Design and synthesis of new potassium channel activators derived from the ring opening of diazoxide: Study of their vasodilatory effect, stimulation of elastin synthesis and inhibitory effect on insulin release

Article abstract of DOI:10.1016/j.bmc.2015.02.043

Benzenesulfonylureas and benzenesulfonylthioureas, as well as benzenecarbonylureas and benzenecarbonylthioureas, were prepared and evaluated as myorelaxants on 30 mM KCl-precontracted rat aortic rings. The most active compounds were further examined as stimulators of elastin synthesis by vascular smooth muscle cells and as inhibitors of insulin release from pancreaticβ-cells. The drugs were also characterized for their effects on glycaemia in rats. Benzenesulfonylureas and benzenesulfonylthioureas did not display any myorelaxant activity on precontracted rat aortic rings. Such an effect could be attributed to their ionization at physiological pH. By contrast, almost all benzenecarbonylureas and benzenecarbonylthioureas displayed a myorelaxant activity, in particular the benzenecarbonylureas with an oxybenzyl group linked to the ortho position of the phenyl ring. The vasodilatory activity of the most active compounds was reduced when measured in the presence of 80 mM KCl or in the presence of 30 mM KCl and 10 μM glibenclamide. Such results suggested the involvement, at least in part, of K_ATP channels. Preservation of a vasodilatory activity in rat aortic rings without endothelium indicated that the site of action of such molecules was located on the vascular smooth muscle cells and not on the endothelial cells. Some of the most active compounds also stimulated elastin synthesis by vascular smooth muscle cells. Lastly, most of the active vasorelaxant drugs, except 15k and 15t at high concentrations, did not exhibit marked inhibitory effects on the insulin releasing process and on glycaemia, suggesting a relative tissue selectivity of some of these compounds for the vascular smooth muscle.

Full text of DOI:10.1016/j.bmc.2015.02.043

Bioorganic & Medicinal Chemistry 23 (2015) 1735–1746
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of new potassium channel activators derived
from the ring opening of diazoxide: Study of their vasodilatory effect,
stimulation of elastin synthesis and inhibitory effect on insulin
release
Nafila Bouider a, Wassim Fhayli b, Zeinab Ghandour b, Marjorie Boyer b, Kamel Harrouche a,
Xavier Florence c, Bernard Pirotte d, , Philippe Lebrun c, Gilles Faury b, Smail Khelili a
⇑
a Laboratoire de Phytochimie et de Pharmacologie, Département de Chimie, Faculté des Sciences Exactes et informatique, Université de Jijel, B.P. 98 Ouled Aissa, 18000 Jijel, Algeria
b Laboratoire ‘Hypoxie: Physiopathologie Cardiovasculaire et Respiratoire’ (HP2), INSERM U1042-Université Grenoble Alpes, F-38042 La Tronche, France
c Laboratoire de Pharmacodynamie et de Thérapeutique, Université Libre de Bruxelles, Faculté de Médecine, 808, Route de Lennik, B-1070 Bruxelles, Belgium
d Laboratoire de Chimie Pharmaceutique, Center for Interdisciplinary Research on Medicines (CIRM), Université de Liège, 1, Avenue de l’Hôpital, B-4000 Liège, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzenesulfonylureas and benzenesulfonylthioureas, as well as benzenecarbonylureas and benzenecar-
bonylthioureas, were prepared and evaluated as myorelaxants on 30 mM KCl-precontracted rat aortic
rings. The most active compounds were further examined as stimulators of elastin synthesis by vascular
smooth muscle cells and as inhibitors of insulin release from pancreaticb-cells. The drugs were also
characterized for their effects on glycaemia in rats. Benzenesulfonylureas and benzenesulfonylthioureas
did not display any myorelaxant activity on precontracted rat aortic rings. Such an effect could be
attributed to their ionization at physiological pH. By contrast, almost all benzenecarbonylureas and ben-
zenecarbonylthioureas displayed a myorelaxant activity, in particular the benzenecarbonylureas with an
oxybenzyl group linked to the ortho position of the phenyl ring. The vasodilatory activity of the most
active compounds was reduced when measured in the presence of 80 mM KCl or in the presence of
Received 19 December 2014
Revised 19 February 2015
Accepted 20 February 2015
Available online 27 February 2015
Keywords:
Potassium channel opener
Diazoxide
Benzenesulfonylurea
Benzenesulfonylthiourea
Benzenecarbonylurea
Benzenecarbonylthiourea
Elastin synthesis
30 mM KCl and 10 lM glibenclamide. Such results suggested the involvement, at least in part, of KATP
channels. Preservation of a vasodilatory activity in rat aortic rings without endothelium indicated that
the site of action of such molecules was located on the vascular smooth muscle cells and not on the
endothelial cells. Some of the most active compounds also stimulated elastin synthesis by vascular
smooth muscle cells. Lastly, most of the active vasorelaxant drugs, except 15k and 15t at high concentra-
tions, did not exhibit marked inhibitory effects on the insulin releasing process and on glycaemia,
suggesting a relative tissue selectivity of some of these compounds for the vascular smooth muscle.
Ó 2015 Elsevier Ltd. All rights reserved.
Myorelaxant activity
Insulin secretion
Glycaemia
1. Introduction
brain under metabolic stress.6 Channel inhibition by ATP and stim-
ulation by nucleotide diphosphates allow the membrane potential
ATP-sensitive potassium (KATP
)
channels control insulin
and cell excitability to be regulated by the cellular metabolic state.
The KATP channel is an octameric complex of 4 Kir6.x and 4 SURx
subunits.7,8 The pore-forming Kir6.x subunit belongs to the
inwardly rectifying family of potassium channels.8–10 There are
two isoforms: Kir6.1, which is found in vascular smooth muscle,
and Kir6.2, which has a widespread tissue distribution. Binding
of ATP to Kir6.x induces KATP channel closure. The sulfonylurea
receptor (SUR) belongs to the ABC transporter family.9 It functions
as a regulatory subunit, which mediates, inter alia, channel inhibi-
tion by sulfonylurea drugs such as glibenclamide.11,12 Differences
in endogenous KATP channel properties and pharmacology13 may
release,1,2 vascular tone3 and may protect heart,4 kidney,5 and
Abbreviations: KATP channel, ATP-sensitive potassium channel; Kir, inwardly
rectifying potassium channel; SUR, sulfonylurea receptor; PCO, potassium channel
opener; ABC transporter, ATP-binding cassette transporter; DMF, dimethylfor-
mamide; d6-DMSO, deuterated dimethyl sulfoxide; TMS, tetramethylsilane; NMR,
nuclear magnetic resonance; TLC, thin layer chromatography; DMEM, Dulbecco’s
modified Eagle medium; FCS, fetal calf serum; HRP, horseradish peroxidase; PBS,
phosphate-buffered saline; BSA, bovine serum albumin; TMB, 3,30,5,50-
tetramethylbenzidine.
⇑
Corresponding author. Tel.: +32 4 366 43 65; fax: +32 4 366 43 62.
E-mail address: b.pirotte@ulg.ac.be (B. Pirotte).
http://dx.doi.org/10.1016/j.bmc.2015.02.043
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

1736
N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
be accounted for by differences in subunit composition: Kir6.2 and
SUR1 in pancreatic insulin-secreting cells, Kir6.2 and SUR2A in car-
diac myocytes, Kir6.2/Kir6.1 and SUR2B in smooth muscle cells,
and Kir6.2 and SUR1 in central neurons.14
Several drugs were found to activate the KATP channels. Such
pharmacological or therapeutic agents, collectively termed potas-
sium channel openers (PCOs), stabilize membrane excitability
and preserve metabolic expenditure.15 As a result, PCOs are able
to inhibit insulin release from pancreatic b-cells16 and exert a
relaxant effect on different smooth muscles.17
non-classical isoster C@O, while NH was replaced by an oxygen
atom. Such structural modifications led to two new series of
ring-opened analogues of diazoxide and 3-alkylamino-4H-1,2,4-
benzothiadiazine 1,1-dioxides (series A and B), as illustrated in
Figure 3.
This strategy was inspired by previous work consisting in the
opening of the pyranic ring of cromakalim, a dihydrobenzopyran,
and the introduction of urea and sulfonylurea groups, which pro-
vided simpler new molecules with substantial vasodilatory
properties.27
Potassium channel openers constitute a family of chemically
diverse compounds that belong to numerous structural classes.
They include benzopyrans (cromakalim, 1), cyanoguanidines
(pinacidil, 2), benzothiadiazines (diazoxide, 3), nicotinamides
(nicorandil, 4), and pyrimidines (minoxidil, 5) (Fig. 1).16,18–20
Cromakalim is a potent myorelaxant but a poor inhibitor of
insulin secretion,21–23 in contrast to diazoxide, which is active on
both insulin-secreting cells (inhibitor) and vascular smooth muscle
cells (vasodilator).24 Optimization of the diazoxide structure pro-
vided compounds such as BPDZ 73 (6) and BPDZ 44 (7), which
potently and selectively activated the KATP channels of pancreatic
b-cells (Fig. 2).25,26
Modification of the benzothiadiazine core structure by ring
opening has never been attempted so far. Thus, in order to develop
new PCOs with improved pharmacological properties, we under-
took the synthesis of some simplified compounds resulting both
from the opening of the thiadiazine ring of 3-alkylamino-4H-
1,2,4-benzothiadiazine 1,1-dioxides and the introduction of urea
or thiourea moieties. The SO2 group has also been replaced by its
It should be pointed out that the A and B series can also be vir-
tually obtained by simplification of glibenclamide, a well-known
KATP channel blocker (Fig. 4).28 However, this observation is only
fortuitous and did not direct our strategy for the development of
new series of compounds expected to be potassium channel open-
ers and not blockers.
The present work reports the synthesis and pharmacological
testing of the vasodilatory activity of compounds belonging to ser-
ies A and B on KCl-precontracted rat aorta rings. Some of the most
potent compounds have also been tested as potential stimulators
of elastin production by vascular smooth muscle cells.
Elastin is the main extracellular matrix protein composing the
elastic fibers, which endow extensible tissues (including skin,
Y
O
O
O
N
O
x
S
R2
x
S
ring opening
N
H
N
H
R
NH
R1
N
H
N
H
isosteric
replacement
H
N
H
N
CH3
O
CH3
CH3
N
O
N
(NH
O)
OH
N
N
CH3
Y
CH3
O
Y
isosteric
O
O
N
replacement
CH3
x
R2
x
S
R2
N
H
N
H
(SO2
CO)
N
H
N
H
Cromakalim (1)
Pinacidil (2)
O
O
O
O
R1
Y= S or O
Cl
S
R1
O-
N
H
N
Series B
Series A
N
N+
N
H
CH3
O
O
Figure 3. Simplification of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides
leading to two new series of compounds (A and B).
O
Nicorandil (4)
Diazoxide (3)
O
N
O
O
S
O
O
N
N
H
N
Cl
H
N
H
H2N
N+
O-
NH2
Glibenclamide
Minoxidil (5)
CH3
Figure 1. Chemical structure of some KATP channels openers.
Removal of
NHCH2CH2C6H4SO2
O
O
N
O
O
N
O
O
O
O
O
Cl
S
S
Replacement of
C=O by SO2
x
x
R2
S
R2
CH3
CH3
N
CH3
N
H
N
H
N
N
H
CH3
H
N
H
N
H
N
H
N
H
O
O
CH3
R1
R1
(6)
(7)
Series B
Series A
Figure 2. Examples of KATP channel openers resulting from structural modifications
of diazoxide.
Figure 4. Structural modulation of glibenclamide leading to series A and B.

N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
1737
lungs and arteries) with elasticity. Elasticity is the essential
mechanical property that allows large arteries to smoothly
transform the pulsatile and discrete blood volumes ejected during
the cardiac cycle into a continuous flow, therefore leading to
appropriate hemodynamics. The elastin gene is only expressed
during the fœtal life and childhood, and elastin is therefore not
replaced and degraded regularly during adulthood and ageing,
leading to a progressive stiffening of the arteries and subsequent
cardiovascular dysfunction.29 Also, abnormal arterial morphogene-
sis, remodelling and function—potentially leading to death—have
been linked to
a genetic heterozygous deficiency in elastin
characterized by low levels of the protein, such as in supravalvular
aortic stenosis (SVAS) or Williams syndrome.30–32 In normal ageing
and the above-cited syndromes, the patients would therefore
benefit from an up regulation of the elastin gene, which would
increase the deficient of elastin content of the arteries and improve
the arterial function.
It has already been demonstrated that the potassium channel
opener minoxidil increases the expression of the elastin gene,
in vitro in skin fibroblasts and vascular smooth muscle cells33,34
and in vivo in rats, leading to an increase in the arterial content
in elastin.35 A similar effect of another potassium channel opener,
nicorandil, has also been demonstrated.36 More recently, it has
been demonstrated that minoxidil, diazoxide, and pinacidil
(PCOs) increased mRNAs encoding proteins and enzymes involved
in elastic fiber formation, whereas glibenclamide had the opposite
effect.37 For these reasons, the investigation of the potential
overexpression of elastin in vascular smooth muscle cells by appli-
cation of the newly synthesized potassium channel openers
described here is of high interest.
Scheme 1. Reagents: (i) K2CO3, R1X, acetone; (ii) HClO3S; (iii) NH4OH, dioxane; (iv)
NaH, R2NCO or R2NCS, DMF.
Scheme 2. Reagents: (i) NaOH, R1X, EtOH; (ii) NaH, R2NCO or R2NCS, DMF.
A series of experiments were also conducted in order to verify
that the biological effects of series A and B compounds were
mediated by KATP channel activation. Selected compounds were
also examined on pancreatic b-cells as putative inhibitors of
insulin secretion, reflecting their KATP channel opening activity on
the pancreatic endocrine tissue, and their effects on glycaemia
were measured. Finally, the ionization constant (pKa) of selected
compounds was determined in order to collect information about
the prominent form of the compounds in physiological media.
elemental analyses to obtain the final materials with the chemical
purity required prior to pharmacological evaluation.
3. Results and discussion
Compounds 12a–o and 15a–u were evaluated for their ability
to relax rat aortic rings precontracted with 30 mM KCl (Table 1).
Diazoxide was used as a reference vasodilator and PCO. On the vas-
cular tissue, diazoxide provoked a marked myorelaxant activity
(EC50 = 21.6 lM).
2. Chemistry
As observed in Table 1, all the sulfonyl(thio)ureas 12 were
found to be inactive on rat aorta rings as vasodilators (EC50
The synthetic pathway to sulfonylureas and sulfonylthioureas
(A) is described in Scheme 1. The compounds were obtained from
the appropriate para-halophenols in four successive steps. Firstly,
an appropriate para-halophenol (8a,b) was alkylated by means of
the appropriate alkyl halide in the presence of potassium
carbonate to provide the 1-halo-4-alkoxybenzenes 9a–c. The ether
intermediates 9a–c were then treated with chlorosulfonic acid in
chloroform, at 0 °C, leading to benzenesulfonyl chlorides 10a–c.
The latters were converted into benzenesulfonamides 11a–c after
reaction with concentrated ammonium hydroxide. Finally, sulfon-
amides 11, after their conversion to the corresponding sodium
salts, provided the final sulfonylureas and sulfonylthioureas 12a–o
by reaction with the appropriate isocyanate (R2N@C@O) or
isothiocyanate (R2N@C@S).
The synthetic route used to prepare carbonylureas and
carbonylthioureas (B) involved two steps (Scheme 2). Starting from
2-hydroxybenzamide, 2-alkoxybenzamides 14a–c were prepared
by reaction with an appropriate alkyl halide in ethanol in the
presence of sodium hydroxide. Treatment of intermediates 14a–c
with sodium hydride, then with an appropriate isocyanate or
isothiocyanate, yielded the final compounds 15a–u.
>300 lM) while most compounds of the second series (car-
boxy(thio)ureas 15) exhibited a substantial vasorelaxant effect,
some of them being equipotent or even more potent than diazox-
ide (such as the most active compound 15k, EC50 = 12.2 lM). It can
also be observed, with the carboxyureas, that the preferred R1
chain for myorelaxant activity was the benzyl chain [see for exam-
ple 15h (EC50 = 16.7 lM), 15j (EC50 = 17.3 lM)]. The car-
boxythioureas analogues were generally found to be equipotent
to their corresponding ureas and some of them reached or slightly
overtook the activity of diazoxide, that is 15n, 15o and 15t, with
EC50 = 15.2 lM, 18.6 lM and 16.5 lM, respectively.
The inactivity of series A compounds (12a–o) could be attribu-
ted to their acidic character, as suggested by their pKa values
(between 4.5 and 5.7; see Table 2). At physiological pH, the nitro-
gen atom between the SO2 and the C@O groups in the A series of
molecules is expected to be mainly ionized, while the analogue
nitrogen atom in the B series (15a–u) is probably not ionized even
at pH = 10. This feature indicates that the presence of a non-ion-
ized NH group linked to the C@O function in the B series could
be essential for the biological activity.
In order to verify whether the vasorelaxant effects were
mediated by KATP channel activation, additional experiments were
performed on: (i) 30 mM K+-depolarized rat aorta rings in the
All compounds from series A (12a–o) and B (15a–u) were pur-
ified by crystallization and characterized by IR, 1H NMR and

1738
N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
Table 1
As shown in Table 3, the presence of glibenclamide in the bath-
ing medium provoked a noticeable increase in the EC50 value of the
selected compounds, suggesting the involvement of KATP channels
in the vasodilatory activity of these molecules. It must be noted
that the glibenclamide-induced increases in the EC50 values were
lower for the B series compounds (about 2 fold) than for diazoxide
(about 8 fold), suggesting that KATP channels only partly mediate
the vasorelaxation.
Moreover, the preservation of myorelaxant effects together
with the increases in the EC50 values recorded on rat aorta rings
precontracted by 80 mM KCl were also indicative for a non-exclu-
sive involvement of KATP channels in the vasorelaxant response.
This was illustrated by the concentration-dependent rightward
Effect of compounds from series A and B on the contractile activity of K+-depolarized
rat aorta rings
R2
HN
X
Z
N
H
Y
R1
O
Compd
X
Z
R1
R2
Y
Vasorelaxant
activity EC50 (lM)
a
12a
12b
12c
12d
12e
12f
12g
12h
12i
Cl SO2 CH3
Cl SO2 CH3
Cl SO2 CH3
Cl SO2 CH3
Cl SO2 CH3
Cl SO2 CH3
Cl SO2 CH3
CH(CH3)2
C6H11
C6H5
p-F-C6H4
C2H5
CH(CH3)2
CH2C6H5
CH(CH3)2
CH(CH3)2
CH(CH3)2
C6H11
O
O
O
O
S
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>300 (6)
>100 (5)
91.4 24.1 (4)
31.8 1.4 (5)
35.9 1.4 (6)
26.7 5.9 (4)
58.2 11.2 (6)
59.6 1.9 (5)
16.7 2.2 (3)
26.8 3.5 (3)
17.3 0.5 (5)
12.2 1.8 (6)
33.8 6.1 (5)
21.1 2.7 (6)
15.2 2.3 (5)
18.6 2.3 (6)
36.6 2.1 (5)
34.5 3.8 (9)
47.2 12.0 (3)
71.1 4.7 (5)
16.5 2.0 (6)
>100 (5)
S
S
Table 3
Myorelaxant effect of selected compounds on 30 mM KCl-precontracted rat aorta
F
F
SO2 CH3
SO2 CH3
O
S
O
O
O
S
rings in the absence or presence of 10
precontracted rat aorta rings
l
M glibenclamide, as well as on 80 mM KCl-
12j
12k
12l
Cl SO2 CH2CH3
Cl SO2 CH2CH3
Cl SO2 CH2CH3
Cl SO2 CH2CH3
Cl SO2 CH2CH3
Cl SO2 CH2CH3
a
Compound
KCl 30 mM
KCl 30 mM + Glib. 10
l
M
KCl 80 mM EC50
M)
C6H5
a
a
EC50
(lM)
EC50
(l
M)
(l
12m
12n
12o
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
15p
15q
15r
15s
15t
15u
Diazoxide
CH(CH3)2
CH2C6H5
p-F-C6H4
C2H5
CH(CH3)2
CH(CH3)CH2CH3
C6H11
15h
15k
15n
15o
15t
Diazoxide
16.7 2.2 (3)
12.2 1.8 (6)
15.2 2.3 (5)
18.6 2.3 (6)
16.5 2.0 (6)
19.5 2.7 (6)b
38.0 3.6⁄ (6)
20.2 2.3⁄ (7)
28.2 6.0 (5)
28.1 4.3 (4)
64.9 14.7⁄⁄ (3)
163.4 41.2 (6)
36.6 3.8$ (6)
45.6 6.6⁄⁄ (5)
57.1 4.6$ (5)
38.1 2.8⁄⁄ (4)
121.0 10.9$ (6)
>300 (6)b
S
S
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C@O CH3
C@O CH3
C@O CH3
C@O CH3
C@O CH3
O
O
O
O
O
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
S
b
C6H5
P <0.05 was considered significant. ⁄p <0.05; ⁄⁄p <0.01; $p <0.001.
C@O CH2CH2CH3 C6H11
C@O CH2CH2CH3 C6H5
a
EC50 is the drug concentration (lM) giving 50% relaxation of the 30 or 80 mM
KCl-induced contraction (mean sem). Number in parentheses refers to the num-
C@O CH2C6H5
C@O CH2C6H5
C@O CH2C6H5
C@O CH2C6H5
C@O CH2C6H5
C@O CH2C6H5
C@O CH3
C@O CH3
C@O CH3
C@O CH3
C@O CH3
C2H5
CH(CH3)2
C5H11
CH(CH3)CH2CH3
C6H11
CH2C6H5
C2H5
CH(CH3)2
CH(CH3)CH2CH3
C6H5
ber of individual experiments performed in each group. Glib: glibenclamide.
b
Published results (Ref. 46). Statistical analysis was performed comparing the
EC50 in the presence of glibenclamide and at KCl 80 mM versus EC50 at KCl 30 mM.
100
KCl 30mM (n=6)
KCl 30mM+ glib 10µM (n=7)
KCl 80mM (n=5)
80
CH2C6H5
C@O CH2CH2CH3 CH2C6H5
C@O CH2CH2CH3 C6H5
C@O CH2C6H5
60
CH2C6H5
40
20
0
21.6 3.0 (9)
a
EC50 is the drug concentration (lM) giving 50% relaxation of the 30 mM KCl-
induced contraction. Results are expressed as means sem; n refers to the number
of experiments.
-7
-6
-5
-4
-3
Log [15k]M
Figure 5. Concentration-response curve of compound 15k on 30 mM KCl-induced
rat aorta rings, in the absence or presence of 10 M glibenclamide (glib), and
Table 2
l
Ionization constants of some compounds of series A
80 mM KCl-precontracted rat aorta rings. Data are expressed as mean sem of (n)
rat aorta rings.
a
Compounds
X
Z
R1
R2
Y
pKa
12a
12b
12c
12f
Cl
Cl
Cl
Cl
F
SO2
SO2
SO2
SO2
SO2
CH3
CH3
CH3
CH3
CH3
CH(CH3)2
C6H11
C6H5
CH(CH3)2
CH(CH3)2
O
O
O
S
5.65
5.46
4.50
5.74
5.52
Table 4
Myorelaxant effect of selected compounds on 30 mM KCl-precontracted rat aorta
rings with and without endothelium
12h
O
a
The pKa values for series B compounds could not be determined because all UV–
visible spectra were identical in this series at all tested pH values.
Compound
Rat aorta rings with
Rat aorta rings without
endothelium EC50 (lM)
a
a
endothelium EC50
(
lM)
15h
15k
15n
15o
15t
16.7 2.2 (3)
12.2 1.8 (6)
15.2 2.3 (5)
18.6 2.3 (6)
16.5 2.0 (6)
17.2 2.6 (6)
15.3 1.7 (5)
17.4 1.9 (4)
15.7 1.2 (5)
12.0 0.5 (5)
continuous presence of 10 lM glibenclamide, a specific blocker of
KATP channels, and (ii) 80 mM K+-depolarized rat aorta rings; two
experimental conditions in which the vasodilator activity of KATP
channel activators is known to be suppressed or markedly
decreased.38
a
EC50 is the drug concentration (lM) giving 50% relaxation of the 30 mM KCl-
induced contraction (mean sem). Number in parentheses refers to the number of
individual experiments performed in each group.

N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
1739
Table 5
Effects of compounds 15h, 15k, 15n, 15o, 15t and diazoxide on insulin secretion from rat pancreatic islets [mean sem (n)]
Compd
X
Z
R1
R2
Y
Residual insulin secretion (%) mean sem (n)
50 10
lM
lM
15h
15k
15n
15o
15t
Diazoxide
H
H
H
H
H
—
C@O
C@O
C@O
C@O
C@O
—
CH2C6H5
CH2C6H5
CH3
CH3
CH2CH2CH3
—
C2H5
CH(CH3)CH2CH3
C2H5
CH(CH3)2
C6H5
—
O
O
S
S
S
113.3 5.8 (24)
21.4 1.9 (22)
99.6 4.8 (24)
104.7 5.4 (24)
44.9 2.1 (23)
28.8 2.4 (21)a
110.2 5.3 (23)
90.1 5.1 (23)
94.1 6.3 (22)
90.9 3.6 (23)
83.8 3.5 (23)
70.0 3.6 (22)a
—
a
Published results (Ref. 46).
Figure 7. Effect of original molecules on elastin production by vascular smooth
muscle cells. (A) Elevation of absorbance as a function of different concentrations of
molecules 15k, 15n and 20o was compared to the effects of the carrier alone
(DMSO), 1 mM minoxidil or 50 lM diazoxide. For minoxidil effect, the control has
been changed because this molecule was solubilized in water at the concentration
used: white bar = control (no DMSO, no minoxidil); black bar = minoxidil. (B)
Figure 6. Time courses for the changes in blood glucose levels in rats orally
administered at time zero with compounds 15 (n, o, t, k, h) (50 mg/kg), diazoxide
(50 mg/kg) or glibenclamide (0.25 mg/kg). Data are expressed as mean values sem
of three rats (n = 3).
Presence of 10
stimulation of elastin production induced by 100
l
M
glibenclamide during the whole experiment abolished the
M 15k or 15o. The absorbance is
l
representative of extracellular elastin quantity (n = 3–4 in each group of test
synthesized molecules, n = 6–9 in each positive or negative control group).
Significance difference with the corresponding control: ⁄P 6 0.05, $P 6 0.07.
shift of the concentration-response curves for B series compounds,
such as 15k (Fig. 5).
As shown in Table 4, most examples of active compounds on
aorta rings (15h, 15k, 15n, 15o, 15t) roughly maintained the same
vasorelaxant potency in the presence or absence of endothelium,
indicating that the mechanism and site of action of these com-
pounds were endothelium-independent.
Compounds 15h, 15k, 15n, 15o, 15t and the reference com-
pound diazoxide were also examined on rat pancreatic islets in
order to evaluate their ability to affect the glucose-induced insulin
release. As shown in Table 5, most compounds were inactive on
insulin secretion except 15t and 15k which exhibited a significant
Compounds 15h, 15k, 15n, 15o and 15t were further evaluated
in vivo to measure blood glucose concentration in rats. Figure 6
revealed that the tested compounds displayed a weak effect on
the blood glucose concentration compared to the reference com-
pounds, diazoxide (hyperglycemic agent) and glibenclamide
(hypoglycemic agent), which induced a hyperglycemic and a hypo-
glycemic effect, respectively. Thus, compounds 15t and 15k, which
were able to inhibit insulin secretion in vitro, were without marked
effect on blood glucose concentration. Such an observation might
result from poor bioavailability or rapid metabolism in vivo after
oral administration.
inhibitory effect at a 50 lM concentration (residual insulin secre-
tion: 44.9% and 21.4%, respectively). Such results suggest a relative
tissue selectivity of some of these compounds for the vascular
smooth muscle.
Finally, some of the most vasoactive compounds were tested in
another biological model, that is, the elastin production by cul-
tured vascular smooth muscle cells. Compounds 15k, 15n and

1740
N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
15o in solution in DMSO were applied to the cells at different con-
centrations: 0 (DMSO alone added to cells), 10, 20, 50 and 100
(Fig. 7). While compound 15n had no effect on elastin production
compared to the effect of DMSO alone, a concentration-dependent
effect was observed with compounds 15k and 15o. Elastin produc-
tion by vascular smooth muscle cells was significantly increased at
5. Experimental section
5.1. Chemistry
lM
Reagents, starting materials and solvents were purchased from
common commercial suppliers and were used as received. Melting
points were determined on Stuart SMP 3 apparatus and were not
corrected. IR spectra were recorded as KBr pellets on a Perkin–
Elmer 1000 FTIR spectrophotometer. The 1H NMR spectra were
recorded at ambient temperature on a Bruker Avance (500 MHz)
instrument using DMSO-d6 as solvent with TMS as an internal
standard. Chemical shifts (d) are given in parts per million and
splitting patterns are designated as follows: s, singlet; d, doublet;
dd, double doublet; t, triplet; q, quadruplet; m, multiplet, b, broad
and bs broad singlet. Elemental analyses (C, H, N, S) were realized
on a Thermo Scientific Flash EA 1112-elemental analyzer and were
within 0.4% of the theoretical values. All reactions were followed
by thin layer chromatography performed on silica gel Merck 60
concentrations as low as 20
lM for 15k and 15o (2-way ANOVA,
P 6 0.05). At 100 M 15k or 15o, the elastin production was
l
increased by about 30% while minoxidil induced the same effect
but at concentration of 1 mM. In additional experiments, com-
pound 15h was found to be unable to enhance elastin production
at concentrations of 10 and 20
ing to note that the stimulatory effects of 15k and 15o on elastin
production were totally abolished by 10 glibenclamide;
indicating a putative involvement of KATP channels in elastin
production (Fig. 7B).
The reference compound diazoxide was also tested on elastin
production by vascular smooth muscle cells. Compared to experi-
mental control conditions (DMSO alone), a significant activation
lM (data not shown). It is interest-
lM
F254
.
of elastin production was observed at 50
ANOVA).
lM (+67%, 1-way
5.1.1. General procedure for preparing 9a–c
p-Halophenol (78 mmol), potassium carbonate (117 mmol) and
methyl iodide (117 mmol) were mixed together in acetone (60 mL)
and refluxed for 3 h. Acetone was removed by evaporation under
reduced pressure and the residue was extracted with Et2O
(3 ꢀ 50 mL). The ether layer was dried over anhydrous MgSO4
and evaporated.
4. Conclusion
Starting from the KATP channel openers 3-alkylamino-4H-1,2,4-
benzothiadiazine 1,1-dioxides, structural modulation by ring
opening led to two series of compounds (series A and B). Series
A molecules (benzenesulfonylureas and benzenesulfonylthioureas)
were found to be devoid of vasorelaxant activity by contrast to
5.1.1.1. 1-Chloro-4-methoxybenzene (9a). Yellow oil (79%); 1H
NMR (DMSO-d6, 500 MHz) d: 3,73 (s, 3H,OCH3), 6.96 (d, 2H, 3,5-H),
7,33 (d, 2H, 2,6-H).
series
B compounds (benzenecarbonylureas and benzenecar-
bonylthioureas); some of the latter drugs being even more active
as vasorelaxants than the reference compound diazoxide (15h,
15j, 15k, 15n, 15o and 15t). Our results strongly suggest that the
lack of activity of series A molecules was dependent on their ion-
ization at physiological pH, as a result of the acidic character linked
to the sulfonylurea function. The vasodilatory activity of selected
series A compounds (15h, 15k, 15n, 15o and 15t) was decreased
5.1.1.2. 1-Fluoro-4-methoxybenzene (9b). The product was
separated on silica gel column chromatography using EtOAc/hex-
ane (10/90) as eluent. Yellow oil (58%); 1H NMR (DMSO-d6,
500 MHz) d: 3.73 (s, 3H,OCH3), 6.75 (d, 2H, 3,5-H), 6.86 (d, 2H,
2,6-H).
in the presence of 10 lM glibenclamide and was reduced on rat
5.1.1.3. 1-Chloro-4-ethoxybenzene (9c). Colorless oil (89%); 1H
NMR (DMSO-d6, 500 MHz) d: 1.32 (t, 3H, OCH2CH3), 4.00 (q, 2H,
OCH2CH3), 6.95 (d, 2H, 3,5-H), 7.30 (d, 2H, 2,6-H).
aorta rings precontracted with 80 mM KCl. Such data suggested,
at least in part, a potential involvement of KATP channels. The pre-
servation of a vasodilatory activity in rat aortic rings deprived of
endothelium indicated that the site of action of the molecules
was located on the vascular smooth muscle cells and not on the
endothelial cells.
5.1.2. General procedure for preparing 11a–c
The solution of 1-halo-4-alkoxybenzene 9 (8.5 g) in chloroform
(50 mL) was introduced in a 250 mL three-necked flask, equipped
with a magnetic stirrer, a thermometer, a calcium chloride guard
and a dropping reagent. The solution was cooled to 0 °C and added
dropwise to chlorosulfonic acid (15 mL). After completion, the
reaction mixture was heated to room temperature, left for 2 h,
and then poured over crushed ice. The resulting mixture was
extracted with chloroform. The organic extracts were dried over
sodium sulfate and concentrated under reduced pressure until
crystallization. The resulting sulfonyl chlorides (7–8 g) (10a–c)
were dissolved in 60 mL dioxane and added dropwise to a 30%
m/v solution of ammonia (100 mL). The reaction mixture was con-
centrated under reduced pressure, leading to a solid which was
collected by filtration, washed with cold water, and dried.
The selected compounds, namely 15h, 15k, 15n, 15o and 15t, at
50 lM, did not display a marked inhibitory effect on insulin secre-
tion from pancreatic b-cells (except 15k and 15t) and on gly-
caemia, which suggested a relative tissue selectivity of some of
these vasorelaxant compounds for the vascular smooth muscle.
In addition, some of the most potent vasorelaxant drugs from
series B (15k and 15o) also exhibited a substantial stimulatory
action on the production of elastin, the main component of the
arterial wall responsible for its elasticity and allowing appropriate
hemodynamics.39 This effect was again abolished by 10
lM gliben-
clamide; indicating a putative involvement of KATP channels in
elastin production.
Taken together, molecules such as 15k and 15o could represent
a class of vasorelaxant drugs, which, in the long term, could
improve arterial elasticity and hemodynamics through their ability
to increase elastin production. Further in vivo tests need to be per-
formed to ascertain such a hypothesis. Additional perspectives also
consist in the re-evaluation of the activity of series A molecules
after methylating the NH group linked to the SO2 group responsible
for their ionization.
5.1.2.1. 5-Chloro-2-methoxybenzenesulfonamide (11a). (45%);
mp 152–153 °C; IR (KBr)
m: 3323, 3259 (N–H); 1329, 1166 (SO2)
cmꢁ1 1H NMR (DMSO-d6, 500 MHz) d: 3.90 (s, 3H, OCH3), 7.24
;
(s, 2H,SO2NH2), 7.25 (d, 1H, 3-H), 7.63 (d, 1H, 4-H), 7.67 (s, 1H, 6-
H). Anal. (C7H8ClNO3S) theoretical: 37.93 C, 3.64 H, 6.32 N, 14.46
S. Found: 38.02 C, 3.64 H, 6.36 N, 14.31 S.

N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
1741
5.1.2.2. 5-Fluoro-2-methoxybenzenesulfonamide (11b). (40%);
mp 174–175 °C; IR (KBr) : 3323, 3259 (N–H); 1329, 1166 (SO2)
cmꢁ1 1H NMR (DMSO-d6, 500 MHz) d: 3.89 (s, 3H, OCH3), 7.22
5.1.3.5.
thiourea (12e). White powder (69%); mp 206–208 °C, IR (KBr)
: 3335 (N–H); 1181 (C@S); 1341,1138 (SO2) cmꢁ1 1H NMR
N-Ethyl-N0-(5-chloro-2-methoxybenzenesulfonyl)
m
;
m
,
(s, 2H,SO2NH2), 7.24 (dd, 1H, 3-H), 7.43 (dd, 1H, 4-H), 7.48 (dd,
1H, 6-H). Anal. (C7H8FNO3S) theoretical: 40.97 C, 3.93 H, 6.83 N,
15.62 S. Found: 41.01 C, 3.92 H, 7.18 N, 15.60 S.
(DMSO-d6, 500 MHz) d: 1,00 (t, 3H, CH3CH2), 3,42 (q, 2H,
CH3CH2), 3,90 (s, 3H, OCH3), 7.29 (d, 1H, arom 3-H), 7.72 (dd, 1H,
arom 4-H), 7.76 (d, 1H, arom 6-H), 8.37 (s, 1H, NHCH2CH3), 11.28
(s, 1H, SO2NHCS). Anal. (C10H13ClFN2O3S2) theoretical: 38.89 C,
4.25 H, 9.07 N, 20.76 S. Found: 38.76 C, 4.25 H, 9.13 N, 20.68 S.
5.1.2.3. 5-Chloro-2-ethoxybenzenesulfonamide (11c). (47%);
mp 156–157 °C; IR (KBr)
m: 3367, 3269 (N–H); 1321, 1163 (SO2)
cmꢁ1 1H NMR (DMSO-d6, 500 MHz) d: 1.36 (t, 3H, OCH2CH3),
;
5.1.3.6. N-Isopropyl-N0-(5-chloro-2-methoxybenzenesulfonyl)
thiourea (12f). White powder (75%); mp 209–210 °C; IR (KBr)
m
500 MHz) d: 1,00 (d, 6H, (CH3)2CH2), 3,55 (st, 1H, (CH3)2CH2),
3,90 (s, 3H, OCH3), 6.25 (d, 1H, NHCH), 7.28 (d, 1H, 3-H), 7.69
(dd, 1H, 4-H), 7.71 (d, 1H, 6-H), 10.28 (s, 1H, SO2NHCS). Anal.
(C11H15ClFN2O3S2) theoretical: 40.92 C, 4.68 H, 8.68 N, 19.86 S.
Found: 41.02 C, 4.78 H, 8.60 N, 19.62 S.
4.21 (q, 2H, OCH2CH3), 7.11 (s, 2H,SO2NH2), 7.25 (d, 1H, 3-H),
7.61 (dd, 1H, 4-H), 7.67 (d, 1H, 6-H). Anal. (C8H10ClNO3S) theoreti-
cal: 40.77 C, 4.28 H, 5.94 N, 13.60 S. Found: 41.97 C, 4.35 H, 6.08 N,
13.74 S.
: 3338 (N–H); 1340, 1150 (SO2) cmꢁ1 1H NMR (DMSO-d6,
,
5.1.3. General procedure for preparing sulfonylurea and
sulfonylthiourea compounds (12a–o)
Sodium hydride (100 mg) was added to a cooled solution of sul-
fonamide 16 (500 mg) in anhydrous DMF. The mixture was stirred
for 15 min, after which an appropriate isocyanate or isothiocyana-
te was added. Stirring was continued at room temperature until
completion of the reaction monitored by TLC. The mixture was
poured into cold water and filtered. The filtrate was acidified with
1 N HCl. The resulting precipitate was collected by filtration,
washed with water and dried.
5.1.3.7.
thiourea (12g). White powder (60%); mp 184 °C, IR (KBr)
3338 (N–H); 1164 (C@S); 1351, 1137 (SO2) cmꢁ1 1H NMR
(DMSO-d6, 500 MHz) d: 3.84 (s, 3H, OCH3), 4.67 (d, 2H, NHCH2),
7.14 (d, 2H, arom 20-H, 60-H), 7.23 (d, 1H, arom 40-H), 7.28 (d, 1H,
arom 3-H), 7.30 (d, 2H, arom 30-H, 50-H), 7.73 (dd, 1H, arom 4-H),
7.77 (d, 1H, arom 6-H), 8.76 (t, 1H, NHCH2), 11.45 (s, 1H,
SO2NHCS). Anal. (C15H15ClFN2O3S2) theoretical: 48.58 C, 4.08 H,
7.55 N, 17.29 S. Found: 48.47 C, 4.14 H, 7.69 N, 16.90 S.
N-Benzyl-N0-(5-chloro-2-methoxybenzenesulfonyl)
m
:
,
5.1.3.1. N-Isopropyl-N0-(5-chloro-2-methoxybenzenesulfonyl)
urea (12a). White powder (76%); mp 198–199 °C, IR (KBr)
m:
3338 (N–H); 1666 (C@O); 1340, 1161 (SO2) cmꢁ1 1H NMR
;
5.1.3.8.
urea (12h). White powder, (85.7%); mp 193,5–194 °C, IR (KBr)
: 3338 (N–H), 1664 (C@O), 1340, 1156 (SO2) cmꢁ1 1H NMR
(DMSO-d6, 500 MHz) d: 1.00 (d, 6H, (CH3)2CH), 3.55 (m, 1H,
(CH3)2CH), 3.90 (s, 3H, OCH3), 6.20 (d, 1H, NHCH2CH3), 7.28 (d,
1H, arom 3-H), 7.51 (dd, 1H, arom 4-H), 7.53 (d, 1H, arom 6-H),
10.20 (s, 1H, SO2NHCO). Anal. (C11H15FN2O4S) theoretical: 45.51
C, 5.21 H, 9.65 N, 11.04 S. Found: 45.50 C, 5.17 H, 9.82 N, 11.16 S.
N-Isopropyl-N0-(5-fluoro-2-methoxybenzenesulfonyl)
(DMSO-d6, 500 MHz) d: 0,98 (d, 6H, CH(CH3)2), 3,55 (st , 1H,
CH(CH3)2), 3,90(s, 3H, OCH3), 6,22 (d, 1H, NHCH), 7,28 (dd, 1H,
arom 3-H), 7.70 (d, 1H, arom 4-H), 7.71 (d, 1H, arom 6-H), 10,25
(s, 1H, SO2NHCO). Anal. (C11H15ClN2O4S) theoretical: 43.07 C,
4.93 H, 9.13 N, 10.45 S. Found: 43.11 C, 4.95 H, 9.30 N, 10.42 S.
m
,
5.1.3.2. N-Pentyl-N0-(5-chloro-2-methoxybenzenesulfonyl)urea
(12b).
White powder (70%); mp 147–148 °C, IR (KBr)
m:
3326 (N–H); 1657 (C@O); 1351, 1160 (SO2) cmꢁ1
;
1H NMR
5.1.3.9.
thiourea (12i).
(KBr)
: 3323 (N–H), 1156 (C@S) , 1341, 1136 (SO2), 1H NMR
(DMSO-d6, 500 MHz) d: 1.10 (d, 6H, (CH3)2CH), 3.89 (s, 3H,
OCH3), 4.19 (m, 1H, (CH3)2CH), 7.30 (dd, 1H, arom 3-H), 7.55 (dd,
1H, arom 4-H), 7.57 (d, 1H, arom 6-H), 8.15 (d, 1H, NHCH), 11.08
(s, 1H, SO2NHCS). Anal. (C11H15FN2O3S2) theoretical: 43.12 C, 4.93
H, 9.14 N, 20.93 S. Found: 43.07 C, 4.94 H, 9.16 N, 20.70 S.
N-Isopropyl-N0-(5-fluoro-2-methoxybenzenesulfonyl)
White powder, (85%) mp 179–180 °C; IR
(DMSO-d6, 500 MHz) d: 0,81 (t, 3H, CH3(CH2)4), 1.11 (m, 2H,
CH3CH2CH2(CH2)2), 1.21 (m, 2H,CH3CH2(CH2)3), 1.30 (m, 2H,
CH3(CH2)2CH2CH2), 2.92 (q, 2H, CH3(CH2)3CH2), 3.90 (s, 3H,
OCH3), 6.37 (t, 1H, NHCH2), 7.28 (d, 1H, arom 3-H), 7.69 (d, 1H,
arom 4-H), 7.71 (d, 1H, arom 6-H), 10.44 (s, 1H, SO2NHCO). Anal.
(C13H19ClN2O4S) theoretical: 46.63 C, 5.72 H, 8.37 N, 9.58 S.
Found: 46.60 C, 5.73 H, 8.59 N, 9.64 S.
m
5.1.3.3. N-Phenyl-N0-(5-chloro-2-methoxybenzenesulfonyl)urea
5.1.3.10.
urea (12j).
N-Isopropyl-N0-(5-chloro-2-ethoxybenzenesulfonyl)
White powder, (49%) mp 173,5–174 °C; IR (KBr)
,
3346 (N–H); 1670 (C@O), 1337, 1163 (SO2) cmꢁ1 1H NMR
(12c).
White powder (43%); mp 176–177 °C; IR (KBr)
m
: 3344
m:
(N–H); 1687 (C@O); 1353, 1164 (SO2) cmꢁ1
;
1H NMR (DMSO-d6,
500 MHz) d: 3.92 (s, 3H, OCH3), 7.02 (t, 1H, arom 40-H), 7.25 (d,
2H, arom 30-H, 50-H), 7.28 (d, 1H, arom 3-H), 7.31 (d, 2H, arom
20-H, 60-H), 7.72 (dd, 1H, arom 4-H), 7.77 (d, 1H, arom 6-H), 8.71
(s, 1H, NHC6H5), 10.40 (s, 1H, SO2NHCO). Anal. (C14H13ClN2O4S)
theoretical: 49.34 C, 3.85 H, 8.22 N, 9.41 S. Found: 49.32 C, 4.08
H, 8.19 N, 9.01 S.
(DMSO-d6, 500 MHz) d: 0.99 (d, 6H, (CH3)3CH), 1.34 (t, 3H,
CH3CH2O), 3.55 (m, 1H, (CH3)3CH), 4.21 (q, 2H, CH3CH2O), 6.25
(d, 1H, NHCH), 7.27 (d, 1H, arom 3-H), 7.66 (dd, 1H, arom 4-H),
7.71 (d, 1H, arom 6-H), 10.04 (s, 1H, SO2NHCO). Anal.
(C12H17ClN2O4S) theoretical: 44.99 C, 5.34 H, 8.73 N, 9.99 S.
Found: 45.21 C, 5.52 H, 8.83 N, 9.53 S.
5.1.3.4.
sulfonyl)urea (12d). White powder (30%); mp 270 °C; IR (KBr)
3334 (N–H); 1638 (C@O); 1315, 1153 (SO2) cmꢁ1 1H NMR
N-(4-Fluorophenyl)-N0-(5-chloro-2-methoxybenzene-
5.1.3.11. N-Cyclohexyl-N0-(5-chloro-2-ethoxybenzenesulfonyl)
m:
urea (12k).
White powder, (72%) mp 189–190 °C, IR (KBr)
m:
;
3357 (N–H), 1667 (C@O), 1340, 1161 (SO2) cmꢁ1
,
1H NMR
(DMSO-d6, 500 MHz) d: 3,92 (s, 3H, OCH3), 7.02 (t, 2H, arom 30-H,
50-H), 7.29 (d, 1H, arom 3-H), 7.34 (dd, 2H, arom 20-H, 60-H), 7.72
(dd, 1H, arom 4-H), 7.77 (d, 1H, arom 6-H), 7.95 (s, 1H, NHC6H5),
10.10 (s, 1H, SO2NHCO). Anal. (C14H12ClFN2O4S) theoretical:
46.87 C, 3.37 H, 7.81 N, 8.94 S. Found: 47.15 C, 3.42 H, 8.01 N,
8.84 S.
(DMSO-d6, 500 MHz) d: 1.05–1.64 (m, 10H, CH2 cyclohexyl), 1.34
(t, 3H, CH3CH2O), 3.29 (b, 1H, NHCH), 4.21 (q, 2H, CH3CH2O), 6.33
(d, 1H, NHCH), 7.27 (d, 1H, arom 3-H), 7.66 (dd, 1H, arom 4-H),
7.70 (d, 1H, arom 6-H), 10.00 (s, 1H, SO2NHCO). Anal.
(C15H21ClN2O4S) theoretical: 49.93 C, 5.87 H, 7.76 N, 8.88 S.
Found: 49.78 C, 5.87 H, 8.10 N, 8.68 S.

1742
N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
5.1.3.12. N-Phenyl-N0-(5-chloro-2-ethoxybenzenesulfonyl)urea
(12l). White powder, (31%) mp 194 °C, IR (KBr) : 3341 (N–H),
1687 (C@O), 1352, 1159 (SO2) cmꢁ1 1H NMR (DMSO-d6,
5.1.4.3. 2-Benzyloxybenzamide (14c).
mp 115–116, IR (KBr) : 3404 (N–H), 3190 (C–H aromatic), 1640
(C@O) cmꢁ1 1H NMR (DMSO-d6, 500 MHz) d: 5.25 (s, 2H, CH2),
White powder (95%);
m
m
,
,
500 MHz) d: 1.32 (t, 3H, CH3CH2O), 4.22 (q, 2H, CH3CH2O), 7.02
(t, 1H, arom 40-H), 7.24 (d, 2H, arom 30-H, 50-H), 7.30 (d, 2H, arom
20-H, 60-H), 7.70 (dd, 1H, arom 4-H), 7.77 (d, 1H, arom 6-H), 8.74 (s,
1H, NHC6H5), 10.30 (s, 1H, SO2NHCO). Anal. (C15H15ClN2O4S) theo-
retical: 50.78 C, 4.26 H, 7.90 N, 9.04 S. Found: 50.76 C, 4.31 H, 8.10
N, 9.53 S.
7.03 (t, 1H, arom 5-H), 7.21 (d, 1H, arom 3-H), 7.42 (m, 5H, arom),
7.44 (t, 1H, arom 4-H), 7.59 (s, 2H, CONH2), 7.78 (dd, 1H, arom 6-H).
Anal. (C14H13NO2) theoretical: 73.99 C, 5.77 H, 6.16 N. Found:
73.63 C, 5.62 H, 6.27 N.
5.1.5. General procedure for preparing 15a–u
Sodium hydride (100 mg) was added to a cooled solution of the
appropriate benzamide 14 (500 mg) in anhydrous DMF. The
mixture was stirred for 15 min, and the appropriate isocyanate
(or isothiocyanate) was added. Stirring was continued at room
temperature until completion of the reaction monitored by TLC.
The mixture was poured into cold water and acidified with 1 N
HCl. The precipitate was collected by filtration, washed with water
and dried.
5.1.3.13.
thiourea (12m).
: 3327 (N–H), 1197 (C@S), 1345, 1158 (SO2) cmꢁ1
(DMSO-d6, 500 MHz) d: 1.09 (d, 6H, (CH3)2CH), 1.33 (t, 3H,
CH3CH2O), 4.16 (m, 1H, (CH3)2CH), 4.20 (q, 2H, CH3CH2O), 7.29
(d, 1H, arom 3-H), 7.70 (dd, 1H, arom 4-H), 7.76 (d, 1H, arom 6-
H), 8.14 (d, 1H, NHCH), 10.90 (s, 1H, SO2NHCS). Anal.
(C12H17ClN2O3S2) theoretical: 42.79 C, 5.09 H, 8.32 N, 19.04 S.
Found: 42.53 C, 5.13 H, 8.52 N, 18.79 S.
N-Isopropyl-N0-(5-chloro-2-ethoxybenzenesulfonyl)
White powder, (35%) mp 150–151 °C, IR (KBr)
1H NMR
m
,
5.1.5.1. N-Ethyl-N0-(2-methoxybenzenecarbonyl)urea (15a). White
powder (50%), mp 70–72 °C; IR (KBr)
m
: 3337 (N–H), 2925 (C–H
;
5.1.3.14.
thiourea (12n).
N-Benzyl-N0-(5-chloro-2-ethoxybenzenesulfonyl)
White powder, (68%) mp 185–186 °C, IR (KBr)
aliphatic), 1706, 1673 (C@O) cmꢁ1
1H NMR (DMSO-d6,
500 MHz) d: 1.11 (t, 3H, CH3CH2), 3.23 (m, 2H, CH3CH2), 3,91
(s, 3H, OCH3), 7.07 (t, 1H, 5-H), 7.19 (d, 1H, 3-H), 7.55 (t, 1H,
4-H), 7.69 (dd, 1H, 6-H), 8.49 (t, 1H, NHCH2CH3), 10.04 (s, 1H,
CONHCO). Anal. (C11H14N2O3) theoretical: 59.45 C, 6.35 H,
12.61 N. Found: 60.59 C, 6.52 H, 12.24 N.
m
: 3311 (N–H), 1165 (C@S), 1355, 1138 (SO2), 1H NMR (DMSO-d6,
500 MHz) d: 1.30 (t, 3H, CH3CH2O), 4.17 (q, 2H, CH3CH2O), 4,66
(d, 2H, CH2C6H5), 7.14 (d, 2H, arom 20-H, 60-H), 7.24 (d, 1H, arom
40-H), 7.25 (d, 1H, arom 3-H), 7.27 (d, 2H, arom 30H, arom 50-H),
7.70 (dd, 1H, arom 4-H), 7.78 (d, 1H, arom 6-H), 8.74 (t, 1H,
NHCH2), 11.33 (s, 1H, SO2NHCS). Anal. (C16H17ClN2O3S2) theoreti-
cal: 49.93 C, 4.45 H, 7.28 N, 16.66 S. Found: 50.14 C, 4.52 H, 7.67
N, 16.63 S.
5.1.5.2.
(15b). White powder (85%); mp 92 °C; IR (KBr)
2925 (C–H aliphatic), 1672 (C@O) cmꢁ1 1H NMR (DMSO-d6,
N-Isopropyl-N0-(2-methoxybenzenecarbonyl)urea
: 3287 (N–H),
m
;
5.1.3.15. N-(4-Fluorophenyl)-N0-(5-chloro-2-ethoxybenzenesul-
500 MHz) d: 1.17 (d, 6H, (CH3)2CH), 3.83 (m, 1H, (CH3)2CH), 3,91
(s, 3H, OCH3), 7.07 (t, 1H, 5-H), 7.19 (d, 1H, 3-H), 7.55 (t, 1H, 4-
H), 7.69 (dd, 1H, 6-H), 8.39 (t, 1H, NHCH(CH3)2), 10.05 (s, 1H,
CONHCO). Anal. (C12H16N2O3) theoretical: 61.00 C, 6.83 H, 11.86
N. Found: 61.27 C, 6.93 H, 11.35 N.
fonyl)thiourea (12o).
White powder, (43%); mp 181–182 °C, IR
(KBr)
m
: 3299 (N–H), 1169 (C@S), 1148 (SO2) cmꢁ1
,
1H NMR
(DMSO-d6, 500 MHz) d: 1.30 (t, 3H, CH3CH2O), 4.23 (q, 2H,
CH3CH2O), 7.18 (t, 2H, arom 20-H, 60-H), 7.29 (d, 1H, arom 3-H),
7.47 (t, 2H, arom 30-H, 50-H), 7.70 (d, 1H, arom 4-H), 7.79 (d, 1H,
arom 6-H), 10.05 (s, 1H, NHC6H5), 11.33 (s, 1H, SO2NHCS). Anal.
(C15H14ClFN2O3S2) theoretical: 46.37 C, 3.63 H, 7.20 N, 16.49 S.
Found: 46.80 C, 3.86 H, 7.41 N, 16.55 S.
5.1.5.3.
(15c). White powder (80%); mp 65–65.5 °C; IR (KBr)
3287 (N–H), 2968 (C–H aliphatic), 1690, 1660 (C@O) cmꢁ1
N-Sec-butyl-N0-(2-methoxybenzenecarbonyl)urea
: 3346,
1H
m
;
NMR (DMSO-d6, 500 MHz) d: 0.88 (t, 3H, (CH3CH2)CH3CH), 1.14
(d, 1H, (CH3CH2)CH3CH), 1.50 (m, 2H, (CH3CH2)CH3CH), 3.72 (m,
1H, (CH3CH2)CH3CH), 3,91 (s, 3H, OCH3), 7.08 (t, 1H, 5-H), 7.19
(d, 1H, 3-H), 7.55 (t, 1H, 4-H), 7.71 (dd, 1H, 6-H), 8.39 (d, 1H,
NHCHCH3(CH2CH3)), 10.05 (s, 1H, CONHCO). Anal. (C31H18N2O3)
theoretical: 62.38 C, 7.25 H, 11.19 N. Found: 62.07 C, 7.32 H,
10.95 N.
5.1.4. General procedure for preparing 14a–c
The mixture of 2-hydroxybenzamide (1 g, 7.3 mmol) and NaOH
(0.4 g, 10 mmol) was stirred in EtOH (40 mL). Then, an appropriate
alkyl halide (10.95 mmol) was added and the reaction medium was
refluxed for 6 h. The excess of solvent was evaporated under
reduced pressure, leading to a residue, which was triturated with
water, filtered and dried.
5.1.5.4.
(15d). White powder (81%); mp 102–102.5 °C; IR (KBr)
3279 (N–H), 2922 (C–H aliphatic), 1705, 1666 (C@O) cmꢁ1
N-Cyclohexyl-N0-(2-methoxybenzenecarbonyl)urea
: 3346,
1H
m
5.1.4.1. 2-Methoxybenzamide (14a).
mp 128 °C, IR (KBr) : 3412 (N–H), 3169 (C–H aromatic), 1644
(C@O) cmꢁ1 1H NMR (DMSO-d6, 500 MHz) d: 3.88 (s, 3H, CH3),
White powder (90%);
;
m
NMR (DMSO-d6, 500 MHz) d: 1.18–1.90 (m, 10H, C6H10), 3.61 (b,
1H, CHNH), 3.91 (s, 3H, OCH3), 7.07 (t, 1H, 5-H), 7.19 (d, 1H, 3-H),
7.55 (t, 1H, 4-H), 7.70 (dd, 1H, 6-H), 8.50 (d, 1H, NHC6H11), 10.06
(s, 1H, CONHCO). Anal. (C15H20N2O3) theoretical: 65.20 C, 7.30 H,
10.14 N. Found: 65.40 C, 7.54 H, 10.22 N.
,
7.02 (t, 1H, arom 5-H), 7.12 (d, 1H, arom 3-H), 7.45 (t, 1H, arom
4-H), 7.62 (s, 2H, CONH2), 7.80 (d, 1H, arom 6-H). Anal.
(C8H6NO2) theoretical: 63.56 C, 6.00 H, 9.27 N. Found: 63.10 C,
5.96 H, 9.38 N.
5.1.5.5.
(15e).
N-Phenyl-N0-(2-methoxybenzenecarbonyl)urea
White powder (40%); mp 134 °C; IR (KBr) : 3238
1H NMR (DMSO-d6, 500 MHz)
5.1.4.2. 2-Propyloxybenzamide (14b).
mp 101 °C, IR (KBr) : 3446 (N–H), 3179 (C–H aromatic), 2965
(C–H aliphatic), 1654 (C@O) cmꢁ1 1H NMR (DMSO-d6,
500 MHz) d: 1.00 (t, 3H, CH3), 1.80 (m, 2H, CH2), 4.09 (q, 2H,
OCH2), 7.02 (t, 1H, arom 5-H), 7.12 (d, 1H, arom 3-H), 7.45 (t,
1H, arom 4-H), 7.62 (s, 2H, CONH2), 7.80 (d, 1H, arom 6-H).
Anal. (C10H13NO2) theoretical: 67.02 C, 7.31 H, 7.82 N. Found:
67.22 C, 7.43 H, 7.78 N.
White powder (92%);
m
m
(N–H), 1703, 1670 (C@O) cmꢁ1
;
,
d: 3.91 (s, 3H, OCH3), 7.11 (t, 2H, 5-H, 40-H), 7.22 (d, 1H, 3-H),
7,36 (t, 2H, 30,50-H), 7.57 (t, 3H, 4, 20, 60-H), 7.73 (dd, 1H, 6-H),
10.46 (d, 1H, NHC6H5), 10.70 (s, 1H, CONHCO). Anal.
(C15H14N2O3) theoretical: 66.66 C, 5.22 H, 10.36 N. Found:
66.57 C, 5.20 H, 9.97 N.

N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
1743
5.1.5.6.
(15f). White powder (80%); mp 73–74 °C; IR(KBr)
(N–H), 2919 (C–H aliphatic), 1707, 1667 (C@O) cmꢁ1
N-Cyclohexyl-N0-(2-propyloxybenzenecarbonyl)urea
: 3355, 3280
1H NMR
7.34–7.51 (m, 5H, CH2C6H5) 7.55 (t, 1H, 4-H), 7.71 (dd, 1H, 6-H),
8.46 (d, 1H, NHC6H11), 10.08 (s, 1H, CONHCO). Anal. (C17H24N2O3)
theoretical: 71.57 C, 6.86 H, 7.95 N. Found: 71.60 C, 7.05 H, 7.74 N.
m
;
(DMSO-d6, 500 MHz) d: 1.03 (t, 3H, CH3CH2CH2), 1.20–1.90
(m, 10H, C6H10), 1.80 (m, 2H, CH3CH2CH2), 3.61 (b, 1H, CHNH),
4.12 (t, 3H, CH3CH2CH2), 7.07 (t, 1H, 5-H), 7.19 (d, 1H, 3-H), 7.55
(t, 1H, 4-H), 7.70 (dd, 1H, 6-H), 8.49 (d, 1H, NHC6H11), 10.08
(s, 1H, CONHCO). Anal. (C17H24N2O3) theoretical: 67.08 C, 7.95 H,
9.20 N. Found: 66.91 C, 8.20 H, 9.07 N.
5.1.5.13.
(15m). White powder (40%); mp 100–101 °C; IR (KBr)
3284 (N–H), 3031 (C–H aromatic), 1692, 1660 (C@O) cmꢁ1
NMR (DMSO-d6, 500 MHz) d: 4.41(s, 2H, NHCH2) 5.27 (s, 2H,
OCH2), 7.09 (t, 1H, 5-H), 7.19 (d, 1H, 3-H), 7.27–7.45(m, 10-H,
CH2C6H5), 7.55 (t, 1H, 4-H), 7.72 (dd, 1H, 6-H), 8.93 (t, 1H,
NHCH2), 10.30 (s, 1H, CONHCO). Anal. (C22H20N2O3) theoretical:
73.32 C, 5.59 H, 7.77 N. Found: 73.72 C, 5.40 H, 7.58 N.
N-Benzyl-N0-(2-benzyloxybenzenecarbonyl)urea
: 3324,
1H
m
;
5.1.5.7.
(15g).
N-Phenyl-N0-(2-propyloxybenzenecarbonyl)urea
White powder (64%); mp 72 °C; IR (KBr) : 3330 (N–H),
1H NMR (DMSO-d6, 500 MHz) d: 1.03 (t,
m
1715, 1664 (C@O) cmꢁ1
;
3H, CH3CH2CH2), 1.84 (m, 2H, CH3CH2CH2), 4.12 (t, 3H,
CH3CH2CH2), 7.13 (t, 2H, 5, 40-H), 7.23 (d, 1H, 3-H), 7.36 (t, 2H, 30,
50-H), 7.59 (t, 3H, 4, 20, 60-H), 7.84 (dd, 1H, 6-H), 10.45 (s, 1H,
NHC6H5), 10.70 (s, 1H, CONHCO). Anal. (C17H18N2O3) theoretical:
68.44 C, 6.08 H, 9.39 N. Found: 68.04 C, 6.50 H, 9.34 N.
5.1.5.14.
(15n). White powder (62%); mp 79.5 °C; IR(KBr)
N-Ethyl-N0-(2-methoxybenzenecarbonyl)thiourea
: 3316 (N–H),
m
2924 (C–H aliphatic), 1661 (C@O) cmꢁ1; 1H NMR (DMSO-d6,
500 MHz) d: 1.15 (t, 3H, CH3CH2), 3.61 (q, 2H, CH3CH2), 3.91 (s,
3H, OCH3), 7.11 (t, 1H, 5-H), 7.24 (d, 1H, 3-H), 7.60 (t, 1H, 4-H),
7.86 (dd, 1H, 6-H), 10.67 (t, 1H, NHCH2CH3), 10.93 (s, 1H,
CONHCS). Anal. (C11H14N2O2S) theoretical: 55.44 C, 5.92 H, 11.76
N, 13.45 S. Found: 55.70 C, 6.12 H, 11.56 N, 13.47 S.
5.1.5.8.
(15h).
N-Ethyl-N0-(2-benzyloxybenzenecarbonyl)urea
White powder (40%); mp 74–75 °C; IR (KBr) : 3335
m
(N–H), 2925 (C–H aliphatic), 1698, 1660 (C@O) cmꢁ1; 1H NMR
(DMSO-d6, 500 MHz) d: 1.09 (t, 3H, CH3CH2), 3.22 (q, 2H,
CH3CH2), 5.27 (s, 2H, CH2), 7.19 (t, 1H, 5-H), 7.28 (d, 1H, 3-H),
7.34–7.54 (m, 5H, CH2C6H5), 7.55 (t, 1H, 4-H), 7.71 (dd, 1H, 6-H),
8.47 (t, 1H, NHCH2CH3), 10.14(s, 1H, CONHCO). Anal.
(C11H14N2O3) theoretical: 68.44 C, 6.08 H, 9.39 N. Found: 68.44 C,
6.28 H, 9.10 N.
5.1.5.15. N-Isopropyl-N0-(2-methoxybenzenecarbonyl)thiourea
(15o).
White powder (60%); mp 109.5–110 °C; IR (KBr)
m:
3317 (N–H), 2929 (C–H aliphatic), 1655 (C@O) cmꢁ1
;
1H NMR
(DMSO-d6, 500 MHz) d: 1.26 (d, 6H, (CH3)2CH), 3.61 (q, 2H,
CH3CH2), 3.98 (s, 3H, OCH3), 4.37 (m, 1H, (CH3)2CH), 7.11 (t, 1H,
5-H), 7.24 (d, 1H, 3-H), 7.60 (t, 1H, 4-H), 7.86 (dd, 1H, 6-H), 10.67
(t, 1H, NHCH2CH3), 10.93 (s, 1H, CONHCS). Anal. (C12H16N2O2S)
theoretical: 57.12 C, 6.39 H, 11.10 N, 12.70 S. Found: 57.51 C,
6.50 H, 11.23 N, 12.78 S.
5.1.5.9. N-Isopropyl-N0-(2-benzyloxybenzenecarbonyl)urea
(15i).
White powder (87%); mp 84–85 °C; IR (KBr) m: 3341,
3286 (N–H), 2925 (C–H aliphatic), 1690, 1664 (C@O) cmꢁ1, 1H
NMR (DMSO-d6, 500 MHz) d: 1.15 (d, 6H, (CH3)2CH), 3.87 (m, 1H,
(CH3)2CH), 5.27 (s, 2H, CH2), 7.09 (t, 1H, 5-H), 7.28 (d, 1H, 3-H),
7.34–7.54 (m, 5H, CH2C6H5), 7.55 (t, 1H, 4-H), 7.71 (dd, 1H, 6-H),
8.36 (d, 1H, NHCH(CH3)2), 10.17 (s, 1H, CONHCO). Anal.
(C18H20N2O3) theoretical: 69.21 C, 6.48 H, 8.97 N. Found: 68.98 C,
6.96 H, 8.59 N.
5.1.5.16. N-Sec-butyl-N0-(2-methoxybenzenecarbonyl)thiourea
(15p). White powder (42%); mp 79 °C; IR (KBr)
m: 3308 (N–H),
2926 (C–H aliphatic), 1656 (C@O) cmꢁ1 1H NMR (DMSO-d6,
;
500 MHz) d: 0.91 (t, 3H, (CH3CH2)CH3CH), 1.22 (d, 3H,
(CH3CH2)CH3CH), 1.63 (m, 2H, (CH3CH2)CH3CH), 3,91 (s, 3H,
OCH3), 4.27 (m, 1H, (CH3CH2)CH3CH), 7.12 (t, 1H, 5-H), 7.25 (d,
1H, 3-H), 7.61 (t, 1H, 4-H), 7.88 (dd, 1H, 6-H), 10.66 (d, 1H,
NHCHCH3(CH2CH3)), 10.94 (s, 1H, CONHCS). Anal. (C13H18N2O2S)
theoretical: 58.62 C, 6.81 H, 10.52 N, 12.04 S. Found: 58.80 C,
6.88 H, 10.66 N, 11.88 S.
5.1.5.10.
(15j).
N-Pentyl-N0-(2-benzyloxybenzenecarbonyl)urea
White powder (45%); mp 74 °C; IR (KBr) : 3350, 3300
1H NMR
m
(N–H), 2900 (C–H aliphatic), 1695, 1652 (C@O) cmꢁ1
,
(DMSO-d6, 500 MHz) d: 0,96 (t, 3H, CH3(CH2)4), 1.29 (m, 2H,
CH3CH2CH2(CH2)2), 1.33 (m, 2H,CH3CH2(CH2)3), 1.50 (m, 2H,
CH3(CH2)2CH2CH2), 3.16 (q, 2H, CH3(CH2)3CH2), 5.27 (s, 2H, CH2),
7.09 (t, 1H, 5-H), 7.28 (d, 1H, 3-H), 7.34–7.51 (m, 5H, CH2C6H5),
7.55 (t, 1H, 4-H), 7.71 (dd, 1H, 6-H), 8.37 (d, 1H, NHCH), 10.15 (s,
1H, CONHCO). Anal. (C20H24N2O3) theoretical: 70.56 C, 7.11 H,
8.23 N. Found: 70.36 C, 6.98 H, 8.34 N.
5.1.5.17.
(15q). White powder (55%); mp 154 °C; IR (KBr)
1655 (C@O) cmꢁ1 1H NMR (DMSO-d6, 500 MHz) d: 3.90 (s, 3H,
N-Phenyl-N0-(2-methoxybenzenecarbonyl)thiourea
: 3325 (N–H),
m
;
OCH3), 6.98–7.50 (m, 9H, H arom), 8.65 (s, 1H, NHC6H5), 9.79 (s,
1H, CONHCS). Anal. (C15H14N2O2S) theoretical: 62.92 C, 4.93 H,
9.78 N, 11.20 S. Found: 62.90 C, 4.91 H, 9.80 N, 11.18 S.
5.1.5.11.
(15k). White powder (65%); mp 62–63 °C; IR (KBr)
N-Sec-butyl-N0-(2-benzyloxybenzenecarbonyl)urea
: 3334, 3270
m
5.1.5.18.
(15r). White powder (62%); mp 93–93.5 °C; IR (KBr)
N-Benzyl-N0-(2-methoxybenzenecarbonyl)thiourea
: 3333
(N–H), 2962 (C–H aliphatic), 1687, 1661 (C@O) cmꢁ1; 1H NMR
(DMSO-d6, 500 MHz) d: 0.87 (t, 3H, (CH3CH2)CH3CH), 1.12 (d,
1H, (CH3CH2)CH3CH), 1.49 (m, 2H, (CH3CH2)CH3CH), 3.71 (m, 1H,
(CH3CH2)CH3CH), 5.27 (s, 2H, CH2), 7.09 (t, 1H, 5-H), 7.28 (d,
1H, 3-H), 7.34–7.51 (m, 5H, CH2C6H5), 7.55 (t, 1H, 4-H), 7.71 (dd,
1H, 6-H), 8.35 (d, 1H, NHCH), 10.18 (s, 1H, CONHCO). Anal.
(C19H22N2O3) theoretical: 69.92 C, 6.79 H, 8.58 N. Found: 70.30 C,
7.13 H, 8.28 N.
m
(N–H), 1657 (C@O) cmꢁ1; 1H NMR (DMSO-d6, 500 MHz) d: 3.99
(s, 3H, OCH3), 4.87 (d, 2H, CH2), 7.14 (t, 1H, 5-H), 7.26 (d, 1H, 3-
H), 7.29–7.42 (m, 5H, CH2C6H5), 7.64 (t, 1H, 4-H), 7.87 (dd, 1H, 6-
H), 7.87 (d, 1H, NHCH2), 11.06 (s, 1H, CONHCS). Anal.
(C16H16N2O2S) theoretical: 63.98 C, 5.37 H, 9.33 N, 10.67 S.
Found: 63.80 C, 5.35 H, 9.61 N, 10.50 S.
N-Cyclohexyl-N0-(2-benzyloxybenzenecarbonyl)urea
White powder (82%); mp 91–92 °C; IR (KBr) : 3273 (N–
1H NMR
5.1.5.19.
(15s). White powder (67%); mp 86–87 °C; IR (KBr)
3238 (N–H), 2966 (C–H aliphatic), 1661 (C@O) cmꢁ1
N-Benzyl-N0-(2-propyloxybenzenecarbonyl)thiourea
: 3320,
1H NMR
5.1.5.12.
(15l).
m
m
;
H), 2929 (C–H aliphatic), 1689, 1666 (C@O) cmꢁ1
;
(DMSO-d6, 500 MHz) d: 1.08 (t, 3H, CH3CH2CH2), 1.92 (m, 2H,
CH3CH2CH2), 4.20 (t, 2H, CH3CH2CH2), 4.88 (d, 2H, NHCH2), 7.15
(DMSO-d6, 500 MHz) d: 1.20–1.90 (m, 10H, C6H10), 3.59 (b, 1H,
CHNH), 5.27 (s, 2H, CH2), 7.09 (t, 1H, 5-H), 7.28 (d, 1H, 3-H),

1744
N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
(t, 1H, 5-H), 7.27 (d, 1H, 3-H), 7.30 (t, 1H, 40-H), 7.38 (m, 4H, 20,30, 50,
60-H), 7.64 (t, 1H, 4-H), 7.96 (dd, 1H, 6-H), 11.11 (t, 1H, NHCH2),
11.18 (s, 1H, CONHCS). Anal. (C18H20N2O2S) theoretical: 65.83 C,
6.14 H, 8.53 N, 9.76 S. Found: 66.03 C, 6.35 H, 8.75 N, 9.97 S.
The relaxation response was expressed as percentage of the
contractile response to KCl. The EC50 values (concentration evoking
50% inhibition of the plateau phase induced by KCl) were assessed
from concentration-response curves.
5.1.5.20. N-Phenyl-N0-(2-propyloxybenzenecarbonyl)thiourea
5.3.2. Aortic smooth muscle cell culture
(15t).
White powder (57%); mp 72–73 °C; IR (KBr)
m
: 3446,
The protocol was adapted from
a previously published
3204 (N–H), 3035 (C–H aromatic), 1651 (C@O) cmꢁ1
;
1H NMR
method.41 6 month-old rats were anaesthetised by intraperitoneal
injection of pentobarbital (60 mg/kg). The abdominal aorta (taken
from the diaphragm to the iliac bifurcation) was harvested and
cleaned of adhering fat before being washed twice in saline buffer
HBSS (Invitrogen-Life Technologies Ltd, Paisley, UK, reference
(DMSO-d6, 500 MHz) d: 0.99 (t, 3H, CH3CH2CH2), 1.80 (m, 2H,
CH3CH2CH2), 4.07 (t, 2H, CH3CH2CH2), 4.87 (d, 2H, CH2), 7.12 (t,
1H, 5-H), 7.25 (d, 1H, 3-H), 7.61 (t, 1H, 4-H), 7.88 (dd, 1H, 6-H),
8.61 (s, 1H, NHCH2), 9.85 (s, 1H, CONHCS). Anal. (C17H18N2O2S)
theoretical: 64.94 C, 5.77 H, 8.91 N, 10.20 S. Found: 65.07 C, 5.77
H, 9.03 N, 9.98 S.
#14025-100) supplemented with amphotericin-B (2.5 lg/mL).
The vessel was pre-digested in a P35 Petri dish containing 2 mL
of a 1 mg/mL collagenase II (Whorthington Biochemical Corp.,
Lakewood, NJ, USA, reference #CLS2 LS004176) for 50 min (37 °C,
5% CO2). The aorta was then washed in HBSS-amphotericin-B solu-
tion, before the enzymatically-altered adventitia was removed by
using tweezers. The rest of the vessel, that is intima + media, was
cut into small pieces which were then digested in a 5 mL tube
for 1 h in 2 mL of a mix containing 0.5 mg/mL sterile elastase
(Whorthington Biochemical Corp., reference # ESL LS002292) and
1 mg/mL collagenase II. The digestion product was centrifuged at
1200 rpm (350ꢀg) for 10 min, and then re-suspended in 3 mL
HBSS-amphotericin-B solution. The digested tissue was then
mechanically dissociated by flushing the solution several times
through a 1 mL pipette, while avoiding the formation of air bub-
bles. The 3 mL digested tissue suspension was then centrifuged
again at 1200 rpm for 10 min, and the pellet was re-suspended in
5.1.5.21.
(15u).
N-Benzyl-N0-(2-benzyloxybenzenecarbonyl)thiourea
White powder (47%); mp 100–101 °C; IR (KBr) : 3290
1H NMR
m
(N–H); 3030 (C–H aromatic); 1648 (C@O), cmꢁ1
;
(DMSO-d6, 500 MHz) d: 4.85 (s, 2H, NHCH2), 5.35 (s, 2H, OCH2),
7.14 (t, 1H, 5-H), 7.19 (d, 1H, 3-H), 7.28–7.45 (m, 10H, CH2C6H5),
7.55 (t, 1H, 4-H), 7.72 (dd, 1H, 6-H), 11.10 (t, 1H, NHCH2), 11.16
(s, 1H, CONHCO). Anal. (C22H20N2O2S) theoretical: 70.19 C, 5.35
H, 7.44 N, 8.52 S. Found: 69.93 C, 5.24 H, 7.54 N, 8.71 S.
5.2. pKa determination
Determination of the pKa value of selected compounds was per-
formed by a spectrophotometric method.43 UV–visible spectra
were recorded on
a
Hitachi
U
3010 Spectrophotometer.
Compounds were dissolved in water to provide mother solutions
at 2.5. 10ꢁ4 M, and a series of borate and phosphate buffers, rang-
ing from pH 3 to pH 10, were prepared according to Sorensen. The
samples were obtained by mixing 5 mL of the mother solution with
20 mL of the appropriate buffer. UV–visible spectra were recorded
at ambient temperature with respect to a control consisting of
5 mL H2O and 20 mL of the same buffer. The wavelength was
selected to record a maximum absorbance difference between
the ionized and the non-ionized forms. The absorbance of the
neutral and ionic species of the drug was determined in a similar
way, using a solution of equal drug concentration in 0.1 N HCl
and 0.1 N NaOH. After each shot spectrum, the exact pH of the
solution was measured by means of a pH meter and the pKa was
calculated using the formula of Albert and Serjeant.40
100 lL culture medium DMEM (Invitrogen-Life Technologies Ltd,
reference #31966021) supplemented with 20% fetal calf serum
(FCS), 1% Penicillin/Streptomycin, in two wells-pre-coated with
0.1% gelatin of a P24 plate (Merck, Darmstadt, Germany, reference
#1.04078.1000). A 30 min period was left for vascular smooth
muscle cell (VSMC) adhesion (at 37 °C, 5% CO2), before the volume
was completed up to 500 lL with culture medium. The well con-
tent was left for 3 days before the first culture medium change.
The culture medium was then replaced every two days, until cell
confluence was reached, which was about 6–7 days after the cells
were seeded. Amplification of the cell culture was then performed
by cell trypsinisation and seeding in several 0.1% gelatin-coated
wells.
5.3.3. Measurement of insulin release from incubated rat
pancreatic islets
5.3. Biological assays
Experiments were performed with pancreatic islets isolated
from adult fed Wistar rats. Groups of 10 islets, each derived from
the same batch of islets, were preincubated for 30 min at 37 °C in
1 mL of a physiological salt medium (in mM: NaCl 115, KCl 5,
CaCl2 2.56, MgCl2 1, NaHCO3 24) supplemented with 2.8 mM
glucose, 0.5% (w/v) dialysed albumin (fraction V, Sigma) and
equilibrated against a mixture of O2 (95%) and CO2 (5%). The islets
were then incubated at 37 °C for 90 min in 1 mL of the same
medium containing 16.7 mM glucose and, in addition, the
reference or the required compound. The release of insulin was
measured radioimmunologically using rat insulin as a standard.42
Residual insulin secretion was expressed as a percentage of the
value recorded in control experiments (100%); that is in the
absence of drug and presence of 16.7 mM glucose.
5.3.1. Measurements of tension in rat aorta rings
Diazoxide was tested as the reference compound. Experiments
were performed with aorta removed from adult Wistar rats
(Elevages Janvier 2010, France). A section of the thoracic aorta
was cleared of adhering fat and connective tissue and was cut into
transverse rings (2–3 mm long). The segments were suspended
under 1.5 g tension by means of steel hooks in an organ bath con-
taining a physiological solution (in mM: NaCl 118, KCl 4.7, CaCl2
2.5, MgCl2 1.2, KH2PO4 1.2, NaHCO3 25, glucose 5, pH = 7.36). The
physiological solution was maintained at 37 °C and continuously
bubbled with a mixture of O2 (95%) and CO2 (5%). Isometric
contractions of the aortic rings were measured with a force-
displacement transducer. After 60 min of equilibration, contractile
activity was induced by increasing the extracellular concentration
of K+: 30 or 80 mM KCl. High KCl was applied until the end of the
experiment. When the tension had stabilized, increasing concen-
trations of the tested drug were cumulatively added to the bath
until maximal relaxation. Some experiments were performed in
5.3.4. Measurement of glucose level in rat blood
Animals (adult fed Wistar albino rats) had free access to water
and received a standard pellet diet. Conscious rats were placed for
60 min prior blood sampling and throughout the duration of the
the presence of 10 lM of the KATP channel blocker glibenclamide.

N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
1745
study in a small cabinet. At zero time, drugs dissolved in DMSO
(0.2 mL) were orally administrated at a dose of 50 mg/kg. Control
animals received an equivalent volume of DMSO (0.2 mL). A drop
of blood was taken from the tail at time 0, 1, 2, 3 and 4 h. Blood
glucose concentration was measured using a reagent strip in
the comparison to the effect of 1 mM minoxidil. Unless otherwise
indicated, P 6 0.05 was considered as statistically significant.
Acknowledgements
combination with
a
glucometer (ACCU-CHEK Active, Roche,
This study was supported by grants from the Algerian Ministry
of Higher Education and Scientific Research, the French CMEP-
Tassili and the National Fund for Scientific Research (F.N.R.S.,
Belgium) from which P. Lebrun is Research Director. The authors
gratefully acknowledge the technical assistance of Stéphane
Counerotte, Fabienne Leleux, Aibech Riad and Bouraoui Hadia.
Mannheim).43
5.3.5. Measurement of the extracellular elastin content by ELISA
The protocol used here was adapted from a previously pub-
lished method.44,45 Vascular smooth muscle cells were deprived
of serum overnight in DMEM containing only 1% FCS, in order to
limit cell proliferation, before trypsinisation and seeding at
25,000 cells/well in a P96 plate, where the cells were left to adhere
for another night. The culture medium was then removed and
replaced by fresh 1% FCS-DMEM into which the molecules to be
tested (in solution in DMSO) were added.For each concentration
of each molecule, 3–9 wells were used. In addition, the cells of 8
wells were bathed in 1% FCS-DMEM containing DMSO only, as
the negative control. The cells of other wells were bathed in 1%
FCS-DMEM containing 1 mM of the KATP channel opener minoxidil
as the positive control which has previously been shown to induce
a strong increase in extracellular elastin.44 The effect of the KATP
References and notes
1. Ashcroft, F. M. Am. J. Physiol. 2007, 293, E880.
2. Ashcroft, F. M. J. Clin. Invest. 2005, 115, 2047.
3. Standen, N. B.; Quayle, J. M.; Davies, N. W.; Brayden, J. E.; Huang, Y.; Nelson, M.
T. Science 1989, 245, 177.
4. Kane, G. C.; Liu, X. K.; Yamada, S.; Olson, T. M.; Terzic, A. J. Mol. Cell. Cardiol.
2005, 38, 937.
5. Quast, U. Naunyn. Schmiedebergs Arch. Pharmacol. 1996, 354, 213.
6. Yamada, K.; Ji, J. J.; Yuan, H. J.; Miki, T.; Sato, S.; Horimoto, N.; Shimizu, T.; Seino,
S.; Inagaki, N. Science 2001, 292, 1543.
7. Shyng, S. L.; Nichols, C. G. J. Gen. Physiol. 1997, 110, 655.
8. Inagaki, N.; Gonoi, T.; Clement, J. P., 4th; Namba, N.; Inazawa, J.; Gonzalez, G.;
Aguilar-Bryan, L.; Seino, S.; Bryan, J. Science 1995, 270, 1166.
9. Aguilar-Bryan, L.; Nichols, C. G.; Wechsler, S. W.; Clement, J. P., 4th; Boyd, A. E.;
Gonzalez, G.; Herrera-Sosa, H.; Nguy, K.; Bryan, J.; Nelson, D. A. Science 1995,
268, 423.
channel opener diazoxide (50 lM), used as a reference in the vas-
cular pharmacology experiments, was also studied. The cells of
additional wells were bathed in 1% FCS-DMEM (without DMSO)
as the negative control for minoxidil effect since minoxidil was sol-
10. Nichols, C. G.; Koster, J. C.; Enkvetchakul, D.; Flagg, T. P. Biol. Membr. 2006, 23,
101.
11. Aguilar-Bryan, L.; Bryan, J. Endocr. Rev. 1999, 20, 101.
12. Ashcroft, F. M.; Gribble, M. Trends Neurosci. 1998, 21, 288.
13. Edwards, G.; Weston, A. H. Annu. Rev. Pharmacol. Toxicol. 1993, 33, 597.
14. Aguilar-Bryan, L.; Clement, J. P., 4th; Gonzalez, G.; Kunjilwar, K.; Babenko, A.;
Bryan, J. Physiol. Rev. 1998, 78, 227.
15. Zingman, L. V.; Hodgson, D. M.; Bast, P. H.; Kane, G. C.; Perez-Terzic, C.;
Gumina, R. J.; Pucar, D.; Bienengraeber, M.; Dzeja, P. P.; Miki, T.; Seino, S.;
Alekseev, A. V.; Terzic, A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13278.
16. Lebrun, P.; Antoine, M.-H.; Herchuelz, A. Life Sci. 1992, 51, 795.
17. Quast, U. Trends Pharmacol. Sci. 1993, 14, 332.
ubilized in water at the concentration used. In some wells, 10 lM
of the KATP channel blocker glibenclamide was added to the tested
molecules during the whole experiment in order to verify that the
effects of the designed compounds were mediated by activation of
the KATP channels. Each experiment, that is, each test performed on
each concentration of each synthesized molecule, was at least tri-
plicated in cells from different cultures (n = 3–4). Control experi-
ments (medium plus DMSO or water, diazoxide, minoxidil) were
performed in each plate used for the ELISA tests, leading to more
wells (n = 8–9). The cells used in our experiments originated from
6 separate cell cultures. In all cases, DMSO concentration exposed
to the cells was <1%. After 48 h, that is, the time to allow for elastin
synthesis and excretion, the cells were washed 3 times with PBS
(Invitrogen-Life Technologies Ltd, reference #14040174), then
fixed for 10 min in 4% paraformaldehyde at room temperature.
The wells were washed again 3 times with PBS, before blockade
of the non-specific binding sites with 2%BSA containing-PBS for
30 min at 37 °C, then washed 3 times with 0.1% Tween-containing
PBS. After this step, the cells were incubated with the primary anti-
body to elastin (Elastin Products Company Inc., Owensville, MO,
USA, reference #RA75) at 1:1000 in 2% BSA containing-PBS for
1 h at 37 °C. The cells were then washed 3 times with 0.1%
Tween-containing PBS before incubation with the secondary anti-
body coupled to horseradish peroxidase (HRP) (Abcam, Paris,
FRANCE, anti-goat antibody, reference #6741) at 1:50,000 for 1 h
at 37 °C in the dark. The cells were then washed 3 times with
18. Coghlan, M. J.; Carroll, W. A.; Gopalakrishan, M. J. Med. Chem. 2001, 44, 1627.
19. Mannhold, R. Med. Res. Rev. 2004, 24, 213.
20. Richer, C.; Pratz, J.; Mulder, P.; Mondot, S.; Giudicelli, J. F.; Cavero, I. Life Sci.
1990, 47, 1693.
21. Dunne, M. J.; Aspinall, R. J.; Petersen, O. H. Br. J. Pharmacol. 1990, 99, 169.
22. Weir, S. W.; Weston, A. H. Br. J. Pharmacol. 1986, 88, 121.
23. Hamilton, T. C.; Weir, S. W.; Weston, A. H. Br. J. Pharmacol. 1986, 88, 103.
24. Quast, U.; Cook, N. S. J. Pharmacol. Exp. Ther. 1989, 250, 261.
25. 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.
26. 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.
27. Khelili, S.; Florence, X.; Bouhedja, M.; Abdelaziz, S.; Mechouch, N.; Yekhlef, M.;
de Tullio, P.; Lebrun, P.; Pirotte, B. Bioorg. Med. Chem. 2008, 16, 6124.
28. Groop, L. C. Diabetes Care 1992, 15, 737.
29. Pezet, M.; Jacob, M. P.; Escoubet, B.; Gheduzzi, D.; Tillet, E.; Perret, P.; Huber, P.;
Quaglino, D.; Vranckx, R.; Li, D. Y.; Starcher, B.; Boyle, W. A.; Mecham, R. P.;
Faury, G. Rejuvenation Res. 2008, 11, 97.
30. Ewart, A. K.; Jin, W.; Atkinson, D.; Morris, C. A.; Keating, M. T. J. Clin. Invest.
1994, 93, 1071.
31. Nickerson, E.; Greenberg, F.; Keating, M. T.; McCaskill, C.; Shaffer, L. G. Am. J.
Hum. Genet. 1995, 56, 1156.
32. Wu, Y. Q.; Sutton, V. R.; Nickerson, E.; Lupski, J. R.; Potocki, L.; Korenberg, J. R.;
Greenberg, F.; Tassabehji, M.; Shaffer, L. G. Am. J. Med. Genet. 1998, 78, 82.
33. Hayashi, A.; Suzuki, T.; Wachi, H.; Tajima, S.; Nishikawa, T.; Murad, S.; Pinnell,
S. R. Arch. Biochem. Biophys. 1994, 315, 137.
34. Tajima, S.; Hayashi, A.; Suzuki, T.; Nishikawa, T. Arch. Dermatol. Res. 1995, 287,
494.
35. Tsoporis, J.; Keeley, F. W.; Lee, R. M.; Leenen, F. H. J. Cardiovasc. Pharmacol.
1998, 31, 960.
0.1% Tween-containing PBS, before 100 l
L 3,30,5,50-tetramethyben-
zidine (TMB) (Millipore, Billerica, MA, USA, reference #ES022), the
substrate of HRP, were added to each well for 30 min in the dark at
room temperature. The reaction was then stopped by addition of
100 lL 0.3 M sulfuric acid to each well. After 10 min, the absor-
bance of each well—a function of extracellular elastin quantity-
36. Raveaud, S.; Mezin, P.; Lavanchy, N.; Starcher, B.; Mecham, R. P.; Verdetti, J.;
Faury, G. Clin. Exp. Pharmacol. Physiol. 2009, 36, 988.
37. Slove, S.; Lannoy, M.; Behmoaras, J.; Pezet, M.; Sloboda, N.; Lacolley, P.;
Escoubet, B.; Buján, J.; Jacob, M.-P. Hypertension 2013, 62, 794.
38. Bray, K. M.; Weston, A. H.; Duty, S.; Newgreen, D. T.; Longmore, J.; Edwards, G.;
Brown, T. J. Br. J. Pharmacol. 1991, 102, 337.
was measured at 450 nm.
Each concentration of each molecule was tested in different cell
cultures. The statistical significance of the differences between
groups was assessed by using 2-way ANOVA for analysis of the
effects of the molecule and concentration, and 1-way ANOVA for
39. Faury, G. Pathol. Biol. (Paris) 2001, 49, 310.

1746
N. Bouider et al. / Bioorg. Med. Chem. 23 (2015) 1735–1746
40. Albert, A.; Serjeant, E.P. In The Determination of Ionization Constants, Chapman &
Hall: London, 1971; pp 44–64.
41. Golovina, V. A.; Blaustein, M. P. Nat. Protoc. 2006, 1, 2681.
42. Lebrun, P.; Antoine, M.-H.; Devreux, V.; Hermann, M.; Herchuelz, A. J. J.
Pharmacol. Exp. Ther. 1990, 255, 948.
44. Giro, M. G.; Hill, K. E.; Sandberg, L. B.; Davidson, J. M. Coll. Relat. Res. 1984, 4, 21.
45. Davidson, J. M.; LuValle, P. A.; Zoia, O.; Quaglino, D., Jr.; Giro, M. J. Biol. Chem.
1997, 272, 345.
46. Pirotte, B.; Ouedraogo, R.; de Tullio, P.; Khelili, S.; Sommers, F.; Boverie, S.;
Dupont, L.; Fontaine, J.; Damas, J.; Lebrun, P. J. Med. Chem. 2000, 43, 1456.
43. Nguyen, Q. A.; Antoine, M. H.; Ouedraogo, R.; Hermann, M.; Sergooris, J.;
Pirotte, B.; Masereel, B.; Lebrun, P. Biochem. Pharmacol. 2002, 63, 515.