: +32 4 366 43 62. E-
mail: p.detullio@ulg.ac.be.
■
*
(
13) Lawson, K. Potassium Channel Activation: A Potential
Therapeutic Approach? Pharmacol. Ther. 1996, 70, 39−63.
14) Lebrun, P.; Antoine, M. H.; Ouedraogo, R.; Herchuelz, A.; de
(
Author Contributions
P.L. and B.P. equally supervised this work.
Tullio, P.; Delarge, J.; Pirotte, B. Pyridothiadiazines as Potent
Inhibitors of Glucose-induced Insulin Release. Adv. Exp. Med. Biol.
1
997, 426, 145−148.
ACKNOWLEDGMENTS
■
(15) Rasmussen, S. B.; Sorensen, T. S.; Hansen, J. B.; Mandrup-
Poulsen, T.; Hornum, L.; Markholst, H. Functional Rest Through
Intensive Treatment with Insulin and Potassium Channel Openers
Preserves Residual β-cell Function and Mass in Acutely Diabetic BB
Rats. Horm. Metab. Res. 2000, 32, 294−300.
This study was supported by grants from the National Fund for
Scientific Research (FNRS, Belgium) for which P. de Tullio is
Senior Research Associate and P. Lebrun is Research Director.
We gratefully acknowledge the technical assistance of Y.
Abrassart, S. Counerotte, P. Fraikin, F. Leleux, A.-M.
Vanbellinghen, A. Van Praet, and V. Hurlet.
(
16) Hansen, J. B. Towards Selective Kir6.2/SUR1 Potassium
Channel Openers, Medicinal Chemistry and Therapeutic Perspectives.
Curr. Med. Chem. 2006, 13, 361−376.
(
17) de Tullio, P.; Becker, B.; Boverie, S.; Dabrowski, M.; Wahl, P.;
ABBREVIATIONS
■
Antoine, M. H.; Somers, F.; Sebille, S.; Ouedraogo, R.; Hansen, J. B.;
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benzothiadiazine 1,1-Dioxides. J. Med. Chem. 2003, 46, 3342−3353.
K
channel, ATP-sensitive potassium channel; SUR,
ATP
sulfonylurea receptor; Kir, inwardly rectifying potassium
channel; PCO, potassium channel opener; RIS, residual insulin
release percentage; IC , half-maximal inhibitory concentration;
(
18) de Tullio, P.; Boverie, S.; Becker, B.; Antoine, M. H.; Nguyen,
Q. A.; Francotte, P.; Counerotte, S.; Sebille, S.; Pirotte, B.; Lebrun, P.
-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides as ATP-
5
0
EC , half-maximal effective concentration; FTIR, Fourier
5
0
3
transform infrared; NMR, nuclear magnetic resonance; TMS,
tetramethylsilane; TLC, thin layer chromatography; SEM,
standard error of the mean; EGTA, ethylene glycol tetraacetic
acid; FOR, fractional outflow rate; NADPH, nicotinamide
adenine dinucleotide phosphate; CE, capillary electrophoresis;
BGE-CD, background electrolyte containing the cyclodextrin
Sensitive Potassium Channel Openers: Effect of 6,7-Disubstitution
on Potency and Tissue Selectivity. J. Med. Chem. 2005, 48, 4990−
5
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(19) Pirotte, B.; de Tullio, P.; Nguyen, Q. A.; Somers, F.; Fraikin, P.;
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Alkylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides as ATP-Sensitive
Potassium Channel Activators: Impact of the Position of the Chlorine
Atom on the Aromatic Ring on Activity and Tissue Selectivity. J. Med.
Chem. 2010, 53, 147−154.
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