Synthesis and pharmacological evaluation of 4H-1,4-benzothiazine-2- carbonitrile 1,1-dioxide and N-(2-cyanomethylsulfonylphenyl)acylamide derivatives as potential activators of ATP sensitive potassium channels

Article abstract of DOI:10.1016/j.bmc.2004.09.051

1,2,4-Thiadiazine derivatives, like 3-methyl-7-chlorobenzo-4H-1,2,4- thiadiazine 1,1-dioxide, diazoxide and 7-chloro-3-isopropylamino-4H-benzo-1,2,4- thiadiazine 1,1-dioxide, BPDZ 73, are potent openers of Kir6.2/SUR1 K _ATP channels. To explore the structure-activity relationship of this series of K_ATP openers, 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide and N-(2-cyanomethylsulfonylphenyl)acylamide derivatives were synthesized from 2-acetylamino-5-chloro-benzenesulfonic acid pyridinium salt or 2-aminobenzenethiols. The 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide derivatives (e.g., 7-chloro-3-isopropylamino-4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide, 3f) were found to activate K_ATP channels as indicated by their ability to hyperpolarize beta cell membrane potential, to inhibit glucose-stimulated insulin release in vitro and to increase ion currents through Kir6.2/SUR1 channel as measured by patch clamp. The potency and efficacy of, for example, 3f is however significantly reduced compared to the corresponding 4H-1,2,4-benzothiadiazine 1,1-dioxide derivatives. Opening of the 4H-1,2,4-thiadiazine ring to get (e.g., 2-cyanomethylsulfonyl-4-fluorophenyl) carbamic acid isopropyl ester (4c) gives rise to compounds, which are able to open K_ATP channels but with considerable reduced potency compared to, for example, diazoxide. Compound 3a, 7-chloro-3-methyl-4H-1,4-benzothiazine-2- carbonitrile 1,1-dioxide, which inhibits insulin release in vitro from beta cells and rat islets, reduces plasma insulin levels and blood pressure in anaesthetized rats upon intravenous administration.

Full text of DOI:10.1016/j.bmc.2004.09.051

Bioorganic & Medicinal Chemistry 13 (2005) 141–155
Synthesis and pharmacological evaluation
of 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide
and N-(2-cyanomethylsulfonylphenyl)acylamide derivatives
as potential activators of ATP sensitive potassium channels
Søren C. Schou,a Holger C. Hansen,a Tina M. Tagmose,a Harrie C. M. Boonen,a
Anne Worsaae,a Michael Drabowski,a Philip Wahl,a Per O. G. Arkhammar,a
a
Thora Bodvarsdottir, Marie-Helene Antoine, Philippe Lebrun
b
b
´ `
and John Bondo Hansena,*
a
˚
Discovery, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maløv, Denmark
Laboratory of Pharmacology, Faculty of Medicine, Universite Libre de Bruxelles,
b
´
Route de Lennik 808-CP 617, Brussels B-1070, Belgium
Received 2 June 2004; accepted 28 September 2004
Abstract—1,2,4-Thiadiazine derivatives, like 3-methyl-7-chlorobenzo-4H-1,2,4-thiadiazine 1,1-dioxide, diazoxide and 7-chloro-3-
isopropylamino-4H-benzo-1,2,4-thiadiazine 1,1-dioxide, BPDZ 73, are potent openers of Kir6.2/SUR1 KATP channels. To explore
the structure–activity relationship of this series of KATP openers, 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide and N-(2-cyano-
methylsulfonylphenyl)acylamide derivatives were synthesized from 2-acetylamino-5-chloro-benzenesulfonic acid pyridinium salt
or 2-aminobenzenethiols. The 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide derivatives (e.g., 7-chloro-3-isopropylamino-4H-
1,4-benzothiazine-2-carbonitrile 1,1-dioxide, 3f) were found to activate KATP channels as indicated by their ability to hyperpolarize
beta cell membrane potential, to inhibit glucose-stimulated insulin release in vitro and to increase ion currents through Kir6.2/SUR1
channel as measured by patch clamp. The potency and efficacy of, for example, 3f is however significantly reduced compared to the
corresponding 4H-1,2,4-benzothiadiazine 1,1-dioxide derivatives. Opening of the 4H-1,2,4-thiadiazine ring to get (e.g., 2-cyano-
methylsulfonyl-4-fluorophenyl) carbamic acid isopropyl ester (4c) gives rise to compounds, which are able to open KATP channels
but with considerable reduced potency compared to, for example, diazoxide. Compound 3a, 7-chloro-3-methyl-4H-1,4-benzothia-
zine-2-carbonitrile 1,1-dioxide, which inhibits insulin release in vitro from beta cells and rat islets, reduces plasma insulin levels and
blood pressure in anaesthetized rats upon intravenous administration.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
lin release and are used in treatment of type 2 diabetes
mellitus. Openers of KATP channels of smooth muscle
(e.g., diazoxide and pinacidil) have been explored as
drugs for treatment of cardiovascular diseases. Recently
it has been suggested that openers of beta cell KATP
channels, which reduce insulin release to induce beta cell
rest, can be used in treatment of metabolic diseases.5–7
ATP sensitive potassium (KATP) channels are found in
different tissues, such as the heart, vascular smooth mus-
cle, central neurons and pancreatic b-cells.1–4 By regulat-
ing the open state probability of the channel, it is
possible to affect cellular membrane potential and there-
by the influx of Ca2+ through the voltage-gated calcium
channels. Blockers of beta cell KATP channels (e.g., rep-
aglinide, tolbutamide and glibenclamide) stimulate insu-
The KATP channels are constructed as 4+4 hetero-
octamers made from the regulatory sulfonylurea recep-
tor SUR and the inward rectifier Kir6.2 or Kir6.1. Dif-
ferent forms of SUR have been cloned and
characterized.4,8 SUR1 have been found to combine
with Kir6.2 to form the KATP channels of beta cells
*
Corresponding author. Tel.: +45 4443 4857; fax: +45 4443 4547;
e-mail: jbha@novonordisk.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.051

142
S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
and neurones whereas SUR2A and SUR2B in combina-
tion with Kir6.1 and Kir6.2 form the channels of the
heart and vascular smooth muscle, respectively.
In the present study the structure–activity relationship
for the KATP channel activity of these diazoxide deriva-
tives have been investigated by a series of 4H-1,4-benzo-
thiazine-2-carbonitrile 1,1-dioxide derivatives (3) and
the ring-opened N-(2-cyanomethylsulfonylphenyl)acyl-
amide derivatives (4) (Fig. 2). One of the objectives of this
study was to examine the consequences of introducing a
substituent in the 2-position of the 4H-1,4-benzothiazine
entity. Using the cyano group, we expected to achieve
this while retaining the acidic proton in position 4 neces-
sary for the activity of the compounds as KATP channel
openers. Another objective was to study the effects of
opening the 4H-1,4-benzothiazine ring to maintain cer-
tain aspects of the parent compound by keeping the rel-
ative positions of the SO2 group versus the NH (position
Ô4Õ) and the hydrophobic side chain.
Pinacidil opens SUR2B KATP channels to induce
vasorelaxation but has nearly no effects on the SUR1
channels of beta cells.9 In contrast, diazoxide is a
moderately potent and non-selective activator of
SUR1 and SUR2B channels with only minimal effects
on SUR2A. Optimizations of diazoxide have given
compounds, for example, BPDZ 62, BPDZ 73 and
NN414 (Fig. 1), which potently and selectively open
the SUR1 KATP channels of beta cells.10–13 In analogy,
it has recently been shown that certain pinacidil
derivatives, for example, 114 and 215 are able to inhibit
insulin release with only minimal effects on smooth
muscle.
Structural requirements for KATP channel openers act-
ing on Kir6.2/SUR1 have been explored. These include
an aromatic group, possibly substituted with one or
more halogen atoms, a space filling alkyl group, an
acidic (hydrogen bond donating) NH group (e.g., N4
of the 1,2,4-thiadiazine derivatives), and an electronega-
tive (hydrogen bond accepting) group (SO2 or NCN)
(Fig. 2).10,16,17 By combining structural component from
diazoxide and pinacidil to substitute the 3-methyl group
of diazoxide with 3-alkylamino groups, it has been
possible to improve potency and change efficacy
highlighting the importance of the exocyclic amino-
group.10,12,13,16,17
2. Chemistry
3-Methylbenzothiazines of the general formula 3 were
synthesized by base catalyzed ring closure of cyano-
methylsulfones 4, which in turn were prepared by differ-
ent routes (Scheme 1 path A, B and C).
Path A: the unstable 2-acetylamino-5-chlorobenz-
enesulfonyl chloride was prepared from the pyridinium
sulfonate 5a18 and reduced without purification to the
sulfinic acid 6a following the general procedure reported
by Lee and Field.19 Alkylation of 6a with iodoacetonit-
rile provided the sulfone 4a.
H
H
N
H
N
H
N
H
N
H
N
H
N
N
Cl
N
N
N
N
Cl
S
O
N
S
S
O
S
O
Cl
N
S
O
O
O
O
O
Diazoxide
NN414
BPDZ 62
BPDZ 73
H
N
H
N
H
N
H
N
Cl
H
N
H
N
Cl
NC
SO2
Cl
N
N
NC
NC
Cl
Cl
1
2
Pinacidil
Figure 1. Openers of KATP channels.
R3
O
H
H
R1
R2
NH
N
R3
R1
R2
N
R3
R1
R2
N
S
CN
S
O
CN
S
O
O
O
O
O
3
4
Figure 2.

S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
143
Path A
O
R3
O
O
R3
H
N
R1
R2
R1
R2
NH
R1
R2
NH
S
R1
R2
NH
a,b
c
d
HN+
O
OH
S
S
S
N
N
O
O
O
O
O
O
O
3
R1, R2, R3
H, Cl, CH3
CF3, H, CH3
F, H, CH3
H, F, CH3
H, OCH3, CH3
5a R1 = H, R2 = Cl
6a R1 = H, R2 = Cl
4
R1, R2, R3
a
b
c
d
e
a
b
c
d
e
H, Cl, CH3
CF3, H, CH3
H, F, O-Pr-i
H, Cl, OEt
CF3, H, OEt
f
F, H, CH3
g
h
H, F, CH3
H, OCH3, CH3
f
Path B
R3
O
R1
R2
NH2
S
R1
R2
NH2
SH
R1
R2
NH
c
e
N
S
N
7
a
b
c
d
R1, R2
9
a
R1, R2, R3
8
R1, R2
a CF3, H
CF3, H
CF3, H, CH3
b H, Cl
c H, F
d F, H
H, Cl
H, F
F, H
b
c
d
H, Cl, OEt
H, F, O-Pr-i
CF3, H, OEt
e
f
g
F, H, CH3
H, F, CH3
H, OCH3, CH3
O
S
g
Path C
NH
R2
R1
S
R1
R2
NH
O
R1 = H, R2 = OCH3, R3 = CH3
Scheme 1. Synthesis of N-(2-cyanomethylsulfonylphenyl)acetamides (4a–h) and 3-methylbenzothiazines (3a–e). Reagents and conditions: (a) PCl5,
60ꢁC, 2h; (b) p-TolSH/Et3N, CH2Cl2; (c) NaH, I–CH2–CN/DMF; (d) (1) 0.5M NaOH, rt 1h, (2) 1M HCl; (e) acetic anhydride or appropriate
chloroformates; (f) m-CPBA/CH2Cl2 or H2O2/HOAc; (g) (1) NaBH4/EtOH, 70ꢁC, 30min, (2) I–CH2–CN, rt, 15min.
Path B: 2-aminobenzenethiols 7, which were either com-
mercially available (e.g., 7a) or prepared by alkaline
hydrolysis of the appropriately substituted 2-amino-
benzothiazoles20 were cyanomethylated to get the
cyanomethylsulfanyl derivatives by a modification of
the procedure previously described for 8b.21 In our
hands, however, a much better yield and a more pure
product was obtained if the aminobenzenethiol was
not isolated but instead generated in situ from the
2-aminobenzothiazole as the thiolate salt and immedi-
ately alkylated with iodoacetonitrile under nitrogen
atmosphere to avoid air oxidation of the thiolate.
Acylation of 8 with acetic anhydride or chloroformates
gave acetamides 9a,e–g, and carbamates 9b–d, respec-
tively, which were oxidized with either m-CPBA in
dichloromethane or, preferentially, with hydrogen per-
oxide in acetic acid to give the sulfones. By treatment
with diluted aqueous base at room temperature, the
Path C: in an attempted synthesis of 2-aminobenzenethiol
7 (R1 = H, R2 = OCH3) from 2-amino-6-methoxy-benzo-
thiazole we only obtained the corresponding disulfide.22
Acetylation gave bis(2-acetylamino-5-methoxyphenyl)-
disulfane,23 which was reduced with sodium borohyd-
ride, and the resulting thiolate alkylated in situ to give 9g.
Spectroscopic data supports the assigned 4H structures
of 3 rather than the 2H tautomer form; for example,
in the H NMR spectrum of 3a the acidic proton was
1
observed at d = 12.11, similar to what has been found
for HN(4) in 1,2,4-benzothiadiazine 1,1-dioxides,10,24
and thus at a much lower field than would be expected
for a 2H tautomer. Furthermore, the 13C NMR spec-
trum showed C2 and C3 signals at d 85.51 and 155.72,
respectively. The predicted values for the 4H tautomer
are d 83.57 and 157.78, whereas for the 2H tautomer val-
ues of d 52.83 and 172.61 are predicted.25 The C–H cor-
relation spectrum showed no interaction of the acidic
proton with any carbon atom, again suggesting a N–H
rather than a C–H proton.
N-(2-cyanomethylsulfonylphenyl)acetamides
4a,b,f–h
underwent a ring closure to give the 4H-1,4-benzothi-
azines 3a–e.

144
S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
R4
O
H
N
H
N
H
N
R1
R2
OH
R1
R2
Cl
R1
R2
R3
R1
R2
NH
S
b
c
a
S
S
S
N
N
N
O
O
O
O
O
O
N
O
O
R1, R2, R3
11 R1, R2
4
R1, R2, R4
10 R1, R2
3
a
b
H, Cl
d
e
H, Cl, OEt
a
b
H, Cl
f
H, Cl, NH-CH(CH3)2
CF3, H
CF3, H, OEt
CF3, H
g
h
i
CF3, H, NH-CH(CH3)2
H, Cl, NH-CH2CH2CH(CH3)2
H, Cl, NH-Cyclobutyl
Scheme 2. Synthesis of 3-alkylamino-4H-1,4-benzothiazine-2-carbonitriles (3f–i). Reagents and conditions: (a) (1) 0.2M NaOH, rt 1h, (2) 1M HCl;
(b) POCl3, pyridine hydrochloride/H3PO4; (c) amine (R3–H) in CH3CN or neat.
In IR, the signal at m 2215cmꢀ1 corresponds to a cyano
group bound to an unsaturated carbon.
moderate yields were obtained, the sterically hindered
amines giving the lowest yields.
3-Alkylamino-4H-1,4-benzothiazine-2-carbonitriles were
prepared starting from the carbamates 4d,e (Scheme 2).
Base catalyzed ring-closure of 4d,e gave 10a,b, which
were treated with phosphoroxychloride using the proce-
dure developed by Andersen and Begtrup26 to get 11a,b.
Prolonged heating of 11 in a closed vial27 with an excess
of the appropriate amine was required in order to obtain
the desired 3-amino substituted derivatives 3f–i. Low to
3. Results and discussion
The synthesized compounds (Table 1) were initially eval-
uated for their ability to repolarize the membrane poten-
tial of bTC3 beta cells depolarized by 10mM glucose; to
inhibit 22mM glucose-stimulated insulin release from
bTC6 beta cells and to relax phenylephrine contracted
Table 1. Structures and biology screening data for compounds 3a–i and 4a–e
O
R3
H
R1
R2
N
R3
R1
R2
NH
S
S
N
N
O
O
O
O
4
3
Compound R1, R2
R3
Membrane potentiala
Inhibition of insulin
releaseb bTC6 cells
Relaxation of
Membrane
bTC3 cells IC50 (lM)
rat aorta ringsc potentiald HEK
EC50 (lM)
293 cells IC50 (lM)
IC50 (lM)
6.78 4.25
Efficacy (%)
3a
3b
3c
3d
3e
3f
H, Cl
CF3, H
F, H
CH3
CH3
CH3
CH3
NA
18 2.39
30.5 5.4
44.3 14.1
>30
14
47.7 23
NA
NA
>100
NA
8
NA
4.71
6
4
35
24
18
9
3
1
NT
NT
NT
13
H, F
3.95 3.25e 25 1.75e
H, OCH3 CH3
NA
NA
42.3 22.85 14 6.22
NA
H, Cl
CF3, H
H, Cl
H, Cl
H, Cl
CF3, H
H, F
NHCH(CH3)2
NHCH(CH3)2
17.6
NA
6
6
4.3 0.8
4.2
18.6 5.4
3g
3h
3i
NA
>100
NA
NA
16 4.77
NA
5
>30
NT
NT
NT
NT
6
NH(CH2)2CH(CH3)2 10.7
NHCyclobutyl
CH3
CH3
NA
NA
NA
NA
NA
NA
9
6
4a
4b
4c
4d
4e
>100
>100
NA
12 5.92
14 6.50
NA
11.4 5.2
84.1 7.3
109 8.6
OCH(CH3)2
OCH2CH3
OCH2CH3
2
H, Cl
H, CF3
>100
0.36 0.20
6
9
2.65
6.75
4.7 2.6
183.0 80.7
NT
NT
Diazoxide
BPDZ 73
Pinacidil
13.7 0.25
0.25 0.02
NA
22.98 4.1
0.46 0.31
NA
12.8 2.5
73 7.5
NA
25 3.8
36.3 2.2f,g
0.8 0.2h
33 11
0.8 0.01
NT
a Effects on membrane potential in bTC3 cells. Values are means of at least three measurements SD.
b Inhibition of glucose-stimulated insulin release from bTC6 cells. Values are means SEM of at least four experiments.
c Relaxation of phenylephrine or KCl induced contraction of rat aorta rings. Values are means SEM of at least three experiments.
d Effects on membrane potential of HEK 293 cells expressing Kir6.2/SUR 1 channels. Values are means SEM of at least three experiments.
e n = 2.
f Values from Lebrun et al.11
g Relaxation of KCl induced contraction.
h Value from Nielsen et al.12 NA = not active. NT = not tested.

S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
145
rat aortic rings. These assays have previously been used
to characterize activators of KATP channels.11,12,14,15
0.10
0.08
0.06
0.04
0.02
0.00
Among the 4H-1,4-benzothiazine-2-carbonitrile 1,1-
dioxide derivatives, compounds 3b, 3f and 3h were able
to hyperpolarize bTC3 cell membranes, whereas com-
pounds 3a,c,d,g,i and the ring-opened N-(2-cyano-
methylsulfonylphenyl)acylamide derivatives (4a–e)
where inactive in this test. Compounds 3b, 3f and 3h
were found to be approximately equipotent with diazox-
ide but considerable less potent than BPDZ 73; efficacies
were moderate (30–60%) and lower than determined for
BPDZ 73 or diazoxide (100%) (data not shown).
-0.02
-0.04
-0.06
-0.08
-0.10
-0.12
-0.14
-0.16
µ
M
Diazoxide, EC50 = 50 19
µ
M
P1075, EC50 = 0.18 0.01
0.01
0.1
1
10
100
1000
The compounds of the present series were in general not
potent inhibitors of glucose-stimulated insulin release
from bTC6 beta cells. Compounds 3a, 3b, 3d, 3f, 3h,
4a, 4b, 4d and 4e significantly inhibit insulin release
although with low efficacy. In comparison are 3a, 3d,
3f and 4e at least as potent as diazoxide and with com-
parable efficacy. Whereas compounds 3b, 3h, 4a, 4b and
4d weakly inhibit insulin release with IC50Õs above
100lM, compounds 3g, 3i and 4c did not affect insulin
release at concentration up to 50lM.
µ
[Drug] M
0.3
0.2
0.1
0.0
3a. EC50 = 16 3.6 µM
3b. EC50= 8.5 4.9 µM
All compounds induced vasodilatation of isolated aorta,
yet with different order of potency. The most potent of
these (i.e., 3f, 3g, 3i and 4d) being more potent than
the non-selective potassium channel opener diazoxide
but equal or less potent than pinacidil.
3f
-0.1
-0.2
3g
4c
4d
0.1
1
10
100
µ
[Drug] M
Overall the screening data suggests that 3-methyl-4H-
1,4-benzothiazine-2-carbonitrile 1,1-dioxide derivatives
with a chlorine or fluorine in position 7 have a profile
similar to that of diazoxide whereas a methoxy substitu-
ent in position 7 seems to reduce potency slightly in
analogy with previous findings.11 Compounds with sub-
stituents in position 6 appear to be less potent. Substi-
tuting the 3-methylgroup with a 3-alkylaminogroup,
which in the 4H-1,4-benzothiadiazine 1,1-dioxide series
strongly increases beta cell activity,10,16 has little benefi-
cial effect on potency.
Figure 3. Concentration-response curves for P1075 and diazoxide
(upper panel) and compounds 3a–d, 4c, 4d (lower panel)-induced
membrane potential responses in HEK293 cells expressing Kir6.2/
SUR2B. Data points are mean SEM from 2 to 3 experiments
performed in triplicate.
ence compound diazoxide and the pinacidil derivative
P1075 potently hyperpolarized the cell membrane with
IC50Õs of 50 19lM and 0.18 0.01lM, respectively,
which are comparable to their ability to relax pre-
contracted rat aorta rings.12,28 Compounds 3a (IC50
=
In order to evaluate their mechanism of action, selected
compounds were examined for effects on HEK 293 cells
expressing Kir6.2/SUR1 (Table 1) or Kir6.2/SUR2B
channels (Fig. 3).
16 3.6lM) and 3b (IC50 = 8.5 4.9lM) significantly
hyperpolarized the membranes, which indicate that
these compounds were able to activate the Kir6.2/
SUR2B channel. At high concentrations compound 3f
and 3g also appeared to hyperpolarize the membranes.
Compounds 4c and 4d were found to be inactive in this
assay. While the ability of compounds 3a and 3b to relax
vascular smooth muscle therefore could be mediated
through a direct activation of the Kir6.2/SUR2B KATP
channels, it is however likely that some compounds of
these series (exemplified by compounds 4c and 4d) affect
smooth muscle through a mechanism not involving
Kir6.2/SUR2B KATP channels.
Compound 3b and 3f, which weakly reduced insulin re-
lease from bTC6 cells and which hyperpolarized bTC3
cell membranes in presence of glucose, did repolarize
the HEK 293 cell membranes depolarized by the KATP
channel blocker tolbutamide (Table 1), suggesting that
these compounds reduce insulin release through an acti-
vation of Kir6.2/SUR1 potassium channels. In conflict
with its inability to reduce glucose-stimulated insulin re-
lease, compound 4c was found to be able to hyperpolar-
ize membrane of cells expressing Kir6.2/SUR1 KATP
channels (IC50 = 6 2lM).
The apparent conflict between the ability of compound
4c to hyperpolarize membranes of HEK 293 cells
expressing Kir6.2/SUR1 and its lack of effects on glu-
cose-stimulated insulin release from bTC6 cells was also
investigated using the more sensitive patch-clamp tech-
nique. Compound 4c potently activates Kir6.2/SUR1
The effect of selected compounds on the membrane
potential of HEK 293 cells expressing Kir6.2/SUR2B
potassium channels were examined (Fig. 3). The refer-

146
S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
channels expressed in HEK293 cells when measured by
patch clamp in the whole cell configuration or Kir6.2/
SUR1 expressed in inside-out macropatches excised
from Xenopus oocytes (Fig. 4). The efficacy of the activa-
tion was, however considerable lower than that of diaz-
oxide. Apparently, the partial agonist effect of 4c as
measured by patch clamp is not sufficient to cause a
hyperpolarization of the cell membrane and subsequent
inhibition of insulin release from the beta cells.
data clearly revealed that micromolar concentrations
of 3a inhibited the glucose-induced insulin release. How-
ever, the capacity of 3a to inhibit the secretory process
was not highly pronounced and the compound appeared
to be less potent than diazoxide.
In the presence of 16.7mM glucose and extracellular
Ca2+ in the perifusing medium, the addition of 10lM
3a provoked a minor reduction in both 45Ca outflow
and insulin output (Fig. 5, left panels). On removal of
3a from the perifusate, a modest increase in 45Ca (frac-
tional outflow rate) FOR and insulin release was no-
ticed. The latter increases could reflect relief from
inhibitory effects of the drug.
Compound 3a, which inhibits glucose-stimulated insulin
release from bTC6 cells with a potency similar to diaz-
oxide was further examined using freshly isolated rat
islets.
Incubation of pancreatic islets in the presence of 3a re-
vealed an inhibitory effect of the drug on 16.7mM glu-
cose-induced insulin release. Residual insulin release
after the addition of 10lM and 50lM 3a represented
88.6 3.8% (n = 16 samples) and 49.6 1.7% (n = 16
samples) of the control value, respectively. Under the
same experimental conditions and after the addition of
10lM and 50lM diazoxide, the residual insulin release
averaged 70.0 3.6% (n = 22 samples) and 28.8 2.5%
(n = 21 samples) of the control value, respectively. The
Under identical experimental conditions, the addition of
a higher concentration (50lM) of 3a provoked more
pronounced inhibitory effects on 45Ca2+ FOR and insu-
lin release from pre-labelled and perifused rat pancreatic
islets (Fig. 5, right panels).
To study the effects of 3a on 45Ca2+ movements in detail,
similar experiments were conducted in the presence of
16.7mM glucose but absence of extracellular Ca2+. In is-
lets exposed to Ca2+-depleted media, the basal rate of
45Ca2+ outflow (min 40–44) was significant lower than
in presence of Ca2+ (P < 0.05) and the addition of either
10lM or 50lM 3a did not affect 45Ca2+ FOR.
800
300µM diazoxide
700
600
These dynamic experiments indicated that the addition
of 3a reduced the 45Ca2+ FOR and insulin release from
pre-labelled islets perifused in the presence of 16.7mM
glucose and extracellular Ca2+. Under the latter experi-
mental conditions, that is, in islets exposed throughout
to Ca2+ and insulinotropic concentrations of glucose,
the 45Ca2+ fractional outflow rate is known to reflect a
500
400
300
200
100
0
4c
10µM 4c
10
1
0,1
sustained stimulation of isotopic exchange between
11,29,30
influent 40Ca2+ and effluent 45Ca2+
.
Thus, the
0
8
17
25
modest inhibitory effect of 3a on 45Ca2+ outflow can
be interpreted as the result of KATP channel opening fol-
lowed by a subsequent reduction in 40Ca2+ entry into the
islet cells. In agreement with such a view, the 3a-induced
decrease in 45Ca2+ outflow did not occur when the islets
were perifused in the absence of extracellular Ca2+. The
decrease in Ca2+ entry mediated by 3a may lead, ulti-
mately, to a reduction in insulin output.
Time (min)
1.00
0.75
0.50
0.25
100 µM MgATP
100 µM MgATP + 1 µM 4c
100 µM MgATP + 10 µM 4c
In order to examine if the effect of 3a in vitro are re-
flected in vivo, 3mg/kg of the compound was adminis-
tered intravenously to anaesthetized rats.12 Compound
3a reduced plasma insulin levels by 48 18% from basal
levels (104 1pM), which is comparable to diazoxide.12
The effect, however, was of shorter duration (less than
20min). Blood pressure was not affected by this dose
(from 78 5 to 76 6mmHg) within the observation
period (0.5h). 3a was also given by gavage (30mg/kg)
to hyperinsulinemic Zucker obese rats12 but no signifi-
cant effect on plasma insulin levels could be observed
(data not shown). Together, these studies support that
3a is able to inhibit insulin release in vivo but that the
duration of action is short and the oral bioavailability
is probably to low to see effects after peroral
administration.
Figure 4. Effects of 4c on Kir6.2/SUR1 channels. Upper panel: whole
cell recording from an HEK293 cell expressing Kir6.2/SUR1. The
points indicate the current obtained in response to a 10mV depolar-
izing pulse applied every 10s from a holding potential of ꢀ80mV.
Diazoxide (300lM) and 4c (0.1, 1 and 10lM) were applied as
indicated by the horizontal bars. Lower panel: effect of 4c on
macroscopic currents through Kir6.2/SUR1 channels in inside-out
patches. Mean macroscopic conductances (G) are expressed as a
fraction of the mean slope conductance in nucleotide and drug free
solution (Gcontrol). Data were obtained in the presence of 100lM
MgATP (white column), 100lM MgATP plus 1lM 4c (grey column)
or 100lM MgATP plus 10lM 4c (black column).

S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
147
µ
3a 50
M
µ
M
3a 10
2.0
1.0
2.0
1.0
0.0
2.0
0.0
4.0
3.0
2.0
1.0
1.0
0.0
µ
30 40 50 60 70 80 90
TIME (min)
30 40 50 60 70 80 90
TIME (min)
Figure 5. Upper panels: effect of 10lM (left panel) and 50lM (right panel) 3a on 45Ca2+ outflow from rat pancreatic islets perifused throughout in
the presence of 16.7mM glucose. Basal media contained extracellular Ca2+ (d, 2.56mM) or were deprived of Ca2+ and enriched with EGTA (
,
ꢁ
0.5mM). Lower panels: effect of 10lM (left panel) and 50lM (right panel) 3a on insulin release from rat pancreatic islets perifused throughout in the
presence of 16.7mM glucose. Basal media contained extracellular Ca2+ (d, 2.56mM). Mean values SEM refer to 4–6 individual experiments.
4. Conclusion
was carried out on a rotary evaporator at bath temper-
atures <40ꢁC and under appropriate vacuum. Flash
chromatography was carried out on a Biotage flash 40
7-Chloro-3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-
dioxide derivatives are potent activators of ATP sensi-
tive potassium (KATP) channels. The 3-alkylamino
group, the acidic proton of position 4 as well as the elec-
tronegative, H-bond accepting SO2-group, are impor-
tant for the activity.10 In the present study, it has been
found that substituents in the 2-position, as found in
the 4H-1,4-benzothiazine-2-carbonitrile 1,1-dioxide
derivatives, retain the ability of the compounds to acti-
vate KATP channels. The potency is however signifi-
cantly reduced compared to the corresponding 3-
alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide deri-
vatives. We furthermore have found that opening of
4H-1,2,4-thiadiazine ring to get N-(2-cyanomethylsulfon-
ylphenyl)acylamide derivatives gives rise to compounds,
which are able to open KATP channels but with consid-
erable reduced potency compared to, for example, diaz-
oxide. The present study also suggests that low efficiency
opening of ATP sensitive K+-currents might not be
enough to inhibit insulin release induced by high
concentrations of glucose.
˚
using Biotage flash columns (KP-SIL 60A, particle size
32–63lm). Melting points were determined with a Buchi
¨
B545 apparatus and are uncorrected. Proton NMR
spectra were recorded at ambient temperature using a
Brucker Avance DPX 200 (200MHz) and Brucker
Avance DPX 300 (300MHz) with tetramethylsilane as
an internal standard for proton spectra, and DMSO-d6
at 39.50ppm or CDCl3 at 77.00ppm as internal stand-
ards for carbon spectra. Chemical shifts are given in
parts per million (d) and splitting patterns are desig-
nated as follows: s, singlet; d, doublet; dd, double dou-
blet; t, triplet; q, quartet; sept, septet; m, multiplet and
br = broad. The 70eV E.I. solid mass spectra were re-
corded on a Finnigan MAT-TSQ 70 mass spectrometer.
Reactions were followed by thin layer chromatography
performed on silica gel 60 F254 (Merck) or ALU-
GRAMꢂSIL G/UV254 (MACHEREY-NAGEL) TLC
aluminium sheets. Elemental analyses (C, H, N, Cl) were
performed by Novo Nordisk, Microanalytical Labora-
tory, Denmark.
5. Experimental section
5.1. Chemistry
5.1.2. 7-Chloro-3-methyl-4H-1,4-benzothiazine-2-carbo-
nitrile 1,1-dioxide (3a). Compound 4a (0.73g, 2.7mmol)
was added at room temperature to 0.5M aqueous
NaOH (10mL, 5mmol) to form a yellow solution. After
45min charcoal (0.1g) was added and the mixture was
filtered through celite. The filtrate was cooled to 0ꢁC
and 1M HCl (7.5mL) was added. After 30min the
precipitate was collected by filtration and dried to
5.1.1. General. Reagents, starting materials and solvents
were purchased from common commercial suppliers and
were used as received. All dry solvents were dried over-
night over molecular sieves (0.3 or 0.4nm). Evaporation

148
S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
give 0.66g of the title compound. Recrystallization from
methanol gave pale crystals (0.45g, 66%); mp
5.1.6. 7-Methoxy-3-methyl-4H-1,4-benzothiazine-2-car-
bonitrile 1,1-dioxide (3e). The compound was prepared
from 4h in 23% yield by a procedure similar to the pro-
cedure described for 3a. White crystals; mp 311–313ꢁC
297–299ꢁC; 1H NMR (CD3OD-d4):
J = 2.8Hz, 1H); 7.68 (dd, 1H); 7.37 (d, J = 9Hz, 1H);
d
7.91 (d,
1
4.88 (br, H2O+NH); 2.51 (s, 3H); H NMR (DMSO-
1
(from methanol); H NMR (DMSO-d6): d 11.89 (br s,
d6): d 12.11 (br, 1H, NH), 8.02 (d, J = 2.3Hz, 1H,
H8), 7.84–7.75 (dd, 1H, H6), 7.48 (d, J = 8.8Hz, 1H,
H5), 2.47 (s, CH3; partly overlapping with the DMSO-
signal); 13C NMR (DMSO-d6): d 155.72 (C3), 133.81
(C4a), 133.72 (C6), 129.38, 124.78, 121.23 and
121.18 (C5 and C8), 112.48, 85.51 (C2), 20.16 (CH3)
(assignments are based on C–H correlation and
HMBC-spectra); IR (KBr) m 2214 (CN) cmꢀ1. Anal.
Calcd for C10H7ClN2O2S: C 47.16%, H 2.77%, N
11.00%, Cl 13.92%. Found: C 47.08%, H 2.71%, N
10.79%, Cl 14.02%. An additional crop of the title com-
pound (0.09g, 13%) was obtained from the mother
liquid.
1H, NH), 7.51–7.27 (m, 3H), 3.86 (s, 3H, CH3O), 2.44
(s, 3H, CH3); IR (KBr) m 3281 (NH), 2207 (CN) cmꢀ1
Anal. Calcd for C11H10N2O3S: C 52.79%, H 4.03%, N
11.19%. Found: C 52.57%, H 3.89%, N 10.95%.
.
5.1.7. 7-Chloro-3-isopropylamino-4H-1,4-benzothiazine-
2-carbonitrile 1,1-dioxide (3f). Compound 11a
(275.1mg, 1mmol) and isopropylamine (95lL,
1.1mmol) was dissolved in 3mL of CH3CN. To the stir-
red solution was added dry Et3N (153lL, 1.1mmol) and
the mixture was stirred in a screw-cap sealed reaction
vessel at 80ꢁC for 52h. The mixture was concentrated
in vacuo and purified by column chromatography
(EtOAc–heptane 2:1, V/V) to give the title compound
(40mg, 13%) as white crystals, mp 291–292ꢁC; 1H
5.1.3. 3-Methyl-6-trifluoromethyl-4H-1,4-benzothiazine-
2-carbonitrile 1,1-dioxide (3b). Following the procedure
described for 3a the title compound was prepared from
4b, white flakes (84%), mp 325–330ꢁC (from MeOH);
1H NMR (CD3OD): d 8.20–8.10 (br d, 1H), 7.78–7.62
(m, 2H), 4.88 (br, H2O+NH), 2.52 (s, 3H); 13C NMR
(DMSO-d6): d 156.26, 135.45, 132.97, 126.46, 124.07,
122.91, 121.81, 116.07, 112.27, 86.21, 20.32; IR (KBr)
m 3301 (NH), 2214 (CN) cmꢀ1; MS m/z (rel abundance):
288 (74) M+, 224 (74), 223 (100), 203 (8), 155 (9), 69 (16).
Anal. Calcd for C11H7F3N2O2S: C 45.84%, H 2.45%, N
9.72%, S 11.12%. Found: C 45.83%, H 2.46%, N 9.66%,
S 11.49%.
NMR (DMSO-d6):
d
10.68 (s, 1H), 7.89 (d,
J = 8.67Hz, 1H), 7.77 (d, J = 2.26Hz, 1H), 7.71 (m,
1H), 7.58 (d, J = 8.67Hz, 1H), 4.10 (m, 1H), 1.24 (d,
J = 6.41Hz, 6H). 13C NMR (DMSO-d6): d 153.88,
133.41, 132.40, 128.35, 128.29, 121.54, 119.38, 114.04,
65.02, 44.98, 22.66. Anal. Calcd for C12H12ClN3O2S:
C 48.41%, H 4.06%, N 14.11%, Cl 11.91%l. Found: C
48.03%, H 4.05%, N 13.83%, Cl 11.70%.
5.1.8. 3-Isopropylamino-6-trifluoromethyl-4H-1,4-benzo-
thiazine-2-carbonitrile 1,1-dioxide (3g). A mixture of 11b
(0.135g, 0.44mmol) and isopropylamine (1mL) was
heated for 42h at 60–65ꢁC in a screw-cap sealed reac-
tion vessel, cooled to room temperature and concen-
trated on a rotary evaporator. The residual yellow oil
was stirred for 1h with H2O (8mL), pH was adjusted
to 1 by 4M HCl (ꢂ10 drops) and the precipitate was fil-
tered off, rinsed with H2O and dried to give a beige pow-
der (0.106g). Purification of the crude product by flash
column chromatography (Rf = 0.8; silica, EtOAc) gave
white voluminous crystals (0.021g, 14%); mp 311–
5.1.4. 6-Fluoro-3-methyl-4H-1,4-benzothiazine-2-carbo-
nitrile 1,1-dioxide (3c). The compound was prepared
from 4f by a procedure similar to the procedure de-
scribed for 3a. White crystals, mp 261–263ꢁC (sinters
at 240–251ꢁC); 1H NMR (DMSO-d6): d 12.03 (br s,
1H, NH), 8.06 (dd, 9.0Hz/5.5Hz, 1H, H8), 7.35 (m, 1H,
H7), 7.20 (dd, 10.0/2.5Hz, 1H, H5), 2.46 (s, 3H, CH3);
13C NMR (DMSO-d6): d 163.89 (d, J = 251Hz),
155.86, 136.98 (d, J = 12Hz), 125.49 (d, J = 11Hz),
120.58 (d, J = 2.7Hz), 114.03 (d, J = 24Hz), 112.46,
104.99 (d, J = 26Hz), 86.31, 20.16; IR (KBr) m 3273
(NH), 2215 (CN) cmꢀ1. Anal. Calcd for C10H7FN2O2S:
C 50.42%, H 2.96%, N 11.76%. Found: C 50.10%, H
2.85%, N 11.45%.
1
311.5ꢁC; H NMR (DMSO-d6): d 10.75 (s, 1H), 8.12–
7.94 (m, 3H), 7.78–7.68 (m, 1H), 4.24–4.01 (m, 1H),
1.26 (d, 6H); MS m/z (rel abundance): 331 (26) M+,
289 (17), 267 (12), 266 8 (12), 252 (83), 225 (100), 199
(22), 176 (15), 160 (34), 156 (23), 107 (14), 67 (16), 58
(22), 43 (43), 41 (39); IR (KBr) m 2196 (CN) cmꢀ1. Anal.
Calcd for C13H12F3N3O2S: C 47.13%, H 3.65%, N
12.68%. Found: C 47.30%, H 3.81%, N 12.28%.
5.1.5. 7-Fluoro-3-methyl-4H-1,4-benzothiazine-2-carbo-
nitrile 1,1-dioxide (3d). The compound was prepared
from 4g in 85% yield by a procedure similar to the pro-
cedure described for 3a. White solid, mp 326–327ꢁC (de-
comp.); 1H NMR (DMSO-d6): d 12.07 (s, 1H, NH),
7.95–7.81 (m, 1H), 7.75–7.45 (m, 2H), 2.47 (s,
CH3+DMSO); 13C NMR (DMSO-d6): d 158.50 (d,
J = 247Hz), 155.47, 131.73 (d, J = 2.3Hz), 124.53 (d,
J = 7.3Hz), 121.89 (d, J = 22Hz), 121.73 (d, J =
9.7Hz), 112.65, 108.02 (d, J = 26Hz), 84.29, 20.08; MS
m/z (rel abundance) 238 (M+, 42), 174 (Mꢀ64, 49),
173 (Mꢀ65, 100); IR (KBr) m 3291 (NH), 2214 (CN)
cmꢀ1. Anal. Calcd for C10H7FN2O2S: C 50.42%; H
2.96%, N 11.76%. Found: C 50.38%, H 2.93%, N
11.60%.
5.1.9. 7-Chloro-3-isopentylamino-4H-1,4-benzothiazine-
2-carbonitrile 1,1-dioxide (3h).
A mixture of 11a
(0.50g, 1.8mmol) and isopentylamine (2mL) was heated
for 24h at 60–65ꢁC in a screw-cap sealed reaction vessel,
cooled to room temperature and condensed on a rotary
evaporator. The residual yellow oil was stirred for 1h
with a mixture of H2O (20mL) and 4M HCl (1mL)
and the precipitate was filtered off, rinsed with H2O
and dried to give a beige powder (0.52g). Recrystalliza-
tion from glacial acetic acid gave the title compound
(0.37g, 63%), mp 252–254ꢁC. Rf = 0.7 (silica, EtOAc);
1H NMR (DMSO-d6): d 10.79 (s, 1H, NH), 7.99 (br t,
1H, NH), 7.81–7.67 (m, 3H, 6H+8H), 7.56 (d,

S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
149
J = 9.2Hz, 1H, 5H), 3.42 (q, ꢂ4H, CH2+H2O), 1.60
(octet, 1H, methin), 1.48 (q, 2H, CH2), 0.90 (d, 6H,
CH3). 13C NMR (DMSO-d6): d 154.77, 133.30, 132.44,
128.31, 121.45, 119.44, 113.94, 65.00, 41.06, 37.49,
25.04, 22.23; MS m/z (rel abundance) 325 (30) M+, 321
(18), 269 (20), 255 (57), 205 (46), 204 (99), 191 (36),
190 (23), 176 (44), 170 (24), 165 (25),156 (47), 148 (21),
126 (22), 70 (25), 43 (100) i-Pr+; IR (KBr) m 3276
(CDCl3): d 9.54 (br, 1H, NH), 9.00 (br, 1H), 8.10 (d,
J = 8.3Hz, 1H), 7.55 (dd, J1 = 8.3Hz, J2 = 1.4Hz,
1H), 4.18 (s, 2H), 2.30 (s, 3H); 13C NMR (CDCl3): d
168.80, 139.07, 138.55 (J = 34Hz), 131.59, 125.72,
122.6 (J = 274Hz), 120.61 (J = 3.7Hz), 120.21 (J =
4.0Hz), 109.26, 45.92, 25.24; MS m/z (rel abundance):
306 (8) M+, 264 (55), 224 (35), 202 (7.6), 183 (5), 176
(12), 160 (28), 140 (6), 43 (100). Anal. Calcd for
C11H9F3N2O3S: C 43.14%, H 2.96%, N 9.15%. Found:
C 43.33% H 2.94% N 8.96%. An additional crop of
the title compound (0.05g, 14%) was obtained from
the mother liquor.
(NH), 2199 (CN) cmꢀ1
.
Anal. Calcd for
C14H16ClN3O2S: C 51.61%, H 4.95%, N 12.90%. Found:
C 51.57%, H 4.90%, N 12.68%.
5.1.10.
7-Chloro-3-cyclobutylamino-4H-1,4-benzothi-
azine-2-carbonitrile 1,1-dioxide (3i). This compound was
prepared from 11a and cyclobutylamine according to
the procedure described for 3h. White crystals (55mg,
10%); mp 316–318ꢁC (decomp.); 1H NMR (DMSO-
d6): d 10.70 (br, 1H), 8.32 (br d, 1H), 7.82–7.65 (d+dd,
2H), 7.61–7.48 (d, 1H), 4.30 (m, 1H), 2.48–1.54 (m,
6H); 13C NMR (DMSO-d6): d 153.50, 133.37 132.44,
128.28, 121.55, 119.41, 113.87, 65.19, 47.68, 30.38,
14.29; IR (KBr) m 2198 (CN) cmꢀ1; MS m/z (rel abun-
dance) 309 (30; M+), 281 (11), 217 (68), 216 (86), 190
(29), 189 (32), 182 (100; MꢀSO2ꢀClꢀC2H4), 155 (38),
55 (24). Anal. Calcd for C13H12ClN3O2S: C 50.41%, H
3.90%, N 13.56%. Found: C 50.27%, H 3.66%, N
13.30%.
5.1.13. (2-Cyanomethylsulfonyl-4-fluorophenyl)carbamic
acid isopropyl ester (4c). To a solution of 9c (0.23g,
0.89mmol) in glacial acetic (1.5mL) acid was added
35% hydrogen peroxide (0.5mL, 5mmol). The solution
was heated at 100ꢁC for 1h 45min, cooled to 0ꢁC and
diluted with H2O (10mL). After stirring at 0ꢁC for
20min the precipitate was collected by filtration, rinsed
with H2O (5mL) and dried to yield the title compound
as white needles (0.21g, 81%); mp 123–123.5ꢁC; 1H
NMR (DMSO-d6): d 9.00 (s, 1H, NH), 8.10–7.95 (m,
1H), 7.85–7.67 (m, 2H), 5.37 (s, 2H), 4.90 (septet, 1H),
1.28 (d, 6H); 13C NMR (DMSO-d6):
d 157.40
(J = 246Hz), 153.08, 134.29 (J = 2.9Hz), 127.96
(J = 6.6Hz), 127.14 (J = 7.6Hz), 124.24 (J = 22Hz),
117.36 (J = 26Hz), 111.57, 68.94, 44.89, 21.74. EI/MS
m/z (rel abundance): 300 (14) M+, 241 (34) M+ꢀPrO,
214 (19), 174 (20), 126 (11), 110 (16), 108 (15), 43
(100); IR (KBr) m 3343 (NH), 2260 (CN), 1712 (C@O)
cmꢀ1. Anal. Calcd for C12H13FN2O4S: C 47.99%, H
4.36%, N 9.33%. Found: C 48.06%, H 4.42%, N 9.13%.
5.1.11. N-(4-Chloro-2-cyanomethylsulfonylphenyl)acet-
amide (4a). NaH (0.10g, 60% dispersion in mineral oil,
2.5mmol) was added at ambient temperature under
nitrogen in small portions to a stirred solution of 6a
(0.55g, 2.4mmol) in dry dimethylformamide (7.5mL).
After stirring for 35min iodoacetonitrile (0.18mL) was
added and the temperature was raised to 70ꢁC for
45min. Then the solvent was removed in vacuo. The res-
idue was triturated with H2O (30mL) and filtered. The
filter cake was dried, stirred with a mixture of ether
(5mL) and petroleum ether (10mL) for 1h, filtered off
and dried to give the title compound as white needles
5.1.14. (4-Chloro-2-cyanomethylsulfonylphenyl)carbamic
acid ethyl ester (4d). Hydrogen peroxide (3mL, 35%)
was added drop wise to a stirred solution of 9b (3.22g,
12mmol) in glacial acetic acid (30mL) at room temper-
ature. After 1h an additional amount of hydrogen per-
oxide (2mL) was added. The mixture was heated at
100ꢁC for 2h. After standing overnight at room temper-
ature voluminous crystals had precipitated. Water
(100mL) was added and the crystals were collected by
filtration. The filter cake was washed on the filter with
portions of H2O (50mL in total) and dried to give the
title compound as pale needles (3.20g, 89%). Recrystal-
lization from 96% ethanol gave white, voluminous nee-
dles (76% recovered), mp 131–133ꢁC; 1H NMR
(DMSO-d6): d 9.09 (s, 1H, NH), 8.18–8.07 (m, 1H),
7.98–7.87 (m, 2H), 5.42 (s, 2H), 4.18 (q, 2H), 1.25 (t,
3H); 13C NMR (DMSO-d6): d 152.95 (C@O), 136.82,
136.38, 130.11, 127.82, 126.71, 125.14, 111.56 (CN),
61.49 (O–CH2), 45.12 (SO2–CH2), 14.26 (CH3); MS m/
z (rel abundance): 302 (34) M+, 230 (17), 190 (38), 170
(26), 142 (47), 126 (100); IR (KBr) m 3358 (NH), 2259
1
(0.52g, 81%); mp 173.5–174ꢁC: H NMR (DMSO-d6):
d 9.69 (s, 1H); 8.06–7.87 (m, 3H); 5.37 (s, 2H), 2.16
(s, 3H); 13C NMR (DMSO-d6): d 169.25, 136.22,
136.02, 130.10, 129.41, 129.13, 128.16, 111.66, 44.57,
23.97ppm; IR (KBr) m 3343(NH), 2258 (CN), 1682
(C@O), 1330 (SO2), 1156 and 1140 (SO2) cmꢀ1
.
Anal. Calcd for C10H9ClN2O3S: C 44.04% H 3.33% N
10.11% Cl 13.00%. Found: C 43.86% H 3.30% N
10.11% Cl 13.09%.
5.1.12. N-(2-Cyanomethylsulfonyl-5-trifluoromethylphen-
yl)acetamide (4b). A solution of 3-chloroperbenzoic
acid (0.78g, purity ꢂ70%, 3.2mmol) in CH2Cl2 was
added to a stirred solution of 9a (0.31g, 1.1mmol) in
CH2Cl2 (10mL) at 0ꢁC. After 6 12 h an additional
amount of 3-chloroperbenzoic acid (0.2g) was added
and the solution was left at room temperature overnight.
Excess of peracid was destroyed by washing the reaction
mixture with a solution of sodium disulfite (0.5g) in
H2O (10mL). The solution was dried over Na2SO4
and evaporated. The residue was recrystallized from
96% ethanol to give the title compound as white volumi-
(CN), 1722 (C@O) cmꢀ1
.
Anal. Calcd for
C11H11N2O4ClS; C 43.64%, H 3.66%, N 9.25%. Found:
C 43.74%, H 3.73%, N 9.12%.
5.1.15.
(2-Cyanomethylsulfonyl-5-trifluoromethylphen-
yl)carbamic acid ethyl ester (4e). Oxidation of 9d
(3.55g) with 3-chloroperbenzoic acid in CH2Cl2 as de-
scribed for 4b gave the title compound as white crystals
1
nous crystals (0.22g, 62%); mp 146–147ꢁC; H NMR

150
S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
(2.61g, 90%; practically pure for further synthesis). An
analytically pure sample was obtained by further recrys-
tallization, mp 109–110ꢁC (from EtOH); 1H NMR
(CDCl3): d 8.93 (br, 1H, NH), 8.82 (br, 1H), 8.10 (d,
J1 = 8.3Hz, 1H), 7.50 (dd, J2 = 1.4Hz, 1H), 4.30 (q,
2H), 4.17 (s, 3H), 1.38 (t, 3H); 13C NMR (CDCl3): d
152.68, 139.50, 138.58 (q, J = 33Hz), 131.94, 124.75,
122.59 (CF3, J = 274Hz), 119.64 (J = 3.6Hz), 118.51
(J = 4.0Hz), 109.13, 62.59, 45.43, 14.31; IR (KBr) m
2258 (CN), 1716 (C@O), cmꢀ1. Anal. Calcd for
C12H11F3N2O4S: C 42.86%, H 3.30%, N 8.05%. Found:
C 42.99%, H 3.26%, N 8.15%.
drop wise with stirring. After 15min an additional
amount of triethylamine (1mL) was added. The mixture
was stirred at ꢀ75ꢁC for 1h, heated to room tempera-
ture and extracted with H2O (5 · 20mL). The combined
aqueous phases were extracted with ether (20mL) and
cooled to 0ꢁC. The solution was acidified with concen-
trated HCl (5mL), NaCl (15g) was added and the solu-
tion was extracted with ether (5 · 20mL). The combined
extracts were dried over Na2SO4, filtered and evapo-
rated to give 6a as a white solid (0.58g, 27%). The crude
product was practically pure and was used without puri-
1
fication; mp 116–119ꢁC; H NMR (DMSO-d6): d 10.07
(br, 1H), 7.7–7.48 (m, 3H), 5.7 (br, –SO2H+H2O), 2.08
(s, 3H).
5.1.16. N-(2-Cyanomethylsulfonyl-5-fluorophenyl)acet-
amide (4f). The title compound was prepared by oxida-
tion of 9e with H2O2 in glacial acetic acid according
to the procedure described for 4c. White crystals,
5.1.20. (2-Amino-4-trifluoromethylphenylsulfanyl)aceto-
nitrile (8a). NaH (2.93g, 60% dispersion in mineral oil
73mmol) was added at 5–10ꢁC under nitrogen in small
portions to a stirred and cooled solution of 2-amino-4-
1
mp 131–132ꢁC; H NMR (DMSO-d6): d 9.65 (s, 1H,
NH), 8.07–7.93 (m, 2H), 7.41–7.28 (m, 1H), 5.32
(s, 2H, CH2), 2.16 (s, 3H, CH3); 13C NMR (DMSO-
d6): d 169.24, 165.91 (d, J = 254Hz), 139.92 (d,
J = 13Hz), 134.12 (d, J = 11.5Hz), 123.19 (d, J =
2.9Hz), 112.28 (d, J = 23Hz), 11.90, 111.60 (d,
J = 24Hz), 44.89, 24.37; IR m 3343 (NH), 2253 (CN),
trifluoromethylbenzenethiol
hydrochloride
(8.00g,
35mmol) dissolved in dry dimethylformamide
(125mL). After stirring for 45min iodoacetonitrile
(2.65mL) was added. Stirring was continued at ꢂ0ꢁC
for 15min and then at room temperature for 1h. The
mixture was concentrated in vacuo at 55ꢁC and the oily
residue was extracted with petroleum ether (3 · 50mL)
in order to remove the mineral oil. The residue was par-
titioned between H2O (50mL) and EtOAc (100mL). The
organic phase was washed with H2O (2 · 50mL) and
then with brine (30mL), dried over Na2SO4, filtered
and concentrated in vacuo to give a yellow oil. The
crude product was purified by flash chromatography
on silica gel eluted with EtOAc–heptane 3:7 (V/V) giving
the title compound as a yellow oil (6.1g, 75%); 1H NMR
(DMSO-d6): d 7.54 (d, J = 8.3Hz, 1H), 7.09 (d,
J = 1.8Hz, 1H), 6.86 (dd, 1H), 5.97 (br, 2H), 4.02 (s,
2H); 13C NMR (DMSO-d6): d 149,.88, 135.66, 130.74
(J = 31Hz), 124.12 (J = 272Hz), 117.58, 116.96
(J = 1.3Hz), 112.04 (J = 3.8Hz), 110.53 (J = 3.9Hz),
17.95.
1693 (C@O) cmꢀ1
.
5.1.17. N-(2-Cyanomethylsulfonyl-4-fluorophenyl)acet-
amide (4g). The title compound was prepared in 58%
yield by oxidation of 9f with H2O2 in glacial acetic
acid according to the procedure described for 4c.
1
White needles, mp 154–157ꢁC (from 96% EtOH); H
NMR (DMSO-d6): d 9.64 (s, 1H, NH), 7.99–7.64
(m, 3H), 5.30 (s, 2H, CH2), 2.08 (s, 3H, CH3);
13C NMR (DMSO-d6):
d
169.43, 158.24 (d,
J = 247Hz), 133.66 (d, J = 3Hz), 130.75 (d, J =
6.5Hz), 130.04 (d, J = 7.3Hz), 123.34 (d, J = 22Hz),
117.39 (d, J = 26Hz), 111.66, 44.35, 23.63; MS m/z
(rel abundance) 256 (M+, 21), 214 (Mꢀ42, 70), 174
(31), 126 (16), 110 (31); IR m 2261 (CN), 1667, 1638
(C@O) cmꢀ1
.
5.1.18. N-(2-Cyanomethylsulfonyl-4-methoxyphenyl)acet-
amide (4h). The title compound was prepared in 58%
yield by oxidation of 9g with H2O2 in glacial acetic acid
according to the procedure described for 4c. Pale yellow
5.1.21.
(2-Amino-5-chloro-phenylsulfanyl)acetonitrile
(8b). A mixture of 2-amino-6-chloro-benzothiazole
(4.90g, 26.5mmol) and a solution of potassium hydrox-
ide (8.9g, 159mmol) in H2O (18mL) was stirred at
160ꢁC under nitrogen for 5.5h. The mixture was cooled
to 0–5ꢁC and acetic acid (6.25mL) in H2O (6mL) was
added in order to neutralize the excess of NaOH but
not the thiolate. To the resulting slurry was added etha-
nol (30mL) and then iodoacetonitrile (1.9mL,
26.5mmol). After 0.5h acetic acid (1.55mL) was added
and the mixture was concentrated in vacuo to approx.
20mL and diluted with H2O (100mL). The precipitated
pale yellow crystals were filtered off, rinsed with H2O
and dried to give the title compound (4.98g, 94%) prac-
tically pure, mp 71–73ꢁC. A sample was recrystalliza-
tion from ethanol to give pure crystals, mp 72–73ꢁC;
1
crystals, mp 189–190ꢁC (from 96% EtOH); H NMR
(DMSO-d6): d 9.54 (br s, 1H, NH), 7.70–7.62 (m, 1H),
7.46–7.35 (m, 2H), 5.20 (s, 2H, CH2), 3.84 (s, 3H,
CH3O), 2.07 (s, 3H, CH3); 13C NMR (DMSO): d
169.46, 156.58, 131.09, ca. 129.62, 129.62, 121.72,
115.07, 111.86, 55.95, 44.21, 23.44.
5.1.19. 2-Acetylamino-5-chloro-benzenesulfinic acid (6a).
A mixture of ground phosphorus pentachloride (2.28g,
11mmol) and 2-acetylamino-5-chloro-benzenesulfonic
acid pyridinium salt (5a) (3.00g, 9mmol) was stirred at
60ꢁC. After 2h the resulting yellow fluid was poured
onto crushed ice/water (100mL) and stirred vigorously
for 10min. The aqueous phase was decanted and the
sticky precipitate was dissolved in CH2Cl2 (75mL) and
dried over Na2SO4 and filtered. The filtrate was cooled
to ꢀ75ꢁC and a solution of p-toluenethiol (2.28g) and
triethylamine (2.55mL) in CH2Cl2 (25mL) was added
1
Rf = 0.39 (EtOAc–heptane 1:1); H NMR (CDCl3): d
7.49 (d, J = 2.3Hz, 1H), 7.18 (dd, 1H), 6.70 (d,
J = 9.2Hz, 1H), 4.42 (br, 2H), 3.47 (s, 2H); 13C NMR
(CDCl3): d 147.49, 136.07, 131.93, 122.87, 116.46,
116.31, 114.75, 19.98; IR (KBr) m 3362 (NH2), 2243
(CN) cmꢀ1
.

S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
151
5.1.22. (2-Amino-5-fluorophenylsulfanyl)acetonitrile (8c).
A mixture of 2-amino-6-fluorobenzothiazole (5.13g,
30.5mmol) and a solution of potassium hydroxide
(10.3g, 183mmol) in H2O (20mL) was stirred at 165–
170ꢁC under nitrogen in a flask with a condenser for
6 12 h. The mixture was cooled to 10ꢁC and acetic acid
(7.2mL) in H2O (7mL) was added. Then pH was ad-
justed to 8 by the addition of 1M NaOH and ethanol
(30mL) and iodoacetonitrile (2.20mL) was added. After
15min the reaction was quenched by the addition of ace-
tic acid (1.8mL) and the mixture was concentrated in
vacuo to approx. 20mL and diluted with H2O
(150mL). A yellow oil separated. The mixture was
extracted with EtOAc (50mL). A white precipitate in
the EtOAc phase was removed by filtration and was
identified as thiocarbamic acid S-(2-amino-5-fluorophe-
nyl) ester, yield 0.35g. The filtrate was dried over
Na2SO4, filtered and the solvent was removed in vacuo
to give the title compound (4.88g, 88%) as a yellow oil
containing a small amount of crystalline precipitate.
ether (100mL) was added and the mixture was washed
with H2O (2 · 25mL) and finally with brine (10mL).
The organic phase was dried over Na2SO4, filtered and
evaporated to give the title compound as a practically
pure off-white solid (3.4g, 75%); mp 93–95ꢁC; 1H
NMR (DMSO-d6): d 9.11 (s, 1H, NH), 7.71–7.56 (m,
1H), 7.48–7.32 (m, 2H), 4.25 (s, 2H), 4.10 (q, 2H),
1.22 (t, 3H); 13C NMR (DMSO-d6): d 154.21 (C@O),
136.10, 131.86, 129.98, 129.03, 127.75, 127.14, 117.54
(CN), 60.68 (O–CH2), 18.09 (S-CH2), 14.46 (CH3); IR
(KBr) m 3362 (NH), 2243 (CN), 1719 (C@O) cmꢀ1
.
5.1.26. (2-Cyanomethylsulfanyl-4-fluorophenyl)carbamic
acid isopropyl ester (9c). To a stirred solution of 8c
(0.20g, 1.1mmol) in dry THF (5mL) at 0ꢁC was added
drop wise a 1M solution of isopropyl chloroformate in
toluene (1.15mL) and then pyridine (93lL, 1.16 mmol).
After 12 h the solvent was stripped off and the oily residue
was triturated with H2O (10mL) at 0ꢁC. A solid precip-
itate was collected by filtration, rinsed with H2O (5mL)
and dried to give the title compound as a beige powder
(0.24g, 81%). The crude product was used without fur-
1
The product was used without further purification. H
NMR (DMSO-d6): d 7.26–7.14 (m, 1H), 7.09–6.95
(m, 1H), 6.85–6.72 (m, 1H), 5.36 (br, 2H, NH2), 3.99
(s, 2H). 13C NMR (DMSO-d6): d 153.53 (d, J =
234Hz), 146.20 (d, J = 1.4Hz), 120.30 (d, J = 22.3Hz),
117.68, 117.54 (d, J = 22.0Hz), 115.69 (d, J = 7.6Hz),
113.29 (d, J = 7.9Hz), 18.19.
1
ther purification. Mp 68–69ꢁC; H NMR (DMSO-d6):
d 9.00 (s, 1H, NH), 7.50–7.28 (m, 2H), 7.22–7.07 (m,
1H), 4.82 (septet, 1H), 4.24 (s, 2H), 1.22 (d, 6H); 13C
NMR (DMSO-d6): d 159.84 (J = 237Hz), 154.15,
132.90 (J = 2.9Hz), 128.34 (J = 9.2Hz), 117.59, 115.22
(J = 25Hz), 114.10 (J = 22Hz), 67.90, 21.92, 17.55; MS
m/z (rel abundance): 268 (30) M+, 226 (6), 209 (9), 184
(7), 182 (20), 168 (9), 154 (22), 142 (92), 114 (15, 98
(10), 45 (16), 43 (100), 42 (43); IR (KBr) m 3304 (NH),
5.1.23. (2-Amino-4-fluorophenylsulfanyl)acetonitrile (8d).
The title compound was prepared in 96% yield from
2-amino-5-fluorobenzothiazole according to the proce-
1
dure described for 8c. Yellow oil; H NMR (DMSO-
2249 (CN), 1693 and 1534 (C@O) cmꢀ1
.
d6): d 7.89 (dd, 1H, H6), 6.56 (dd, 1H, H3), 6.37 (ddd,
1H, H5), 5.88 (br, 2H, NH2), 3.83 (s, 2H, CH2); 13C
NMR (CDCl3): d 164.41 (d, J = 244Hz), 152.24 (d,
J = 12.8Hz), 138.67 (d, J = 11.0Hz), 117.75, 107.76 (d,
J = 2.1Hz), 103.27 (d, J = 22.4Hz), 100.40 (d, J =
25.1Hz), 18.93.
5.1.27.
(2-Cyanomethylsulfanyl-5-trifluoromethylphen-
yl)carbamic acid ethyl ester (9d). Ethyl chloroformate
(1.67mL, 17.4mmol) and subsequently triethylamine
(2.41mL, 17.4mmol) were added drop wise to a stirred
solution of 8a (3.68g, 17.4mmol) in dry THF (25mL)
at 0ꢁC. The ice bath was removed and stirring was con-
tinued at ambient temperature for 30min and then 1 12 h
at 60ꢁC. The solvent was removed on a rotary evapora-
tor and the residue was partitioned between ether
(75mL) and H2O (25mL). The organic layer was
washed with H2O (2 · 20mL) and finally with brine
(20mL), dried over Na2SO4, filtered and evaporated to
give the title compound as yellow crystals (3.06g,
63%). Recrystallization from 96% ethanol gave pale
prisms (2.42g, 50%), pure enough for further synthesis.
A pure sample was obtained from ether–petroleum ether
5.1.24. N-(2-Cyanomethylsulfanyl-5-trifluoromethylphen-
yl)acetamide (9a). Compound 8a (0.66g, 2.8mmol) was
stirred on an ice bath at 0ꢁC. Acetic acid anhydride
(2.5mL) was added, and the resulting solution was
allowed to reach room temperature. After 1h the mixture
was concentrated in vacuo and the solid residue was tri-
turated with petroleum ether (2 · 2mL), filtered off and
dried to give the title compound as a white solid (0.75g,
1
96%), mp 103–103.3ꢁC; H NMR (CDCl3): d 8.79 (br,
1H), 8.36 (br, 1H), 7.78 (d, 1H), 7.38 (dd, 1H, J: 8Hz/
2Hz), 3.50 (s, 2H), 2.29 (s, 3H); 13C NMR (DMSO-d6):
d 169.06, 137.02, 134.25, 129.49, 127.56 (J = 33Hz),
123.81 (J = 272Hz), 122.61 (two very close signals),
1
1:1; mp 87–88ꢁC, H NMR (DMSO-d6): d 9.30 (s, 1H),
7.85–7.59 (m, 3H), 4.29 (s, 2H), 4.14 (q, 2H), 1.25 (t,
3H); 13C NMR (DMSO-d6): d 154.22 (C@O), 137.29,
134.31, 129.84, 127.82 (q, J = 32Hz, CF3), 123.78 (q,
J = 272Hz, CF3), 122.38 (J = 3.8Hz), 121.90
(J = 3.6Hz), 117.49 (CN), 60.88, 17.70, 14.42; EI-MS:
m/z (rel abundance): 304 (100) M+, 285 (18), 258 (14),
232 (50), 204 (53), 192 (99), 45 (16), 40 (22); IR (KBr)
117.49, 23.06, 17.46; IR (KBr) m 2244 (C„N) cmꢀ1
.
5.1.25. (4-Chloro-2-cyanomethylsulfanyl-phenyl)carbamic
acid ethyl ester (9b). Compound 8b (3.1g, 15.5mmol)
was dissolved dry THF (in 25mL) and cooled in an
ice bath at 0ꢁC. To the stirred solution was added first
a solution of ethyl chloroformate (1.50mL) in THF
(5mL) and then a solution of pyridine (1.25mL) in
THF (5mL). The ice bath was removed after completion
of the addition and the mixture was stirred for 2h. The
solvent was removed in vacuo on a rotary evaporator,
m 3361 (NH), 2247 (CN), 1725 (C@O) cmꢀ1
.
5.1.28. N-(2-Cyanomethylsulfanyl-5-fluorophenyl)acet-
amide (9e). Acetyl chloride (1.5mL) in THF (10mL)
and subsequently pyridine (1.7mL) in THF (10mL)
was added to a stirred solution of 8d (3.5g) in THF

152
S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
(30mL) at 0ꢁC. The mixture was stirred at 0ꢁC for
50min. Then the solvent was evaporated and the residue
was triturated with water (80mL). The precipitate was
filtered off and dried yielding 3.7g (86%) of the title com-
pound. Yellow crystals; mp 113–115ꢁC; 1H NMR
(DMSO-d6): d 9.59 (s, 1H, NH), 7.73–7.57 (m, 2H),
7.19–7.05 (m, 1H), 4.05 (s, 2H, CH2), 2.12 (s, 3H,
CH3); 13C NMR (DMSO-d6): d 169.03, 162.16 (d,
J = 245Hz), 140.67 (d, J = 12Hz), 135.36 (d,
J = 9.7Hz), 120.34 (br), 117.64, 112.35 (d, J = 22Hz),
111.05 (d, J = 26Hz), 23.60, 19.79; IR (KBr) m 3314
126.27, 124.65, 119.82, 118.75, 117.24, 66.44ppm; MS
m/z (rel abundance) 256 (100) M+, 189 (31), 137 (25),
129 (19), 126 (22), 125 (78), 90 (20), 67 (31), 63 (53);
IR (KBr) m 3253 (NH), 2222 (CN) cmꢀ1
.
5.1.32.
3-Hydroxy-6-trifluoromethyl-4H-1,4-benzothi-
azine-2-carbonitrile 1,1-dioxide (10b). Compound 4e
(2.61g, 7.8mmol) was stirred at room temperature in
0.2M NaOH (50mL). During the next 6 12 h additional
amounts of aqueous base were added: After 1.5h
0.2M NaOH (10mL), and after 3h 1M NaOH (2mL).
The turbid mixture was filtered through celite and acid-
ified to pH = 1 by the addition of 4M HCl (3mL) giving
a white precipitate. The water was removed by evapora-
tion and the white residue was extracted with two por-
tions (30 + 10mL) of EtOAc. The combined extracts
were dried over Na2SO4 and the solvent was evaporated
to give an amorphous residue. Trituration with a mix-
ture of petroleum ether (20mL) and ether (5mL) gave
white crystals, which were filtered off and dried to give
the title compound, practically pure (2.08g, 95%). The
compound may be purified further by recrystallization
from glacial acetic acid; mp 222–224ꢁC (ebullition; dis-
(NH), 2250 (CN), 1686 (C@O) cmꢀ1
.
5.1.29. N-(2-Cyanomethylsulfanyl-4-fluorophenyl)acet-
amide (9f). This compound was prepared in 82% crude
yield by acetylation of 8c according to the procedure
for 9e. Recrystallization from 96% ethanol gave white
crystals, mp 100.5–101.5ꢁC; 1H NMR (DMSO-d6): d
9.68 (s, 1H, NH), 7.50–7.30 (m, 2H), 7.23–7.07 (m,
1H), 4.26 (s, 2H, CH2), 2.05 (s, 3H, CH3); 13C NMR
(DMSO-d6) 168.80 (C@O), 159.90 (d, J = 244Hz),
132.82 (d, J = 2.8Hz), 132.11 (d, J = 8.6Hz), 128.42 (d,
J = 8.9Hz), 117.60, 115.18 (d, J = 25.3Hz), 114.05 (d,
J = 22.3Hz), 22.84, 17.50; IR (KBr) m 3239 (NH), 2244
1
colourization started at ꢂ200ꢁC); H NMR (DMSO-
(CN), 1641 and 1579 (amide) cmꢀ1
.
d6): d 10.20 (br, 1H, NH), 7.89–7.76 (ꢂd at 7.82,
J = 8.3Hz,1H), 7.51–7.30 (m, 2H, d at 7.82 and dd at
7.38), 6.6–6.2 (br, two peaks: 6.44 and 6.39; 1H+H2O);
13C NMR (CDCl3): d 162.46 (C3), 136.32, 131.48
(J = 32Hz, C6), 128.16, 123.52 (J = 273Hz, CF3),
122.10, 117.51 (CN), 116.86 (J = 3.7Hz), 113.19
(J = 4.1Hz), 66.06 (C2); EI-MS: m/z (rel abundance):
290 (100) M+, 271 (18) M+ꢀF, 247 (4) M+ꢀCNꢀOH,
226 (28) M+ꢀSO2, 223 (73) M+ꢀ67, 206 (38), 171
(60), 159 (78), 132 (67), 113 (31), 109 (29),88 (22), 75
(34) CF2C2H, 69 (32) CF3, 67 (49) NCCCOH, 63 (85);
5.1.30. N-(2-Cyanomethylsulfanyl-4-methoxyphenyl)acet-
amide (9g). Sodium borohydride (0.19g) was added in
small portions over 30min to a stirred solution of
bis(2-acetylamino-5-methoxyphenyl)disulfane (1.0g) in
dry ethanol (100mL) at 70ꢁC under nitrogen. The mix-
ture was cooled to room temperature and iodoacetonit-
rile (355lL) was added. After stirring for 15min the
solvent was evaporated. The residue was partitioned be-
tween ethyl acetate (50mL) and water (50mL); the or-
ganic phase was washed with water (2 · 50mL) and
dried over sodium sulfate. Removal of the solvent gave
1.06g (91%) of the pure title compound. White crystals,
mp 130.5–131.5ꢁC; 1H NMR (DMSO-d6): d 9.48 (s, 1H,
NH), 7.23 (d, J = 9Hz, 1H), 7.08 (d, J = 3Hz, 1H), 6.88
(dd, 1H), 4.18 (s, 2H, CH2), 3.78 (s, 3H, CH3O), 2.01 (s,
3H, CH3); 13C NMR (DMSO-d6): d 168.73, 157.44,
130.86, 129.64, 128.00, 117.85, 114.41, 112.84, 55.49,
22.83, 17.68; IR (KBr) m 3286 (NH), 2250 (CN), 1658
IR (KBr) m 3278 (NH), 2233 and 2215 (CN) cmꢀ1
.
5.1.33. 3,7-Dichloro-4H-1,4-benzothiazine-2-carbonitrile
1,1-dioxide (11a). Compound 10a (1.75g, 6.8mmol)
was added to a stirred solution of phosphoric acid
(0.2mL) in phosphorus oxychloride (6mL). Then pyr-
idine hydrochloride (1.98g) was added. The mixture
was heated at 100ꢁC overnight. The resulting dark solu-
tion was cooled on an ice bath and crushed ice (50mL)
was added with vigorous stirring. The mixture was fil-
tered after 10min, the beige filter cake washed on the fil-
ter with H2O and dried to give the title compound (1.78g,
95%). The product was almost pure and was used with-
(C@O) cmꢀ1
.
5.1.31. 7-Chloro-3-hydroxy-4H-1,4-benzothiazine-2-car-
bonitrile 1,1-dioxide (10a). Compound 4d (5.47g,
18mmol) was stirred at room temperature with 0.25M
NaOH (180mL). After 2h the resulting yellow solution
was extracted with EtOAc (3 · 50mL). The extracts
were discarded and the aqueous phase was acidified with
4M HCl (11.5mL). The solvent was removed on a ro-
tary evaporator and the yellow solid residue was ex-
tracted with petroleum ether–ether (3:1 (V/V),
25 + 40mL)). The undissolved material was extracted
with EtOAc (50 + 20 + 20mL). The combined EtOAc
extracts were evaporated to give the title compound as
a beige solid (3.70g, 80%); mp 204–206ꢁC (ebullition);
1H NMR (DMSO-d6): d 10.09 (br, 1H, NH), 7.54 (d,
J = 2.3Hz, 1H), 7.47 (dd, 1H, C(6)–H), 7.12 (d,
J = 8.7Hz, 1H, C(5)–H), 5.31 (br, H2O and possibly
OH); 13C NMR (DMSO-d6): d 162.51, 134.50, 131.74,
1
out further purification; mp 269–271ꢁC (decomp.); H
NMR (DMSO-d6): d 8.01 (d, J = 2.8Hz, 1H), 7.79 (dd,
1H), 7.50 (d, J = 9.2Hz, 1H), 6.33 (br, NH+H2O); 13C
NMR (DMSO-d6): d 146.59, 134.87, 133.61, 129.75,
125.53, 123.23, 120.94, 112.02 (apparently two aromatic
signals are coinciding); IR (KBr) m 2217 (CN) cmꢀ1
.
5.1.34. 3-Chloro-6-trifluoromethyl-4H-1,4-benzothiazine-
2-carbonitrile 1,1-dioxide (11b). Compound 10b (0.52g,
1.8mmol) was added to a stirred solution of phosphoric
acid (0.05mL) in phosphorus oxychloride (2.5mL).
Then pyridine hydrochloride (0.5g) was added. The
mixture was heated at 100ꢁC overnight. The resulting
yellow solution was concentrated in a rotary evaporator
in vacuo at 50–60ꢁC for 0.5h. The oily residue was

S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
153
cooled on an ice bath at 0ꢁC and stirred with ice/water
(10mL) for 0.5h and the beige precipitate was filtered
off, washed on the filter with water and dried to give
the title compound (0.51g, 93%). The product was al-
most pure and was used without further purification;
mp 275–285ꢁC (decomp.); 1H NMR (DMSO-d6): d
8.20–8.10 (m, 1H), 7.83–7.69 (m, 2H), 5.75 (br,
NH+H2O); 13C NMR (DMSO-d6): d 147.87, 138.05,
132.67 (J = 33Hz), 127.52, 127.31, 123.08 (J = 273Hz),
121.58 (J = 3.4Hz), 119.53, 112.62; MS m/z (rel abun-
dance): 310 (11) M++2, 308 (31) M+, 289 (<1) M+ꢀF,
244 (100) M+ꢀSO2, 225 (25), 209 (11), 194 (25); IR
parameter logistic curve fit was made (Hill slope locked
at ꢀ1.5) for each run of eight points separately. The IC50
values were extracted and put into MS Excel to calculate
a mean estimate of IC50 with SD. If the curves showed
no slope the abbreviation is NA. If the IC50 value was
above 100 the notation is >100 and no SD is given.
Three or more observations from one or two assay
runs were used for the compilation of data given in
Table 1.
5.2.3. Membrane potential studies in HEK 293 cells
expressing Kir6.2/SUR1 or Kir6.2/SUR2B. KATP chan-
nel activity was monitored by evaluating changes in
membrane potential, using a membrane potential kit
(Molecular Devices, USA). Assays were carried out in
black clear-bottomed 96-well plates at 34ꢁC on a Novo-
StarTM machine (BMG, Germany) and KATP channels
activated by the addition of the KATP channel opener
in a mixed assay medium containing 10lM tolbutamide.
Changes in fluorescence were measured using an excita-
tion wavelength of 490nm and an emission wavelength
of 520nm, before and 5–10min after addition of test
compounds.
(KBr) m 2218 (CN) cmꢀ1
.
5.2. Biology
5.2.1. Cell culture and transfection. HEK 293 cells were
cultured at 37ꢁC in a humidified atmosphere of 95%
air and 5% CO2 in DulbeccoÕs modified Eagles medium
with 4.5g/L glucose supplemented with 10% FCS, peni-
cillin (100units/mL) and streptomycin (0.1mg/mL).
Transient transfections were performed using Fugene 6
Transfection reagent (Roche), according to the manu-
facturerÕs instruction. Cells were seeded at 50% conflu-
ency and transfected with murine SUR2B (GenBank
D86038) and human Kir6.2 (GenBank D50582). Cells
were co-transfected with green fluorescent protein
(GFP) to enable visual identification of transfected cells.
Experiments were performed 2days after transfection.
HEK293 cells stably expressing human Kir6.2 and hu-
man SUR1 (Genbank L78207) were used for some
experiments.31
5.2.4. Inhibition of glucose-stimulated insulin release from
bTC6 cells. bTC6 cells32 were cultured at 5 · 104 cells/
microtitre well in DMEM+10% FCS, 11mM glucose,
1% Glutamax and 20mM Hepes for 3days (95% humid-
ity, 37ꢁC, 5% CO2). Cells were washed twice with NN
buffer (all in mM: NaCl 114; KCl 47; KH2PO4, 1.21;
MgSO4, 1.16; NaHCO3, 25.5; CaCl, 2H2O, 2.5; HEPES,
10) supplemented with 0.1% BSA and incubated 60min
in this buffer. All wells were aspirated and the cells incu-
bated for 3h with NN buffer, 22mM glucose, 0.1mM
IBMX and serial dilutions of the compounds. A refer-
ence compound (BPDZ 73)11 served as positive control.
A test for responsiveness towards a series of glucose
concentrations was included in every assay to ensure
functionality. The supernatant from each well was har-
vested and insulin content was measured by an in-house
ELISA using guinea pig anti-insulin antibodies and a rat
insulin as standard. ELISA microtitre plates were coated
with anti-guinea pig IgG (Dako Z 108, 1:1000) and incu-
bated overnight at 4ꢁC in PBS pH7.2. All plates were
washed five times with washing buffer (PBS diluted 1:4
in H2O+0.05% Tween20) and incubated 30min in this
buffer at rt. Wells were aspirated and anti-insulin anti-
bodies (polyclonal GP4 (Novo Nordisk 1:75.000) was
added, followed by an incubation for 2h at room tem-
perature (rt). Plates were washed five times in washing
buffer and 10lL test samples were added together with
60lL Peroxidase-labelled (PO)-insulin (Sigma I 2133
1:2400) and incubated for another 2h at rt in Assay buf-
fer (washing buffer+0.5% BSA). A series of standards of
rat insulin (NN) were made to cover a range from 1000
down to 1ng/mL insulin and the standards were incu-
bated with PO-insulin as well. Eventually, 120lL
TMB-substrate (3.30, 5,50-tetramethylbenzidine hydro-
gen peroxide) was added to all wells and the enzyme
reaction stopped after 5min by adding 120lL 4M
H3PO4. Absorption was measured in an Elisa reader
(450nm with 620nm as reference) and converted
into nanogram per millilitre insulin. The results were
5.2.2. Measurements of effects on membrane potential.
Two days before experiments 50,000 cells/well, in 100lL
culture medium, were seeded out in black walled 96-well
plates (Greiner). At the day of experiment, Loading
Buffer from the Membrane potential kit (Molecular
Devices) made in accordance with the kit prescription
(100lL/well) was added to all wells.
Plates were then incubated at 37ꢁC, 5.0% CO2 for
30min. Polypropylene plates (Greiner) were used to
plate out 5· concentrated dilution series (starting at a
final assay concentration of 30lM at a 1:3 dilution).
Compounds were tested as triple dilution series, each
with eight points in 1:3 dilution steps. Both cell plate
and compound plate were put in a NovoStarTM fluores-
cence plate reader for about 30min at 34ꢁC and the
measurements were subsequently begun. An initial read-
ing was taken from the plate. Compounds were added
manually from the compound plate with an eight-chan-
nel multipipette (50lL/well) and the assay was then
incubated for 10min. A second reading was taken using
the same measurement protocol without any adjustment
of the signal gain. Measurement data were ported over
to MS Excel where a before–after subtraction and nor-
malization to the measurement level preceding any addi-
tion was made. Concentrations of test and control
(BPDZ 73)11 compounds were compared and Emax val-
ues were determined using BPDZ 7311 as the reference
at 30lM. Data from three parallel runs on the same
plate were copied to Graph Pad Prism where a four-

154
S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
analyzed in Prism (Graph Pad software) and expressed
as IC50 and Emax. The IC50 value was calculated as the
concentration of test compound needed to inhibit the
insulin release half of the maximal inhibition for that
particular compound. The Emax was defined as the per-
centage inhibition of a compound tested at 10lM rela-
tive to the maximum release when no compound was
added. SEM was calculated for all compounds.
groups of 100 islets were incubated for 60min in a bicar-
bonate buffered medium (in mM: 115 NaCl, 5 KCl, 2.56
CaCl2, 1 MgCl2, 24 NaHCO3) containing 16.7mM glu-
cose and 45Ca ion (0.02–0.04mM; 100lCi/mL). After
incubation, the islets were washed four times with a
non-radioactive medium and then placed in a perifusion
chamber. The perifusate was delivered at a constant rate
(1.0mL/min). From the 31st to the 90th min of peri-
fusion, the effluent was continuously collected over suc-
cessive periods of 1min each. An aliquot of the effluent
(0.5mL) was used for scintillation counting while the
remainder was stored at ꢀ20ꢁC for further radioimmu-
nological measurement of insulin release.33 At the end of
the experiment, the radioactive content of the islets was
also determined. The outflow of 45Ca2+ ion (cpm/min)
was expressed as a fractional outflow rate (% of instan-
taneous islet content/min, FOR). Insulin release was ex-
pressed as lU/islet/min. Results are expressed as the
mean ( SEM). The statistical significance of the differ-
ences between mean data was assessed by use of Stu-
dentÕs t-test.
5.2.5. Relaxation of rat aorta rings. Female Wistar rats
weighing approximately 150–200g were killed by cervi-
cal dislocation, and the thoracic aorta was removed.
Aorta was cut into rings of approximately 5mm wide.
Ring preparations were mounted in 5mL organ baths
(Danish Myo Technology, Aarhus, Denmark) with a
resting tension of 2g, and bathed in Krebs Ringer solu-
tion with the following composition (in mM: 118.5
NaCl; 25 NaHCO3; 4.7 KCl; 6.8 CaCl2; 2.4 MgCl2;
and 11.1 glucose in double distilled water). The Ringer
solution was continuously aerated with 95% O2/5%
CO2 at 37ꢁC. Cumulative concentration response curves
(0.1–300lM) were constructed for potassium channel
openers on top of a precontraction induced by 0.3lM
phenylephrine. All test compounds were freshly dis-
solved in dimethylsulfoxide. Minimum values of each
concentration step of potassium channel openers were
measured. Sensitivity of vasodilating effect is expressed
as the concentration inducing half of the maximal effect
(EC50), estimated using a four parameter logistic regres-
sion (Graphpad Prism, San Diego, CA, USA). Phenyl-
ephrine was obtained from Sigma Chemicals, St.
Louis, MA, USA.
5.2.8. Patch-clamp measurements
5.2.8.1. RNA preparation. Mouse Kir6.2 (Genbank
D50581) and rat SUR1 (Genbank L40624) cDNAs were
cloned into the pBF expression vector. Capped mRNA
was prepared using the mMESSAGE mMACHINE in
vitro transcription kit (Ambion, Austin, USA).
5.2.8.2. Patch-clamp recordings. Whole cell currents
were recorded from HEK 293 cells stably expressing
human Kir6.2/SUR1 channels, using an EPC9 patch-
clamp amplifier (HEKA Electronic GmbH, Lambrecht,
Germany). Cells were clamped at ꢀ70mV and currents
evoked by repetitive 250ms, 10mV depolarizing voltage
steps. Currents were filtered at 2kHz and sampled at
10kHz. The internal solution contained (in mM): 120
KCl, 1 MgCl2, 5 EGTA, 2 CaCl2, 20 Hepes, 0.3
MgADP, 5 MgATP (pH7.3). The external solution
was (in mM): 140 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 20
mannitol, 10 HEPES (pH7.2 with NaOH).
5.2.6. Measurements of insulin release from incubated rat
pancreatic islets. Experiments were performed with pan-
creatic islets isolated by the collagenase method from fed
female Wistar rats (Iffa Credo, Belgium).
Groups of 10 islets were preincubated for 30min in a
bicarbonate-buffered solution (in mM: 115 NaCl, 5
KCl, 2.56 CaCl2, 1MgCl2, 24 NaHCO3) supplemented
with 2.8mM glucose and 0.5% (w/v) dialyzed albumin
(fraction V, Sigma Chemical Co.). The islets were then
incubated for a further 90min in 1mL of the same bicar-
bonate-buffered medium containing 16.7mM glucose
and, in addition, the required compound. Individual
experiments were repeated on different islets populations
with the values corresponding to the number of samples
pooled. Due to the islet-related variations, insulin re-
lease was expressed as a percentage of the value re-
corded in control experiments (100%), that is, in the
absence of drug and presence of 16.7mM glucose. The
release of insulin was measured radioimmunologically
as reported previously.33 Compound 3a and diazoxide
were dissolved in dimethylsulfoxide, which was added
to both control and test media. At the final concentra-
tions used, dimethylsulfoxide fails to affect islet
function.29,34
5.2.8.3. Xenopus oocyte macropatches. Currents were
recorded from giant inside-out patches excised from
oocytes expressing various types of KATP channel using
an EPC7 patch-clamp amplifier. Currents were evoked
by repetitive 3s voltage ramps from ꢀ110 to +100mV,
filtered at 0.2kHz and digitized at 0.4kHz. The external
(pipette) solution contained (in mM): 140 KCl, 1.2
MgCl2, 2.6 CaCl2, 10 HEPES (pH7.4 with KOH). The
intracellular (bath) solution contained (in mM): 110
KCl, 2 MgCl2, 1 CaCl2 10 EGTA, 10 HEPES (pH7.2
with KOH); final [K+]: 140mM). The slope conductance
(G) was measured by fitting a straight line to the cur-
rent–voltage relation between ꢀ20mV and ꢀ100mV.
Drug effects were calculated as the conductance in the
presence of drug (G) relative to the conductance in drug
and nucleotide-free solution (Gc).
5.2.7. Measurements of 45Ca outflow and insulin release
from perifused rat pancreatic islets. The methods used to
measure 45Ca outflow and insulin release from perifused
islets have been described previously.11,29,30 Briefly,
5.2.9. Beta cell selectivity studies after intravenous
administration to rats. Post-prandial, adult male Sprague
Dawley rats, weighing 280–320g (M&B Breeding Cen-

S. C. Schou et al. / Bioorg. Med. Chem. 13 (2005) 141–155
155
10. de Tullio, P.; Pirotte, B.; Lebrun, P.; Fontaine, J.; Dupont,
L.; Antoine, M. H.; Ouedraogo, R.; Khelili, S.; Maggetto,
C.; Masereel, B.; Diouf, O.; Podona, T.; Delarge, J. J.
Med. Chem. 1996, 39, 937.
11. Lebrun, P.; Arkhammar, P.; Antoine, M. H.; Nguyen, Q.
A.; Hansen, J. B.; Pirotte, B. Diabetologia 2000, 43, 723.
12. Nielsen, F. E.; Bodvarsdottir, T. B.; Worsaae, A.; Mac-
Kay, P.; Stidsen, C. E.; Boonen, H. C. M.; Pridal, L.;
Arkhammar, P. O. G.; Wahl, P.; Ynddal, L.; Junager, F.;
Dragsted, N.; Tagmose, T. M.; Mogensen, J. P.; Koch, A.;
Treppendahl, S. P.; Hansen, J. B. J. Med. Chem. 2002, 45,
4171.
13. Pirotte, B.; de Tullio, P.; Lebrun, P.; Antoine, M. H.;
Fontaine, J.; Masereel, B.; Schynts, M.; Dupont, L.;
Herchuelz, A.; Delarge, J. J. Med. Chem. 1993, 36, 3211.
14. Tagmose, T. M.; Mogensen, J. P.; Agerholm, P. C.;
Arkhammar, P. O. G.; Wahl, P.; Worsaae, A.; Hansen, J.
B. Bioorg. Med. Chem. Lett. 2001, 11, 1749.
15. Tagmose, T. M.; Zaragoza, F.; Boonen, H. C.; Worsaae,
A.; Mogensen, J. P.; Nielsen, F. E.; Jensen, A. F.; Hansen,
J. B. Bioorg. Med. Chem. 2003, 11, 931.
16. de Tullio, P.; Becker, B.; Boverie, S.; Dabrowski, M.;
Wahl, P.; Antoine, M. H.; Somers, F.; Sebille, S.;
Ouedraogo, R.; Hansen, J. B.; Lebrun, P.; Pirotte, B. J.
Med. Chem. 2003, 46, 3342.
17. Cosgrove, K. E.; Antoine, M. H.; Lee, A. T.; Barnes, P.
D.; de Tullio, P.; Clayton, P.; McCloy, R.; De Lonlay, P.;
Nihoul, F.; Robert, J. J.; Saudubray, J. M.; Rahier, J.;
Lindley, K. J.; Hussain, K.; Aynsley-Green, A.; Pirotte,
B.; Lebrun, P.; Dunne, M. J. J. Clin. Endocrinol. Metab.
2002, 87, 4860.
18. Barco, A.; Benetti, S.; Pollini, G. P.; Taddia, R. Synthesis
1974, 877.
19. Lee, C.; Field, L. Synthesis 1990, 391.
20. Mital, R. L.; Jain, S. K. J. Chem. Soc. (C) 1969, 2148.
21. Shridhar, D. R.; Sastry, C. V. R.; Bansal, O. P.; Rao, P. P.
Indian J. Chem. Sect. B 1986, 25, 874.
ter, Ll. Skensved, Denmark), were anaesthetized subcu-
taneously with 2mL/kg of a 1:1:1 mixture of Hypnorm
(fentanyl citrate 0.315mg/mL and fluanisone 10mg/
mL, Janssen Pharmaceutica, Belgium), Dormicum (mid-
azolam, 5mg/mL, Hoffmann-La Roche AG, Switzer-
land) and 0.9% NaCl aqueous solution, receiving
supplemental anesthetic (1mL/kg) every 20min.35 Body
temperature was maintained at 37ꢁC by a homeother-
mic blanket system (Harvard Homeothermic Blanket
System, Harvard Apparatus Ltd, UK). A polythene
cannula (PE50) coupled to a pressure transducer (Stat-
ham P23XC) and a pressure coupler (HSE 561, Hugo
Sachs Elektronik Kg, Germany) was introduced into
the carotid artery to measure mean arterial blood pres-
sure, which was recorded continuously and fed to a plot-
ter (Linearcorder Mark VII, WR 3310, Hugo Sachs
Elektronik Kg, Germany) and a computer and analyzed
offline. A polythene cannula (PE50) was inserted into
the jugular vein for drug infusion and blood sampling.
Cannulas were filled with 25u/mL heparin (LEO Pharma
Nordic, Sweden) dissolved in 0.9% NaCl aqueous solu-
tion. Due to the short time frame of the experiment
(<1h), the trachea was not cannulated. After a stabiliza-
tion period, the test substances were injected as a bolus
lasting for 20s. Blood samples were drawn at 5–10min
intervals, from 15min pre-injection until 30min post-
injection. Compounds were dissolved in 5% NaOH
(0.15% in 0.9% NaCl aqueous solution) and 95% in
0.9% NaCl aqueous solution, and were administered in
a fixed dose of 3mg/kg rat. Plasma insulin levels were
determined using a mono/polyclonal ELISA assay
with monoclonal antibodies as catching antibodies.
Standards were rat insulin (Novo Nordisk, Bagsvaerd,
Denmark). Catching antibody was HUI-018 (Novo
Nordisk, Bagsvaerd, Denmark) raised against the
A-chain of the insulin. Detecting antibody, a pool of
polyclonal guinea pig antibodies from GP4042D (Novo
Nordisk, Bagsvaerd, Denmark), used as non-purified
serum.
22. White, E. H.; Bursey, M. M.; Roswell, D. F.; Hill, J. H. J.
Org. Chem. 1967, 32, 1198.
23. Hodgson, H. H.; Handley, F. W. J. Chem. Soc. 1928, 625.
24. Jacobsen, P.; Treppendahl, S. P. Tetrahedron 1979, 35,
2151.
25. 13C NMR spectra were predicted using the ACD Spec-
Manager CNMR Predictor, ACD/Labs Version 7.08
23Apr 2003, Advanced Chemistry Development Inc.,
Toronto, Canada, 2004.
References and notes
26. Andersen, K.; Begtrup, M. Acta Chem. Scand. 1992, 46,
1130.
27. Begtrup, M. J. Chem. Educ. 1987, 64, 974.
28. Manley, P. W.; Quast, U. J. Med. Chem. 1992, 35, 2327.
29. Lebrun, P.; Malaisse, W. J.; Herchuelz, A. Am. J. Physiol.
Endocrinol. Metab. 1982, 5, E59.
1. Aguilar-Bryan, L.; Bryan, J. Endocr. Rev. 1999, 20, 101.
2. Ashcroft, F. M.; Gribble, F. M. Trends Neurosci. 1998, 21,
288.
3. Coghlan, M. J.; Carroll, W. A.; Gopalakrishnan, M. J.
Med. Chem. 2001, 44, 1627.
4. Seino, S.; Inagaki, N.; Namba, N.; Wang, C. H.; Kotake,
K.; Nagashima, K.; Miki, T.; Aguilar-Bryan, L.; Bryan, J.;
Gonoi, T. Jpn. J. Physiol. 1997, 47, S3.
5. Aizawa, T.; Taguchi, N.; Sato, Y.; Nakabayashi, T.;
Kobuchi, H.; Hidaka, H.; Nagasawa, T.; Ishihara, F.;
Itoh, N.; Hashizume, K. J. Pharmacol. Exp. Ther. 1995,
275, 194.
30. Lebrun, P.; Antoine, M. H.; Ouedraogo, R.; Kane, C.;
Dunne, M.; Hermann, M.; Herchuelz, A.; Masereel, B.;
Delarge, J.; de Tullio, P.; Pirotte, B. J. Pharmacol. Exp.
Ther. 1996, 277, 156.
31. Dabrowski, M.; Wahl, P.; Holmes, W. E.; Ashcroft, F. M.
Diabetologia 2001, 44, 747.
32. Efrat, S.; Linde, S.; Kofod, H.; Spector, D.; Delannoy,
M.; Grant, S.; Hanahan, D.; Baekkeskov, S. Proc. Natl.
Acad. Sci. U.S.A. 1988, 85, 9037.
6. Carr, R. D.; Brand, C. L.; Bo¨dvarsdottir, T. B.; Hansen, J.
B.; Sturis, J. Diabetes 2003, 52, 2513.
7. Alemzadeh, R.; Slonim, A. E.; Zdanowicz, M. M.;
Maturo, J. Endocrinology 1993, 133, 705.
8. Seino, S.; Miki, T. Prog. Biophys. Mol. Biol. 2003, 81, 133.
9. Gribble, F. M.; Reimann, F.; Ashfield, R.; Ashcroft, F. M.
Mol. Pharmacol. 2000, 57, 1256.
33. Lebrun, P.; Antoine, M. H.; Devreux, V.; Hermann, M.;
Herchuelz, A. J. Pharmacol. Exp. Ther. 1990, 255, 948.
34. Lebrun, P.; Atwater, I. Biophys. J. 1985, 48, 919.
35. Brand, C. L.; Rolin, B.; Jorgensen, P. N.; Svendsen, I.;
Kristensen, J. S.; Holst, J. J. Diabetologia 1994, 37, 985.