Synthesis and activity on rat aorta rings and rat pancreatic β-cells of ring-opened analogues of benzopyran-type potassium channel activators

Article abstract of DOI:10.1016/j.bmc.2008.04.043

Ring-opened analogues of dihydrobenzopyran potassium channel openers (PCOs) were prepared and evaluated as putative PCOs on rat aorta rings (myorelaxant effect) and rat pancreatic β-cells (inhibition of insulin secretion). These derivatives are characterized by the presence of a sulfonylurea, a urea or an amide function. Some compounds bearing an arylurea moiety provoked vasorelaxant effects and a marked inhibition of insulin release. Derivatives bearing a sulfonylurea or an amide function were, however, poorly active on both tissues. Structure-activity relationships and apparent tissue selectivity are discussed.

Full text of DOI:10.1016/j.bmc.2008.04.043

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6124–6130
Synthesis and activity on rat aorta rings and rat pancreatic
b-cells of ring-opened analogues of benzopyran-type
potassium channel activators
Smail Khelili,a Xavier Florence,b Mourad Bouhadja,a Samia Abdelaziz,a
Nadia Mechouch,a Yekhlef Mohamed,a Pascal de Tullio,b
Philippe Lebrunc, and Bernard Pirotteb,*,
aLaboratoire de chimie pharmaceutique, de pharmacologie et d’ecotoxicologie, Faculte´ des sciences, Universite´ de Jijel,
B.P. 98 Ouled Aissa, 18000 Jijel, Algeria
b
`
Laboratoire de chimie pharmaceutique, Centre Interfacultaire de Recherche du Me´dicament, Universite´ de Liege, C.H.U.,
ˆ
`
1 Avenue de l’Hopital, B-4000 Liege, Belgium
cLaboratoire de pharmacodynamie et de the´rapeutique, Universite´ Libre de Bruxelles, Faculte´ de Me´decine, 808, Route de Lennik,
B-1070 Bruxelles, Belgium
Received 27 March 2008; accepted 16 April 2008
Available online 25 April 2008
Abstract—Ring-opened analogues of dihydrobenzopyran potassium channel openers (PCOs) were prepared and evaluated as
putative PCOs on rat aorta rings (myorelaxant effect) and rat pancreatic b-cells (inhibition of insulin secretion). These deriva-
tives are characterized by the presence of a sulfonylurea, a urea or an amide function. Some compounds bearing an arylurea
moiety provoked vasorelaxant effects and a marked inhibition of insulin release. Derivatives bearing a sulfonylurea or an amide
function were, however, poorly active on both tissues. Structure–activity relationships and apparent tissue selectivity are
discussed.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
as myorelaxants.3 On the other hand, ureas with R’
corresponding to a m- or a p-chlorophenyl moiety
(see Fig. 1), were identified as potent inhibitors of
insulin release.5 The biological effects of these drugs
were related to the activation of ATP-sensitive potas-
sium channels.3,5
ATP-sensitive potassium channel openers consist of a
class of compounds with a wide range of chemical struc-
tures, among which the most known belong to benzopy-
rans (i.e., cromakalim), cyanoguanidines (i.e., pinacidil)
and arylthiadiazine dioxides (i.e., diazoxide).1 Cromaka-
lim derivatives bearing amide, urea, thiourea, sulfonyl-
urea and carbamate moieties have been recently
described (Fig. 1).2–5 Some of these compounds pro-
voked a marked inhibition of insulin secretion from
rat pancreatic b-cells, and/or exhibited smooth muscle
myorelaxant activity on rat aorta and rat uterus.2–5
In order to develop new drugs, we undertook the syn-
thesis and pharmacological evaluation of some simpli-
fied compounds resulting from both the opening of
the pyran ring of dihydrobenzopyrans and the intro-
duction of amide, urea or sulfonylurea moieties
(Fig. 2). These original drugs should be more flexible,
which might lead to additional pharmacological
properties.
In particular, amides with a short R alkyl chain (see
Fig. 1) were found to be more active than diazoxide
Moreover, benzopyran-type arylureas devoid of a sub-
stituent at the 6-position of the heterocycle were also
synthesized. These compounds constitute the link be-
tween the ring-opened drugs and previously described
6-halo-substituted benzopyran-type arylureas.
Keywords: Ring-opened cromakalim analogues; Inhibition of insulin
secretion; Vasorelaxant effect; Potassium channel opener.
*
Corresponding author. E-mail: B.Pirotte@ulg.ac.be
The two last authors assumed equal supervision.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.043

S. Khelili et al. / Bioorg. Med. Chem. 16 (2008) 6124–6130
6125
ortho-alkoxybenzylamines 1a and 1b, by reacting the lat-
ter intermediates with alkyl or aryl isocyanates, acyl
chlorides and arylsulfonyl isocyanates, respectively.
The resulting compounds were isolated in good yields
after vacuum filtration.
R
H
O
N
X
N
5
4
6
Y
NC
OH
3
7
2
O
O
The synthesis of the non-substituted benzopyran-type
arylureas 9a–b is depicted in Scheme 2. Starting from
2-hydroxyacetophenone 5, the classical ring closure
reaction using acetone in the presence of pyrrolidine
led to the expected 2,2-dimethylchroman-4-one 6. Ac-
cess to 4-amino-3,4-dihydro-2,2-dimethyl-2H-1-benzo-
pyran 8 was achieved, in excellent yields, by means of
an unusual route consisting in the reduction of the
oxime intermediate 7 by hydrogenation in presence of
Raney Nickel.8 The phenylureas 9a–b were finally ob-
tained after the reaction of the primary amine 8 with
the appropriate chloro-substituted phenyl isocyanate.
8
1
cromakalim
R = alkyl (X = O: amides)
NHR' (X = O or S: ureas or thioureas)
NHSO2R' (X = O: sulfonylureas)
OR' (X = O: carbamates)
Y = F, Cl, Br, NO2
Figure 1. Structure of cromakalim and of some recently described
dihydrobenzopyans bearing miscellaneous functional groups at the
4-position.
R
R
1
1
H
H
N
O
O
N
O
O
2.2. Biological evaluation
X
molecular
2.2.1. Inhibitory activity on insulin secretion from rat
pancreatic islets. Compounds 2a–f, 3a–d, 4a–b and 9a–b
were evaluated as inhibitors of insulin secretion from rat
pancreatic islets incubated in the presence of an insuli-
notropic glucose concentration (16.7 mM). As observed
in Table 1, urea derivatives 2c–f for which R2 corre-
sponded to p-ClC6H5 or m-ClC6H5 exhibited a marked
inhibitory effect on the insulin releasing process. Such
arylurea derivatives were much more potent than cro-
makalim and pinacidil at reducing the glucose-induced
insulin release. Most drugs were found to be equipotent
or even more potent than the KATP channel opener diaz-
oxide. Thus, the estimated IC50 value (drug concentra-
tion inducing a 50% reduction in the insulin secretory
rate) amounted to 18.9 lM for compound 2d, 22.0 lM
simplification
R
2
R
1 = alkyl or aryl (amide), NHR' (urea),
NHSO2Ar (sulfonylurea)
2 = Me, Et
X = F, Cl, Br, NO2
R1 = alkyl or aryl (amide), NHR' (urea),
NHSO2Ar (sulfonylurea)
R
Figure 2. General structure of the target compounds after molecular
simplification of dihydrobenzopyrans.
2. Results
2.1. Synthesis
Scheme 1 illustrates the synthetic route used to prepare
the target compounds 2a–f, 3a–d and 4a–b from the
H
N
R2
O
R2
N
H
H
N
O
O
NH2
R2NCO
CH2Cl2
R2COCl
toluene
R1
R1
R1
O
O
3a-d
1a : R1 = CH3
2a-f
1b : R1 = CH2CH3
R2SO2NCO
diethylether
O
O
H
N
S
N
R2
H
O
R1
O
4a-b
Scheme 1. Synthetic route to the target compounds 2a–f, 3a–d and 4a–b.

6126
S. Khelili et al. / Bioorg. Med. Chem. 16 (2008) 6124–6130
OH
O
O
N
NH
NH2OH.HCl
CH3
K2CO3
CH3COCH3
OH
O
O
6
7
5
X
H2, Raney Ni
H
N
N
NCO
H
O
NH2
X
O
O
8
9a-b
Scheme 2. Synthetic route to the target compounds 9a–b.
Table 1. Effects of compounds 2a–f, 3a–d, 4a–b, 9a–b and 10a–b as well as several reference PCOs on insulin secretion from rat pancreatic islets and
on the contractile activity of K+-depolarized rat aorta rings (results expressed as means SEM (n))
No
R1
R2
Residual insulin secretion %
Myorelaxant activity ED50 (lM)
(50 lM)
(10 lM)
2a
2b
CH3
CH2CH3
CH3
CH2CH3
CH2CH3
p ꢀ ClC6H5
p ꢀ ClC6H5
m ꢀ ClC6H5
m ꢀ ClC6H5
p ꢀ NitroC6H5
p ꢀ NitroC6H5
2-Thienyl
2-Thienyl
p ꢀ ClC6H5
p ꢀ ClC6H5
—
97.0 5.4 (24)
95.7 4.2 (16)
38.8 2.2 (16)
23.7 1.9 (15)
25.9 2.3 (22)
12.7 0.9 (15)
86.6 5.8 (15)
86.5 8.0 (16)
83.3 5.8 (14)
93.3 6.6 (15)
112.6 5.9 (15)
107.7 3.1 (16)
nd
nd
nd
>300 (4)
>300 (4)
2c
2d
90.8 3.8 (15)
72.6 4.1 (16)
79.5 3.3 (16)
64.1 3.2 (15)
nd
>30 (4)b
CH2CH3
CH3
>30 (4)b
2e
10.3 1.4 (4)
7.8 0.8 (4)
>30 (4)b
2f
3a
CH2CH3
CH3
3b
3c
CH2CH3
CH3
nd
nd
>30 (4)b
>30 (4)b
3d
4a
CH2CH3
CH3
nd
nd
>30 (4)b
>30 (4)b
4b
9a
CH2CH3
—
—
nd
>30 (4)b
63.6 4.6 (15)
45.5 2.5 (16)
34.6 1.9 (21)a
25.0 1.3 (24)a
71.7 2.8 (38)a
96.0 4.2 (20)a
94.7 4.3 (24)a
12.0 1.7 (4)
10.0 2.1 (5)
> 300 (4)a
>30 (4)a
23.8 2.4 (10)a
0.35 0.02 (11)a
0.13 0.01 (7)a
9b
10a
—
—
nd
nd
—
—
10b
—
—
nd
Diazoxide
Pinacidil
Cromakalim
—
—
27.9 1.5 (37)a
92.1 5.5 (21)a
77.2 4.3 (22)a
—
—
—
3–7
.
a Published results: Refs.
b Precipitates at 10ꢀ4 M.
for compound 2e, 14.1 lM for compound 2f and
19.7 lM for diazoxide, respectively.
Looking at the effect of compounds 9a–b on the insulin
releasing process, their potency was intermediate be-
tween that of the ring-opened arylureas 2c–f and the pre-
Ureas 2a and 2b with an ethyl group at R2 were inactive,
indicating that an aromatic group with an electron-with-
drawing substituent is more suitable than an aliphatic
group to promote a reduction of the insulin secretory
rate. It can also be observed that ureas with R1 = ethyl
were more potent than ureas bearing a methyl group
at R1. Lastly, and in contrast to most of their ring-closed
analogues,3,4 amide and sulfonylurea derivatives, 3a–d
and 4a–b, respectively, were poorly active (Table 1).
viously
described
ring-closed
6-halo-substituted
arylureas 10a–b.5 Interestingly, there was a progressive
improvement of the inhibitory activity on pancreatic
b-cells from the simplest methoxy compounds 2c and
2e to the ethoxy derivatives 2d and 2f, followed by the
non-substituted ring-closed compounds 9a–b, and final-
ly the 6-chloro-substituted ring-closed compounds 10a–
b (Fig. 3 and Table 1). In any case, the meta-chlorophe-
nylureas were found to be slightly more potent than the

S. Khelili et al. / Bioorg. Med. Chem. 16 (2008) 6124–6130
6127
X
X
X
X
H
N
H
N
H
N
H
N
N
N
N
N
H
H
H
H
O
O
O
O
Cl
O
O
O
O
9a: X = p-Cl
9b: X = m-Cl
10a: X = p-Cl
10b: X = m-Cl
2c: X = p-Cl
2e: X = m-Cl
2d: X = p-Cl
2f: X = m-Cl
Figure 3. From ring-opened to ring-closed benzopyran-type arylureas.
corresponding para-chlorophenylureas (Table 1). These
results further highlight the critical importance of a sub-
stituent (i.e., a halogen atom) at the 6-position of the
benzopyran ring.
displaying weaker vasorelaxant activities than the refer-
ence molecules.
However, and in contrast to the reference compounds
cromakalim and pinacidil, the biologically active ring-
opened arylurea derivatives did not express a clear-cut
tissue selectivity (pancreatic endocrine versus vascular
tissue). As a result, these new compounds are rather
comparable to the KATP channel opener diazoxide.
2.2.2. Myorelaxant effects on precontracted rat aorta
rings. The myorelaxant effects of compounds 2a–f, 3a–d,
4a–b and 9a–b were examined on rat aorta rings precon-
tracted by 30 mM KCl (Table 1). The new ring-opened
cromakalim analogues were less potent than cromaka-
lim and pinacidil at reducing the vascular tone. Except
for the m-chlorophenyl compounds 2e and 2f and for
compounds 9a–b, the vasorelaxant activity of these
new analogues was also less pronounced than that of
diazoxide. By comparing compounds 9a–b with com-
pounds 10a–b, it clearly appeared that the introduction
of a halogen atom at the 6-position of the benzopyran
ring decreased the myorelaxant activity and enhanced
the inhibitory effect on the insulin releasing process.
Moreover, the biological results obtained with several
ring-closed analogues devoid of any substituent at the
6-position of the benzopyran ring also highlight the crit-
ical importance of the presence of such a substituent
(i.e., a halogen atom) for activity and selectivity on pan-
creatic b-cells.
Since the chemical structure of arylureas such as 2f is
closely related to that of pinacidil (Fig. 4), another KATP
channel opener, these new compounds may also be re-
garded as structural analogues of N-alkyl-N0-aryl-N00-
cyanoguanidine KATP channel openers. It is tempting
to speculate that the new arylureas, like their ‘isostere’
pinacidil, could act as KATP channel openers, but with
a different affinity for the target tissues. The latter
assumption remains to be demonstrated by further
pharmacological investigations.
IC50/ED50 ratios have been determined to assess the
apparent tissue selectivity (pancreatic endocrine versus
vascular tissue) of the most active ring-opened cromaka-
lim analogues. The IC50/ED50 ratio was found to be
>0.63 for 2d, 2.14 for 2e and 1.80 for 2f, respectively.
Previous investigations indicated that cromakalim
exhibited an IC50/ED50 selectivity ratio above 769,
pinacidil above 285 and diazoxide around 1.5
In conclusion, we succeeded to develop novel ring-
opened analogues of cromakalim that are much more
3. Discussion and conclusion
The present results clearly revealed that the molecular
simplification of dihydrobenzopyrans, by opening the
pyran ring, removing the substituent on the benzene ring
and introducing arylurea moieties led to a new class of
compounds expressing identical biological effects than
their ring-closed analogues.2–4 The introduction on the
ring-opened analogues of alkylurea, arylamide or aryl-
sulfonylurea moieties failed to generate biologically ac-
tive compounds.
Cl
N
H
N
H
N
N
N
H
H
CN
O
N
O
Arylurea derivatives bearing an aromatic group with an
electron-withdrawing substituent at the m- or p-position
were shown to be much more potent than cromakalim
and pinacidil at reducing the insulin secretory rate whilst
2f
pinacidil
Figure 4. Chemical structure of 2f and pinacidil.

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S. Khelili et al. / Bioorg. Med. Chem. 16 (2008) 6124–6130
active on the pancreatic insulin-secreting tissue than the
reference molecules. Such data call for further structural
modification to identify original ring-opened analogues
of cromakalim with improved insulin-secreting cell
selectivity. Additional investigations are also required
to ascertain that the biological effects of these cromaka-
lim (or pinacidil)-derived compounds are related to the
activation of ATP-sensitive K+ channels.
(t, 1H, NHCH2CH3), 6.05 (t, 1H, C6H5CH2NH), 6.89
(t, 1H, CHarom), 6.96 (d, 1H, CHarom), 7.16 (d, 1H,
CHarom), 7.21 (t, 1H, CHarom). Calculated (%) for
C11H16N2O2: C, 63.44; H, 7.74; N, 13.45. Found (%):
C, 63.42; H, 7.73; N, 13.44.
4.1.3.2. 1-(2-Ethoxybenzyl)-3-ethylurea (2b). White
powder (93%); mp 130–131 °C; IR (KBr) m: 3320,
3120, 1540 (NH); 1635 (CO); 3040 (CHarom); 2970
1
(CHaliph) cmꢀ1. H NMR (d6-DMSO) d (ppm): 0.99 (t,
4. Experimental
3H, NHCH2CH3), 1,35 (t, 3H, OCH2CH3), 3.02 (q,
2H, NHCH2CH3), 4.03 (q, 2H, OCH2CH3), 4.15 (d,
2H, C6H5CH2NH), 5.93 (t, 1H, NHCH2CH3), 6.02 (t,
1H, C6H5CH2NH), 6.88 (t, 1H, CHarom), 6.93 (d, 1H,
CHarom), 7.14 (d, 1H, CHarom), 7.18 (t, 1H, CHarom).
Calculated (%) for C12H18N2O2: C, 64.84; H, 8.16; N,
12.60. Found (%): C, 64.85; H, 8.15; N, 12.59.
4.1. Chemistry, general procedures
Melting points were determined on a Buchi–Tottoli cap-
¨
illary apparatus and are uncorrected. IR spectra were re-
corded as KBr pellets on a Perkin-Elmer 1750 FT
spectrophotometer. The H NMR spectra were taken
1
on a Bruker (500 MHz) instrument in DMSO-d6 or in
CDCl3 with hexamethyldisiloxane (HMDS) as an inter-
nal standard. Chemical shifts are reported in d values
(ppm) relative to internal HMDS. The abbreviation
s = singlet, d = doublet, t = triplet, q = quadruplet,
m = multiplet, CHarom = aromatic CH, CHaliph = ali-
phatic CH, and b = broad are used throughout. Elemen-
tal analyses (C, H, N, S) were realized on a Carlo-Erba
EA 1108-elemental analyser and were within 0.4% of
theoretical values. All reactions were routinely checked
by TLC on silica gel Merck 60F 254.
4.1.3.3. 1-(2-Methoxybenzyl)-3-(4-chlorophenyl)urea
(2c). White powder (93%); mp 202–203 °C; IR (KBr)
m: 3350, 3280, 3200, 3170, 1540 (NH); 1640 (CO); 3025
1
(CHarom) cmꢀ1; 2835 (CHaliph). H NMR (d6-DMSO)
d (ppm): 3.83(s, 3H, OCH3), 4.24 (d, 2H, C6H5CH2NH),
6.46 (t, 1H, C6H5CH2NH), 6.91 (t, 1H, CHarom),
6.98 (d, 1H, CHarom), 7.22 (m, 2H, CHarom), 7.25 (d,
2H, CHarom), 7.41 (d, 2H, CHarom), 8.71 (s, 1H, p-
ClC6H5NH). Calculated (%) for C15H15ClN2O2: C,
61.97; H, 5.20; N, 9.64. Found (%): C, 62.00; H, 5.21;
N, 9.61.
4.1.1. General procedure for compounds 2a–f. An appro-
priate alkyl or aryl isocyanate (11 mmol; 1.1 equiv) was
added to a solution of 1a or 1b (10 mmol; 1 equiv) in
dichloromethane (20 mL). The mixture was stirred at
room temperature for two hours. The resulting white
precipitate was then filtered under vacuum and washed
with a small amount of diethyl ether. In the absence of
precipitate, the solvent was evaporated under reduced
pressure and the residue was washed with a small
amount of diethyl ether and dried.
4.1.3.4. 1-(2-Ethoxybenzyl)-3-(4-chlorophenyl)urea (2d).
White powder (92%); mp 196–197 °C; IR (KBr) m: 3330,
3160, 1520 (NH); 1635 (CO); 3040 (CHarom) cmꢀ1; 2960
1
(CHaliph). H NMR (d6-DMSO) d (ppm):1,33 (t, 3H,
OCH2CH3), 4.03 (q, 2H, OCH2CH3), 4.42 (d, 2H,
C6H5CH2NH), 6.46 (t, 1H, C6H5CH2NH), 6.90 (t, 1H,
CHarom), 6.97 (d, 1H, CHarom), 7.21 (m, 2H, CHarom),
7.26 (d, 2H, CHarom), 7.42 (d, 2H, CHarom), 8.90 (s,
1H, p-ClC6H5NH). Calculated (%) for C16H17ClN2O2:
C, 63.05; H, 5.62; N, 9.19. Found (%): C, 63.02; H,
5.59; N, 9.18.
4.1.2. General procedure for compounds 3a–d. An appro-
priate acyl chloride (11 mmol; 1.1 equiv) was added to a
solution of 1a or 1b (10 mmol; 1 equiv) in toluene
(20 mL). The mixture was refluxed for two hours and
then cooled. The white precipitate was filtered under
vacuum, washed with a small amount of diethyl ether
and dried.
4.1.3.5. 1-(2-Methoxybenzyl)-3-(3-chlorophenyl)urea
(2e). White powder (94%); mp 188–189 °C; IR (KBr)
m: 3330, 3280, 3160, 3120, 1535 (NH); 1640 (CO); 3015
1
(CHarom); 2835 (CHaliph) cmꢀ1. H NMR (d6-DMSO)
d
(ppm): 3.73 (s, 3H, OCH3), 4.42 (d, 2H,
C6H5CH2NH), 6.44 (t, 1H, C6H5CH2NH), 6.90 (t, 1H,
CHarom), 6.96 (d, 1H, CHarom), 7.21 (m, 2H, CHarom),
7.26 (d, 2H, CHarom), 7.42 (d, 1H, CHarom), 8.70 (s,
1H, p-ClC6H5NH). Calculated (%) for C15H15ClN2O2:
C, 61.97; H, 5.20; N, 9.64. Found (%): C, 62.01; H,
5.19; N, 9.62.
4.1.3. General procedure for compounds 4a and 4b. An
appropriate arylsulfonyl isocyanate (11 mmol; 1.1
equiv) was added to a solution of 1a or 1b (10 mmol;
1 equiv) in diethyl ether (20 mL). The mixture was stir-
red at room temperature for one hour. The resulting
white precipitate was then filtered under vacuum,
washed with a small amount of diethyl ether and dried.
4.1.3.6. 1-(2-Ethoxybenzyl)-3-(3-chlorophenyl)urea (2f).
White powder (92%); mp 174–178 °C; IR (KBr) m: 3330,
1520 (NH); 1640 (CO); 3040 (CHarom); 2970 (CHaliph
)
4.1.3.1. 1-(2-Methoxybenzyl)-3-ethylurea (2a). White
powder (92%); mp 138–140 °C; IR (KBr) m: 3340,
3320, 3120, 1525 (NH); 1635 (CO); 3010 (CHarom);
cmꢀ1 1H NMR (d6-DMSO) d (ppm): 1.33 (t, 3H,
.
OCH2CH3), 4.07 (q, 2H, OCH2CH3), 4.26 (d, 2H,
C6H5CH2NH), 6.45 (t, 1H, C6H5CH2NH), 6.89 (t, 1H,
CHarom), 6.92 (d, 1H, CHarom), 6.97 (d, 1H, CHarom),
7.17 (d, 1H, CHarom), 7.22 (m, 4H, CHarom), 8.78 (s,
1H, m-ClC6H5NH). Calculated (%) for C16H17ClN2O2:
2840 (CHaliph) cmꢀ1 1H NMR (d6-DMSO) d (ppm):
.
0.99 (t, 3H, NHCH2CH3), 3.02 (q, 2H, NHCH2CH3),
3.79 (s, 3H, OCH3), 4.15 (d, 2H, C6H5CH2NH), 5.89

S. Khelili et al. / Bioorg. Med. Chem. 16 (2008) 6124–6130
6129
C, 63.05; H, 5.62; N, 9.19. Found (%): C, 63.01; H, 5.60;
N, 9.20.
NMR (d6-DMSO) d (ppm): 1,35 (t, 3H, OCH2CH3); 4
(q, 2H, OCH2CH3); 4,15 (s, 2H, C6H5CH2NH); 7 (m,
4H, CHarom); 7,7 (d, 2H, CHarom); 7,9 (d, 2H, CHarom);
10,85 (s, 1H, SO2NH). Calculated (%) for
C16H17ClN2O4S: C, 52.10; H, 4.65; N, 7.60; S, 8.69.
Found (%): C, 52,07; H, 4,63; N, 7,55; S, 8,73.
4.1.3.7. N-(2-Methoxybenzyl)-4-nitrobenzamide (3a).
White powder (94%); mp 135–136 °C; IR (KBr) m: 3349
(NH); 1650 (CO); 3084 (CHarom); 2830 (CHaliph), 1450
1
(NO2) cmꢀ1. H NMR (d6-DMSO) d (ppm): 3.65 (s,
3H, OCH3), 4.48 (d, 2H, C6H5CH2NH), 6.91 (t, 1H,
CHarom), 7.08 (d, 1H, CHarom), 7.21 (d, 1H, CHarom),
7.25 (t, 1H, CHarom), 8.14 (d, 2H, CHarom), 8.32 (d,
2H, CHarom), 9.18 (t, 1H, C6H5CH2NH). Calculated
(%) for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79. Found
(%): C, 62.90; H, 4.92; N, 9.80.
4.1.4. Synthesis of the ring-closed analogues 9a and 9b
4.1.4.1. 3,4-Dihydro-2,2-dimethyl-2H-1-benzopyran-4-
one (6). A solution of 2-hydroxyacetophenone (1.76 mL,
14.7 mmol), acetone (3.62 mL, 49 mmol) and pyrroli-
dine (1.72 mL, 21 mmol) in methanol (20 mL) was stir-
red at 25 °C overnight. The mixture was then
concentrated to dryness by evaporation of the solvents
under reduced pressure. Water was added to the residue,
and the mixture was adjusted to pH 1 with concentrated
hydrochloric acid. The insoluble material was collected
by filtration and dissolved in a small volume of metha-
nol. The solution was treated with activated charcoal
and the filtrate was supplemented with water until pre-
cipitation of the title compound. The precipitate was
collected by filtration, washed with water and dried.
The product was purified by chromatography on silica
gel eluted with chloroform to give the title compound:
pale yellow powder (54%); mp 85–86 °C; IR (KBr) m:
4.1.3.8. N-(2-Ethoxybenzyl)-4-nitrobenzamide (3b).
White powder (95%); mp 130–132 °C; IR (KBr) m: 3330
(NH); 1645 (CO); 3083 (CHarom); 2886 (CHaliph), 1430
(NO2) cmꢀ1 1H NMR (d6-DMSO) d (ppm): 1.35 (t,
.
3H, OCH2CH3), 4.15 (q, 2H, OCH2CH3), 4.45 (d,
C6H5CH2NH), 6.89 (t, 1H, CHarom), 7.09 (d, 1H,
CHarom), 7.22 (d, 1H, CHarom), 7.27 (t, 1H, CHarom),
8.13 (d, 2H, CHarom), 8.3O (d, 2H, CHarom), 9.20 (t,
1H, C6H5CH2NH). Calculated (%) for C16H16N2O4:
C, 63.99; H, 5.37; N, 9.33. Found (%): C, 64.00; H,
5.38; N, 9.30.
1
2977, 2934 (CHaliph), 1687 (CO) cmꢀ1. H NMR (d6-
4.1.3.9. N-(2-Methoxybenzyl)thiophene-2-carboxam-
ide (3c). White powder (91%); mp 157–1159 °C; IR
(KBr) m: 3320 (NH); 1651 (CO); 3093 (CHarom); 2830
DMSO) d (ppm) : 1.39 (s, 6H, CH3), 2.79 (s, 2H,
CH2), 7.00 (m, 2H, CHarom), 7.55 (dd, 1H, CHarom),
7.72 (d, 1H, CHarom). Calculated ((%): C, 74.67; H, 6.96.
1
(CHaliph) cmꢀ1. H NMR (d6-DMSO) d (ppm): 3.81 (s,
3H, OCH3), 4.42 (d, C6H5CH2NH), 6.91(t, 1H,
CHarom), 6.99 (d, 1H, CHarom), 7.17 (m, 2H, CHarom),
7.24 (t, 1H, CHarom), 7.76 (d, 1H, CHarom), 7.85 (d,
1H, CHarom), 8.86 (t, 1H, C6H5CH2NH). Calculated
(%) for C13H13NO2S: C, 63.13; H, 5.30; N, 5.66; S,
12.97. Found (%): C, 63.10; H, 5.29; N, 5.65; S, 12.98.
4.1.4.2. 3,4-Dihydro-2,2-dimethyl-2H-1-benzopyran-4-
hydroxyimine (7). A mixture of 3,4-dihydro-2,2-di-
methyl-2H-1-benzopyran-4-one (6) (1 g, 5.68 mmol),
hydroxylamine hydrochloride (0.7934 g, 11.42 mmol),
potassium carbonate (1.587 g 11.48 mmol) and ethanol
(40 mL) was refluxed for 3 h. After completion of the
reaction, the mixture was poured onto crushed ice.
The precipitate was collected by filtration, washed with
water, and dried: white powder (92%); mp 123–125 °C
(litt. 121–123 °C8); IR (KBr) t: 3264 (NH), 2977, 2935
4.1.3.10. N-(2-Ethoxybenzyl)thiophene-2-carboxamide
(3d). White powder (95%); mp 133–135 °C; IR (KBr) m:
3323 (NH); 1667 (CO); 3098 (CHarom); 2892 (CHaliph),
1
1450 (NO2) cmꢀ1. H NMR (d6-DMSO) d (ppm): 1.35
(CHaliph), 1650 (CN) cmꢀ1 1H NMR (d6-DMSO) d
.
(t, 3H, OCH2CH3), 4.05(q, 2H, OCH2CH3), 4.43 (d,
C6H5CH2NH), 6.89 (t, 1H, CHarom), 6.97 (d, 1H,
CHarom), 7.16 (m, 2H, CHarom), 7.25 (t, 1H, CHarom),
7.75 (d, 1H, CHarom), 7.87 (d, 1H, CHarom), 8.92 (t,
1H, C6H5CH2NH). Calculated (%) for C14H15NO2S:
C, 64.34; H, 5.79; N, 5.36; S, 12.27. Found (%): C,
64.35; H, 5.80; N, 9.37; S, 12.28.
(ppm): 1.30 (s, 6H, CH3), 2.77 (s, 2H, CH2), 6.82 (d,
1H, CHarom), 6.89 (dd, 1H, CHarom), 7.24 (dd, 1H,
CHarom), 7.74 (d, 1H, CHarom), 11.19 (s, 1H, @N–
OH). Calculated (%) for C11H13NO2: C, 69.09; H,
6.85; N, 7.32. Found (%): C, 69.40; H, 6.77; N, 7.34.
4.1.4.3. R/S-4-Amino-3,4-dihydro-2,2-dimethyl-2H-
1-benzopyran (8). A mixture of 3,4-dihydro-2,2-di-
methyl-2H-1-benzopyran-4-hydroxyimine (7) (10 g,
52.29 mmol), Raney Ni (2 g) and methanol (130 mL)
was stirred under hydrogen pressure (5bar) at 25 °C
overnight. The catalyst was filtered off, washed with
methanol and the filtrate was evaporated under reduced
pressure. The oily residue was purified by chromatogra-
phy on silica gel eluted with chloroform and ethyl ace-
tate (15:5 v/v) to give the title compound as a
colourless oil (90%). For analytical purposes, the hydro-
chloride was obtained as a white powder; mp 268 °C
4.1.3.11. 1-(2-Methoxybenzyl)-3-(4-chlorophenyl)sul-
fonylurea (4a). White powder (90%); mp 163–164 °C; m
(NH) = 3340, 3310, 3180, 3120, 1555; m (CO) =ł1655,
1700;
m
(SO2-N) = 1345;
m
(SO2) = 1160, 1245,
1
1280 cmꢀ1. H NMR (d6-DMSO) d (ppm): 3,75 (s, 3H,
OCH3); 4,1 (s, 2H, C6H5CH2NH); 7 (m, 4H, CHarom);
7,8 (m, 4H, CHarom); 10,75 (s, 1H, SO2NH). Calculated
(%) for C15H15ClN2O4S: C, 50.78; H, 4.26; N, 7.90; S,
9.04. Found (%): C, 50,75; H, 4,25; N, 7,92; S, 9,07.
1
4.1.3.12. 1-(2-Ethoxybenzyl)-3-(4-chlorophenyl)sulfo-
nylurea (4b). White powder (90%); mp 142–143 °C; m
(NH) = 3340, 3310, 3110, 1550; m (CO) = 1665, 1700; m
(dec); IR (KBr) t: 3044, 2978, 2894 (NH+) cmꢀ1. H
NMR (d6-DMSO) d (ppm): 1.21 (s, 3H, CH3), 1.42
(s, 3H, CH3), 1.81 (t, 1H, CH2), 2.32 (m, 1H, CH2),
4.49 (m, 1H, 4-H), 6.80 (d, 1H, CHarom ), 6.96 (dd,
(SO2-N) = 1345; m (SO2) = 1160, 1240, 1285 cmꢀ1 1H
.

6130
S. Khelili et al. / Bioorg. Med. Chem. 16 (2008) 6124–6130
1H, CHarom), 7.24 (dd, 1H, CHarom), 7.69 (d, 1H, CHar-
om), 8.80 (bs, ꢁ3H, ꢀNH3þ). Calculated (%) for
C11H15NO.HCl: C, 61.82; H, 7.55; N, 6.55. Found
(%): C, 61.52; H, 7.59; N, 6.63.
the reference compound or the ring-opened cromakalim
derivative. The release of insulin was measured radioim-
munologically using rat insulin as a standard9. Residual
insulin release was expressed as a percentage of the value
recorded in control experiment (100%), that is, in the ab-
sence of drug and presence of 16.7 mM glucose.
4.1.4.4. R/S-4-(4-Chlorophenylaminocarbonylamino)-
3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (9a). 4-Chlo-
rophenyl isocyanate (413 lL, 3.38 mmol) was added to
a solution of 8 (500 mg, 2.82 mmol) in methylene chlo-
ride (5 mL). After completion of the reaction, the result-
ing precipitate was collected by filtration, washed with
n-hexane, and dried: white powder (93%); mp 214–
216 °C; IR (KBr) t: 3328 (NH), 2978, 2929 (CHaliph),
4.3. Rat aorta rings
All experiments were performed on aorta removed from
fed Wistar rats (180–220 g), as previously described.2,6,9
The ED50 was assessed for each dose–response curve as
the concentration evoking 50% inhibition of the plateau
phase induced by KCl 30 mM. Results are expressed as
mean values ( SEM).
1
1633 (CO) cmꢀ1. H NMR (d6-DMSO) d (ppm): 1.27
(s, 3H, CH3), 1.38 (s, 3H, CH3), 1.73 (t, 1H, CH2),
2.11 (m, 1H, CH2), 4.96 (m, 1H, 4-H), 6.53 (d, 1H, –
NH–CO–NH–C6H4Cl), 6.73 (d, 1H, CHarom), 6.87
(dd, 1H, CHarom), 7.13 (dd, 1H, CHarom), 7.25 (d, 1H,
CHarom), 7.28 (d, 2H, CHarom), 7.46 (d, 2H, CHarom),
8.63 (s, 1H, –NH–CO–NH–C6H4Cl). Calculated (%)
for C18H19ClN2O2: C, 65.35; H, 5.79; N, 8.47. Found
(%): C, 65.13; H, 5.76; N, 8.43.
Acknowledgments
`
This work was supported in part by ‘Le Ministere de
l’Enseignement Superieur et de la Recherche Scientifi-
´
que’ of Algeria and by the National Fund for Scientific
Research (Belgium) from which P.L. is a Research
Director, P. de T. a Senior Research Associate and
X.F. a F.R.I.A. Researcher. The technical assistance
of S. Counerotte, F. Leleux, A.M. Vanbellinghen and
A. Van Praet is gratefully acknowledged.
4.1.4.5. R/S-4-(3-Chlorophenylaminocarbonylamino)-
3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (9b). The title
compound was obtained as described for 9a starting
from 8 (500 mg, 2.82 mmol) and 3-cyanophenyl isocya-
nate (413 ll, 3.38 mmol): white powder (92%); mp
193–195 °C; IR (KBr) t: 3322 (NH), 2980, 2928 (CHa-
1
liph), 1631 (CO) cmꢀ1. H NMR (d6-DMSO) d (ppm):
References and notes
1.27 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.75 (t, 1H,
CH2), 2.12 (m, 1H, CH2), 4.96 (m, 1H, 4-H), 6.59 (d,
1H, –NH–CO–NH–C6H4Cl), 6.73 (d, 1H, CHarom),
6.87 (dd, 1H, CHarom), 6.96 (d, 1H, CHarom), 7.13 (dd,
1H, CHarom), 7.24 (m, 3H, CHarom), 7.73 (s, 1H, CHar-
om), 8.71 (s, 1H, –NH–CO–NH–C6H4Cl). Calculated
(%) for C18H19ClN2O2: C, 65.35; H, 5.79; N, 8.47.
Found (%): C, 65.39; H, 5.89; N, 8.53.
1. Mannhold, R. Curr. Top. Med. Chem. 2006, 6, 1031.
2. Khelili, S.; Nguyen, Q.-A.; Lebrun, P.; Delarge, J.; Pirotte,
B. Pharm. Pharmacol. Commun. 1999, 5, 189.
3. Sebille, S.; de Tullio, P.; Becker, B.; Antoine, M.-H.;
Boverie, S.; Pirotte, B.; Lebrun, P. J. Med. Chem. 2005, 48,
614.
4. Khelili, S.; Lebrun, P.; de Tullio, P.; Pirotte, B. Bioorg.
Med. Chem. 2006, 14, 3530.
5. Sebille, S.; Gall, D.; de Tullio, P.; Florence, X.; Lebrun, P.;
Pirotte, B. J. Med. Chem. 2006, 49, 4690.
4.2. Rat pancreatic b-cells
6. Becker, B.; Antoine, M.-H.; Nguyen, Q.-A.; Rigo, B.;
Cosgrove, K. E.; Barnes, P. D.; Dunne, M. J.; Pirotte, B.;
Lebrun, P. Br. J. Pharmacol. 2001, 134, 375.
7. 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.
8. Sebok, P.; Levai, A.; Timar, T. Heterocyclic Commun. 1998,
4, 547.
9. Lebrun, P.; Arkhammar, P.; Antoine, M.-H.; Nguyen,
Q.-A.; Bondo Hansen, J.; Pirotte, B. Diabetologia 2000,
43, 723.
Pancreatic islets were isolated by collagenase method
from fed Wistar rats (180–220 g). Groups of 10 islets,
each derived from the same batch of islets, were preincu-
bated for 30 min at 37 °C in 1 mL of a physiological salt
medium (in mM: NaCl 115, KCl 5, CaCl2 2.56, MgCl2 1,
NaHCO3 24) supplemented with 2.8 mM glucose, 0.5%
(w/v) dialyzed albumin (Sigma) and equilibrated against
a mixture of O2 (95%) and CO2 (5%). The islets were
then incubated at 37 °C for 90 min in 1 mL of the same
medium containing 16.7 mM glucose and, in addition,