Influence of the alkylsulfonylamino substituent located at the 6-position of 2,2-dimethylchromans structurally related to cromakalim: From potassium channel openers to calcium entry blockers?

Article abstract of DOI:10.1016/j.ejmech.2014.04.024

The present study described the synthesis of original R/S-6- alkylsulfonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans bearing a 3- or 4-substituted phenylthiourea or phenylurea moiety at the 4-position. Their biological effects were evaluated both on insulin-secreting and smooth muscle cells and were compared to those of reference K_ATP channel activators such as (±)-cromakalim, diazoxide and previously synthesized cromakalim analogues. The study aimed at exploring the influence of the introduction of an alkylsulfonylamino substituent at the 6-position of 2,2-dimethylchromans in order to improve biological activity, tissue selectivity but also hydrophilicity of dihydrobenzopyran derivatives. Several compounds were found to be equipotent or even more potent than (±)-cromakalim and diazoxide at inhibiting the insulin releasing process. Most of the newly synthesized and more hydrophilic dihydrobenzopyrans also exhibited a marked vasorelaxant activity although they were less potent than (±)-cromakalim. Additional pharmacological and radioisotopic investigations suggested that R/S-N-3-chlorophenyl-N-(3,4-dihydro- 6-methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (21) did not act as a potassium channel opener but rather as a Ca²⁺ entry blocker.

Full text of DOI:10.1016/j.ejmech.2014.04.024

European Journal of Medicinal Chemistry 80 (2014) 36e46
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Influence of the alkylsulfonylamino substituent located at the 6-
position of 2,2-dimethylchromans structurally related to cromakalim:
From potassium channel openers to calcium entry blockers?
,
c
b
b
b,1
Xavier Florence a , Vincent Desvaux , Eric Goffin , Pascal de Tullio , Bernard Pirotte
,
*
Philippe Lebrun a
,
1
a Laboratoire de Pharmacodynamie et de Thérapeutique, Université Libre de Bruxelles, Faculté de Médecine, 808 Route de Lennik, B-1070 Bruxelles, Belgium
b Laboratoire de Chimie Pharmaceutique, Centre Interfacultaire de Recherche du Médicament (C.I.R.M.), Université de Liège, C.H.U., 1 Avenue de l’Hôpital,
B-4000 Liège, Belgium
c Centre de Recherches du Cyclotron, Université de Liège, 8 Allée du 6 Août, 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:
The present study described the synthesis of original R/S-6-alkylsulfonylamino-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyrans bearing a 3- or 4-substituted phenylthiourea or phenylurea moiety at the
4-position. Their biological effects were evaluated both on insulin-secreting and smooth muscle cells and
were compared to those of reference KATP channel activators such as (ꢀ)-cromakalim, diazoxide and
previously synthesized cromakalim analogues. The study aimed at exploring the influence of the
introduction of an alkylsulfonylamino substituent at the 6-position of 2,2-dimethylchromans in order to
improve biological activity, tissue selectivity but also hydrophilicity of dihydrobenzopyran derivatives.
Several compounds were found to be equipotent or even more potent than (ꢀ)-cromakalim and diaz-
oxide at inhibiting the insulin releasing process. Most of the newly synthesized and more hydrophilic
dihydrobenzopyrans also exhibited a marked vasorelaxant activity although they were less potent than
(ꢀ)-cromakalim. Additional pharmacological and radioisotopic investigations suggested that R/S-N-3-
chlorophenyl-Nʹ-(3,4-dihydro-6-methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
(21) did not act as a potassium channel opener but rather as a Ca2þ entry blocker.
Received 11 October 2013
Received in revised form
28 March 2014
Accepted 5 April 2014
Available online 13 April 2014
Keywords:
Benzopyran derivatives
ATP-sensitive potassium channels
Potassium channel openers
Cromakalim analogues
Insulin secretion
Smooth muscle contractile activity
Ó 2014 Published by Elsevier Masson SAS.
1. Introduction
KATP channels are distributed in many tissues where they play a
large variety of physiological roles [5]. KATP channels are involved in
the control of the insulin secretory process from pancreatic B-cells
[6e8] and have also been depicted as participating in the control of
the smooth muscle vascular tone [9].
KATP channels are octameric complexes comprising four Kir6.x
subunits (Kir6.1 or Kir6.2), members of the inwardly rectifying Kþ
channel family, and four sulfonylurea receptor (SUR) subunits
(SUR1, SUR2A, or SUR2B). The Kir6.0 subunit forms the pore of the
Potassium channels represent a wide family of proteins involved
in various cellular processes. Such channels are ubiquitously
expressed and play a key role in the control of the membrane po-
tential [1]. Among this family of ionic channels, ATP-sensitive po-
tassium channels (KATP channels) have been shown to link cell
metabolism to membrane excitability. Indeed, the activity of KATP
channels is mainly coupled to the intracellular concentration ratio
of adenosine triphosphate (ATP) to adenosine diphosphate (ADP).
As the ratio increases, the KATP channel activity is reduced. At the
opposite, a decrease of the ATP/ADP ratio activates the KATP chan-
nels [2e4].
KATP channel complex whereas the SUR subunit acts as a regulator
of KATP channel activity [5]. The combination of the different sub-
units leads to tissue-specific KATP channels, potential target sites for
drugs. For example, four SUR1 subunits combine with four Kir6.2
subunits to form the SUR1/Kir6.2 KATP channel subtype as found in
the endocrine pancreas and the brain tissue [10] whereas a SUR2A/
Kir6.2 channel subtype is expressed in cardiac and skeletal muscle
cells [11]. A SUR2B/Kir6.1 combination has been found in vascular
smooth muscle cells while a SUR2B/Kir6.2 assembly has been
characterized in various smooth muscle cells [11e13].
Abbreviations: Kir, inwardly rectifying potassium channel; SUR, sulfonylurea
receptor; KATP channel, ATP-sensitive potassium channel.
* Corresponding author.
E-mail address: xavier.florence@gmail.com (X. Florence).
Philippe Lebrun and Bernard Pirotte equally supervised this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.04.024
0223-5234/Ó 2014 Published by Elsevier Masson SAS.

X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
37
According to their wide physiological functions, KATP channels
represent a promising target to develop new therapeutic agents.
Such an objective can theoretically be reached through the syn-
thesis of compounds exhibiting a marked specificity and a high
selectivity for a single KATP channel subtype.
position in order to develop 4,6-disubstituted R/S-2,2-
dimethylchromans displaying improved hydrophilicity and selec-
tivity towards insulin secreting cells. This approach allowed us to
synthesize more hydrophilic 4-phenylthiourea-substituted R/S-6-
acetamido-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans
(com-
The potential and recognized therapeutic indications for ATP-
sensitive potassium channel openers (PCOs) include, among
others, the treatment of arterial hypertension [14,15], angina pec-
toris [14,16], cardiac arrhythmias [14,17], bronchial asthma [14,18],
urinary incontinence [14,19] and androgenic alopecia [14,20]. PCOs
have also been proposed for the prevention and/or management of
type I, type II diabetes, obesity [14,15,21,22], nesidioblastosis [14],
insulinomas [14] and polycystic ovary syndrome [21,23].
(ꢀ)-Cromakalim (1) and diazoxide (2) are well known PCOs
belonging to different chemical families (Fig. 1). (ꢀ)-Cromakalim
(1), leader of the dihydrobenzopyran-type PCOs, has been found to
exert a marked myorelaxant activity [24,25] although the drug has
also been reported to be slightly active as inhibitor of the insulin
secretory process [26]. By contrast, diazoxide (2), leader of the
benzothiadiazine 1,1-dioxide-type PCOs, has been reported to be
equipotent on the vascular smooth muscle and the insulin
secreting-cells [26,27].
pounds 7e10, Fig. 1) but the latter compounds were less active on
the endocrine pancreatic tissue than previously described de-
rivatives [30].
According to the structureeactivity relationships deduced from
our previous investigations, the present work aimed at further
exploring the influence of the nature of the substituent at the 6-
position in order to develop 4,6-disubstituted R/S-2,2-
dimethylchromans displaying a marked inhibitory activity on the
insulin secretory process but also an improved hydrophilicity.
Therefore, the newly synthesized compounds were bearing, at the
6-position, a sulfonamide function as a bioisosteric group of the
amide function characterizing previously described compounds
(7e10) [31].
2. Chemistry
The common intermediate, R/S-6-amino-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-one (15), giving access to the target
compounds (19e34) was synthesized as previously described [30]
in four steps; starting from 4-methoxyaniline (Scheme 1).
Firstly, 4-methoxyaniline (11) was acetylated by acetic anhy-
dride to provide N-(4-methoxyphenyl)acetamide (12). This reac-
tion was followed by a FriedeleCrafts acylation to give N-(3-acetyl-
4-hydroxyphenyl)acetamide (13). It should be noted that the re-
action conditions led to the demethylation of the methoxy group
located at the para-position of the acetamido moiety. Treatment of
compound 13 with acetone, in the presence of pyrrolidine, led to
chromanone intermediate 14.
The acetamido group of intermediate 14 was hydrolyzed in an
alcoholic solution of diluted hydrochloric acid.
The amine function of the resulting intermediate (15) was
substituted by methanesulfonyl chloride or ethanesulfonyl chloride
which provides compound 16a or 16b, respectively.
We have previously identified original R/S-3,4-dihydro-2,2-
dimethyl-6-halo-2H-1-benzopyrans
(ꢀ)-cromakalim, among which four derivatives exhibiting
structurally
related
to
a
modified tissue selectivity profile have been characterized [28,29].
These original 6-bromo-substituted R/S-3,4-dihydro-2,2-dimethyl-
2H-1-benzopyrans bearing a 3- or 4-substituted phenylthiourea
moiety at the 4-position (compounds 3e6, Fig. 1) were found to be
less effective as vasorelaxants but more potent as inhibitors of the
insulin secretory process than the reference molecule (ꢀ)-croma-
kalim (1) [28]. Structureeactivity relationships further indicated
that the nature of the substituent at the 4-position of the benzo-
pyran nucleus played a crucial role in the expression of an inhibi-
tory effect on insulin release. Unfortunately, such compounds
exhibited a weak solubility in the physiological medium. Thus, in
another study, we kept identical substitutions at the 4-position and
explored the influence of the nature of the substituent at the 6-
In order to obtain the key intermediates 18a, b, the two ketonic
compounds 16a and 16b were treated with hydroxylamine hydro-
chloride and potassium carbonate to give the corresponding ox-
imes 17a, b, which were further hydrogenated in the presence of
RaneyeNickel.
R/S-N-(m/p-substituted)phenyl-Nʹ-(6-methylsulfonylamino-
3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thioureas (19e
22) or ureas (23e26) as well as R/S-N-(m/p-substituted)phenyl-N0-
(6-ethylsulfonlamino-3,4-dihydro-2,2-dimethyl-2H-1-
benzopyran-4-yl)thioureas (27e30) or ureas (31e34) were ob-
tained from the reaction of the amines 18aeb with the appropriate
m/p-cyano/chlorophenyl isothiocyanate (ReN]C]S) or isocyanate
(ReN]C]O) (Scheme 1).
3. Results and discussion
3.1. Insulin secretion
The ability of the newly synthesized compounds (Table 1,
compounds 19e34) to inhibit the insulin releasing process was
evaluated on isolated rat pancreatic islets incubated in the presence
of an insulinotropic glucose concentration (16.7 mM).
(ꢀ)-Cromakalim and diazoxide were used as reference PCOs
(Table 1). The biological activity of the original 2,2-
dimethylchroman derivatives was also compared to that of previ-
ously described molecules (7e10 and 3e6) (Table 1) [28e30].
Fig. 1. Chemical structure of (ꢀ)-cromakalim (1), diazoxide (2) and previously
described compounds 3e10 [30,32].

38
X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
Scheme 1. Synthetic pathway to (ꢀ)-cromakalim analogues 19e34.
Biological data obtained with (ꢀ)-cromakalim, tested at a 10
m
M
process (p < 0.05). The inhibitory effect on the insulin releasing
process was more marked for the phenylthiourea derivatives
bearing a chlorine atom than for the corresponding chloro-
substituted phenylurea compounds (21, 22 vs 25, 26, p < 0.05).
However, the isosteric replacement of the 6-acetamido group (7e
10) by the 6-methylsulfonylamino group (19e22) did not improve
the inhibitory activity on the pancreatic endocrine tissue. Com-
pounds 19 and 20 were found to be less potent than 7 and 8
(p < 0.05) and compounds 21 and 22 were equipotent to 9 and 10
(p > 0.05).
Among the compounds bearing a 6-ethylsulfonylamino group
(27e34), the thiourea derivatives (27e30) were more potent than
the corresponding urea derivatives (31e34, p < 0.05) and than
diazoxide at inhibiting the insulin releasing process (p < 0.05).
Drugs bearing a chlorine atom (29 and 30) at the meta or para
position of the phenyl group of the thiourea chain were again more
potent than those bearing a cyano group (27 and 28, p < 0.05).
concentration, indicated that this reference PCO was roughly
inactive on the pancreatic endocrine tissue (Table 1). Diazoxide
(10
m
M), however, provoked a ꢀ20% reduction in the insulin
secretory rate (Table 1).
By contrast, previously synthesized 2,2-dimethylchromans (7e
10 and 3e6), although being only slightly water soluble, were much
more potent than (ꢀ)-cromakalim and diazoxide at inhibiting the
glucose-induced insulin release (p < 0.05) (Table 1). Structuree
activity relationships suggested that the presence of an electron-
withdrawing group at the meta or para position of the phenyl
ring of R/S-N-phenyl-Nʹ-(6-bromo-3,4-dihydro-2,2-dimethyl-4-2H-
1-benzopyran-4-yl)thioureas improved the inhibitory effect on the
insulin releasing process [28,29]. Therefore, an electron-
withdrawing group, such as a chlorine atom or a cyano group,
was held at the 3- or 4-position and new moieties located at the 6-
position were explored in order to develop original
dimethylchroman-type
hydrophilicity.
analogues
with
an
enhanced
Table 1 indicated that several compounds bearing a 6-
ethylsulfonylamino moiety (27e34) were more active (p < 0.05)
than those bearing a 6-methylsulfonylamino group (19e26) (except
23 vs 31 and 24 vs 32).
Unfortunately, all these newly synthesized 6-methyl- and 6-
ethylsulfonylamino-substituted 2,2-dimethylchromans remained
less active at reducing the insulin secretory rate than the previously
described 6-bromo-substituted analogues (3e6, p < 0.05).
The secretory data showed that the new chroman derivatives
(20e34, except 19 and 25), by contrast to (ꢀ)-cromakalim, inhibited
the insulin-releasing process. Among these newly synthesized an-
alogues, some compounds were found to be equipotent (p > 0.05)
(20, 23, 24, 26, 31e34) or even more potent than diazoxide (21, 22,
27e30) at reducing the glucose-induced insulin response (p < 0.05)
(Table 1).
6-Methylsulfonylamino-substituted benzopyran derivatives
bearing a chlorine atom on the phenyl ring linked to the thiourea
function (21, 22) were more potent than the corresponding com-
pounds bearing a cyano group (19, 20) (p < 0.05). Drugs 21, 22 were
even more potent than diazoxide at inhibiting the insulin secretory
3.2. Myogenic activity
The potential vasorelaxant activity of the original derivatives
was determined on 30 mM Kþ-depolarized rat aorta (endothelium-
free) rings and was compared to that of (ꢀ)-cromakalim and

X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
39
Table 1
AC log P estimated values and effects of original dimethylchromans on insulin secretion from rat pancreatic islets and on the contractile activity of rat aorta rings.
Compounds
Y
R
R3
R4
AC log Pa
Residual insulin secretion (%)b
10
Myorelaxant activity
EC50
M)c
m
M
(m
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
7
8
9
10
3
4
5
6
S
CH3e
CN
Cl
2.91
2.91
3.71
3.71
2.61
2.61
3.41
3.41
3.42
3.42
4.22
4.22
3.12
3.12
3.92
3.92
3.54
3.54
4.34
4.34
5.43
5.43
5.14
5.14
1.82
n.d.
91.7 ꢀ 4.8 (29)
81.7 ꢀ 3.3 (30)
69.2 ꢀ 2.9 (24)
63.9 ꢀ 3.8 (22)
84.4 ꢀ 4.6 (30)
81.6 ꢀ 4.0 (29)
94.5 ꢀ 4.3 (23)
88.5 ꢀ 4.0 (23)
64.7 ꢀ 2.4 (16)
57.9 ꢀ 2.1 (16)
54.3 ꢀ 2.3 (24)
49.1 ꢀ 2.2 (23)
75.6 ꢀ 3.2 (24)
73.6 ꢀ 4.3 (24)
72.1 ꢀ 3.6 (31)
78.1 ꢀ 3.0 (30)
60.9 ꢀ 3.3 (21)d
55.8 ꢀ 3.4 (21)d
63.1 ꢀ 2.9 (21)d
60.4 ꢀ 3.3 (21)d
23.0 ꢀ 2.4 (31)d
12.2 ꢀ 1.2 (20)d
28.6 ꢀ 1.7 (22)e
25.7 ꢀ 1.5 (23)e
94.4 ꢀ 4.1 (32)d
80.8 ꢀ 3.7 (32)
9.8 ꢀ 2.4 (4)
11.6 ꢀ 3.1 (4)
9.8 ꢀ 1.6 (11)
11.6 ꢀ 1.7 (4)
9.3 ꢀ 1.5 (4)
19.8 ꢀ 3.1 (5)
15.2 ꢀ 1.1 (4)
>30.0 (8)
CN
Cl
O
S
CH3e
CN
Cl
CN
Cl
CH3eCH2e
CH3eCH2-
CN
Cl
12.1 ꢀ 4.3 (6)
12.0 ꢀ 2.1 (6)
9.8 ꢀ 2.1 (4)
5.1 ꢀ 0.4 (3)
9.5 ꢀ 2.1 (5)
11.8 ꢀ 3.2 (6)
17.2 ꢀ 1.6 (4)
>10.0 (8)
CN
Cl
O
CN
Cl
CN
Cl
S
S
S
O
CH3eCOeNHe
CH3eCOeNHe
Bre
CN
Cl
11.9 ꢀ 1.4 (4)d
CN
Cl
>10.0 (4)d
>10.0 (6)d
8.3 ꢀ 1.7 (4)d
Cl
>10 (5)d
Cl
>10 (4)d
Bre
Cl
>30 (4)e
Cl
>300 (4)e
(ꢀ)-Cromakalim
0.13 ꢀ 0.01 (7)d
26.1 ꢀ 2.9 (4)
Diazoxide
a
Ref [33].
b
Percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7 mM glucose (mean ꢀ SEM (n)).
EC50: drug concentration giving 50% relaxation of the 30 mM KCl-induced contraction of rat aorta rings (mean ꢀ SEM (n)).
Published compound and results (Ref [30]).
c
d
e
Published compound and results (Ref [32]).
diazoxide. The myorelaxant properties of the newly synthesized
compounds were also compared with those of previously described
molecules (7e10 and 3e6) [28e30].
On the vascular tissue, (ꢀ)-cromakalim displayed a marked
myorelaxant activity while diazoxide exhibited moderate vaso-
relaxant properties (Table 1). Several previously synthesized 6-
their corresponding urea analogues (23e26 and 31e34). Moreover,
the nature and the position of the substituent on the phenyl ring
did not markedly affect the myorelaxant activity of the alka-
nesulfonamides (19e34) (p > 0.05). Compound 30 was found to be
the most potent 6-alkanesulfonamide derivative.
The biological data reported on Table 1 further suggest that the
myorelaxant capacity of the newly synthesized alkanesulfonamide
analogues was more pronounced than that of the reference 2,2-
dimethylchroman compounds, namely the acetamide analogues
(7e10) and the 6-bromo-substituted 2,2-dimethylchromans (3e6).
In accordance with the predicted AC log P values [33], the new
molecules were also found to be more hydrophilic and therefore
should be more soluble in the physiological medium than the
previously described 6-bromo-substituted and 6-acetamido-
substituted 2,2-dimethylchromans (Table 1) [30].
acetamido-substituted
and
6-bromo-substituted
2,2-
dimethylchromans (7e10 and 3e6) also produced a vasorelaxant
effect but such compounds were less potent than (ꢀ)-cromakalim
(Table 1). It should be noticed that compounds 8, 9 and 3e6
precipitated in the bathing medium before reaching their maximal
myorelaxant activity [30,32], making it difficult to accurately
determine their EC50 values (Table 1).
The new 6-methyl- and ethylsulfonylamino-substituted 2,2-
dimethylchroman analogues (19e34), which did not precipitate
into the experimental medium (except for compounds 26 and 34)
and displayed accurately quantifiable EC50 values, exhibited a
weaker vasorelaxant activity than (ꢀ)-cromakalim. However, most
of these original compounds exerted a more pronounced myor-
elaxant activity than diazoxide (p < 0.05). Compound 24 was found
to be equipotent to diazoxide (p > 0.05).
3.3. Effects of 21 on the contractile activity of rat aorta rings
incubated in the presence of different glibenclamide or KCl
concentrations
In most cases, the thiourea derivatives (19e22 and 27e30)
appeared to elicit a slightly more potent myorelaxant effect than
Compound 21, a methanesulfonamide derivative more active on
the smooth muscle than on the insulin-secreting cells, was selected

40
X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
Fig. 2. Left panel: concentrationeresponse curves for the myorelaxant effect of compound 21 on KCl (30 mM)-induced contraction of rat aorta rings incubated in the absence or
presence of different glibenclamide concentrations. Mean values ꢀ S.E.M refer 7e8 individual experiments. Right panel: concentrationeresponse curves for the myorelaxant effect
of compound 21 on KCl (30 mM or 80 mM)-induced contraction of rat aorta rings. Mean values ꢀ S.E.M refer to 7e9 individual experiments.
to perform additional experiments aiming at characterizing the
mechanism of action of this original compound.
First, the effect of the methanesulfonamide derivative 21 was
evaluated on 86Rb (42K substitute) fractional outflow rate (FOR)
from prelabeled and perifused rat aorta rings exposed throughout
to 30 mM extracellular Kþ. Under such experimental conditions,
which mimicked the conditions used to measure muscle tension,
In rat aortic rings exposed to 30 mM Kþ, the cumulative appli-
cation of 21 induced concentration-dependent relaxations (Fig. 2).
When the same experiment was conducted on rat aorta rings
exposed throughout to either 1 or 10
m
M of the KATP channel
the addition of 25 or 100 mM 21 to the perifusing medium failed to
blocker glibenclamide [5,34], the vasorelaxant capacity of 21 was
unaffected (p > 0.05, Table 2 and Fig. 2 left panel). By contrast, the
presence of glibenclamide in the physiological medium provoked a
concentration-dependent rightward shift of the doseeresponse
curves for the reference KATP channel openers diazoxide and/or
(ꢀ)-cromakalim (Table 2). Indeed, diazoxide was found to be 4.4
affect 86Rb FOR (data not shown), suggesting that the drug did not
interfere with the membrane permeability to Kþ.
In another series of experiments, we quantified the effects of 21
on 45Ca outflow from prelabelled and perifused rat aortic rings.
As previously described, a rise in the extracellular concentration
of Kþ from 5 to 80 mM provoked a rapid and marked increase in
45Ca FOR (Fig. 3) [36]. This cationic response to high Kþ is known to
reflect a process of 40Cae45Ca exchange into which 40Ca influent,
flowing through activated voltage-sensitive Ca2þ channels, dis-
places 45Ca from intracellular binding sites [36,37].
fold less potent in the presence of 1
mM glibenclamide (p < 0.05)
and 8.0 fold less potent in the presence of 10
(p < 0.05) in the physiological medium (Table 2). Likewise, 1
glibenclamide provoked a 10 fold reduction (p < 0.05) whilst 10
m
M glibenclamide
m
m
M
M
glibenclamide induced a 130 fold reduction (p < 0.05) in the
(ꢀ)-cromakalim response (Table 2).
When compound 21 (30 mM) was present in the perifusing
medium, the 45Ca response to 80 mM Kþ was reduced (Fig. 3). Thus,
In the next series of experiments, contractile activity was eli-
cited by high concentrations of extracellular Kþ (80 mM). Under
such experimental conditions, the vasorelaxant properties of 21
remained unchanged (p > 0.05, Table 2 and Fig. 2 right panel)
whereas the myorelaxant effects of diazoxide and (ꢀ)-cromakalim
were dramatically reduced (p < 0.05, Table 2).
the peak 45Ca outflow averaged 6.06 ꢀ 0.36% per min. (n ¼ 5) in the
absence and 3.68 ꢀ 0.43% per min. (n ¼ 6) in the presence of 30
mM
21 (p < 0.05). Such an inhibitory effect of 21 on the 45Ca response to
80 mM Kþ was reminiscent of that provoked by the Ca2þ entry
blocker verapamil [37].
These radioisotopic data indirectly suggest that the effects of 21
result from a direct inhibition of Ca2þ entry into smooth muscle
cells and that 21 behaves as a Ca2þ entry blocker.
It should be stressed that the Ca2þ entry blocker verapamil [35]
also provoked concentration-dependent relaxations of the
myogenic responses induced by 30 mM extracellular Kþ (Table 2).
As indicated in Table 2, the myorelaxant effects of verapamil
4. Conclusion
were unaffected by the presence of 1
mM (p > 0.05) or 10 mM
(p > 0.05) glibenclamide in the bathing medium and persisted in
In search of more hydrosoluble 4,6-disubstituted R/S-3,4-
dihydro-2,2-dimethyl-2H-1-benzopyrans acting as pancreatic
PCOs, we have synthesized 16 original 4-substituted R/S-6-
alkylsulfonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans
(19e34) structurally related to R/S-6-acetamido-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyrans bearing a substituted phenylthiourea
moiety at the 4-position (7e10). All these new dihydrobenzopyran
derivatives were bearing, at the 4-position, a phenylthiourea (19e
80 mM Kþ media (p > 0.05, Table 2).
Altogether, these data reveal that compound 21 displays a
pharmacological profile different from that of KATP channel openers
and similar to that of the Ca2þ entry blocker verapamil.
3.4. Effects of 21 on 86Rb outflow and on KCl-induced changes in
45Ca outflow from perifused rat aortic rings
22 and 27e30) or
a phenylurea chain (23e26 and 31e34)
substituted on the phenyl ring by a meta or a para electron with-
drawing group such as a cyano group or a chlorine atom. Some of
these new 2,2-dimethylchromans included, at the 6-position, a
methylsulfonylamino (19e26) or an ethylsulfonylamino group
Radioisotopic experiments were further conducted to charac-
terize, indirectly, the effects of compound 21 on transmembrane
ionic movements.

X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
41
Table 2
Myorelaxant effects of compound 21, diazoxide, (ꢀ)-cromakalim and verapamil on 30 mM and 80 mM KCl-precontracted rat aorta rings incubated in the absence or presence of
glibenclamide.
Compounds
Myorelaxant activity
EC50
M)a
KCl 30 mM
(
m
KCl 30 mM þ 1
m
M Gliben.b
KCl 30 mM þ 10
m
M Gliben.b
KCl 80 mM
21
12.0 ꢀ 2.0 (7)
16.6 ꢀ 3.3 (8)
2.5 ꢀ 0.7 (9)
15.7 ꢀ 2.9 (7)
28.8 ꢀ 6.0 (5)
15.8 ꢀ 0.7 (7)
0.22 ꢀ 0.07 (6)
0.17 ꢀ 0.01 (4)
34.8 ꢀ 2.7 (6)
16.8 ꢀ 1.2 (9)
(ꢀ)-Cromakalim
Diazoxide
Verapamil
137.7 ꢀ 10.4 (10)
>300 (8)
153.7 ꢀ 8.0 (7)
0.038 ꢀ 0.007 (6)
279.8 ꢀ 11.8 (4)
0.035 ꢀ 0.003 (5)
23.8 ꢀ 5.0 (6)
0.031 ꢀ 0.004 (4)
0.045 ꢀ 0.004 (5)
0.052 ꢀ 0.006 (7)
a
EC50: drug concentration giving 50% relaxation (mean ꢀ SEM (n)).
b
Gliben.: glibenclamide.
(27e34). The in vitro biological activity (inhibition of insulin release
from pancreatic B-cells and myorelaxant effect on vascular smooth
muscle) of these original compounds was compared to that of
(ꢀ)-cromakalim, diazoxide and previously reported compounds 3e
10.
The huge majority of the new 6-alkylsulfonylamino-substituted
dihydrobenzopyran derivatives was more potent than the reference
compound (ꢀ)-cromakalim at inhibiting the insulin releasing
process. Some compounds were found to be equipotent or even
more potent than diazoxide.
The newly synthesized dihydrobenzopyrans, although being
less potent than (ꢀ)-cromakalim, exhibited marked vasorelaxant
properties. The myorelaxant activity of most original alka-
nesulfonamide derivatives was more pronounced than that of
diazoxide and than that of previously described 6-bromo- or 6-
acetamido-substituted 2,2-dimethylchromans.
Whether on the glucose-stimulated insulin releasing process or
the KCl-induced myogenic activity, the phenylthiourea derivatives
appeared to be more active than their corresponding phenylurea
analogues.
Biological results further indicated that the bioisosteric
replacement of the acetamide group at the 6-position by an alkyl-
sulfonylamino substituent allowed to identify compounds rather
acting on the vascular smooth muscle tissue. Moreover, it should
also be pointed out that, compared to the previously described R/S-
6-bromo- and R/S-6-acetamido-3,4-dihydro-2,2-dimethyl-2H-1-
benzopyrans, the hydrophilicity of the new compounds was
notably increased.
Additional pharmacological and radioisotopic experiments
conducted
with
R/S-N-3-chlorophenyl-N’-(3,4-dihydro-6-
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)thio-
urea (21) suggested that the myorelaxant properties of the 6-
alkylsulfonylamino-substituted dihydrobenzopyran mainly resul-
ted from a direct Ca2þ entry blockade. In such a way, compound 21
behaved as a Ca2þ entry blocker rather than as a specific potassium
channel opener.
5. Experimental
5.1. Chemistry
Reagents and solvents were purchased from usual commercial
suppliers and were used without further purification. Yields re-
ported refer to purified products. All reactions were routinely
checked by thin-layer chromatography (TLC) on silica gel 60 F254
(Merck) and visualization was performed by UV light (254 nm).
Melting points were determined on a Stuart SMP3 apparatus in
open capillary tubes and were uncorrected. IR spectra were recor-
ded as KBr pellets on a PerkineElmer 1000 FTIR spectrophotom-
eter. The 1H NMR spectra were recorded on a Bruker Avance 500
instrument (1H: 500 MHz; 13C 125 MHz) using DMSO-d6 as solvent
and tetramethylsilane (TMS) as internal standard; chemical shifts
were reported in
d values (ppm) relative to internal TMS. The
abbreviation s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quadruplet,
m ¼ multiplet and b ¼ broad signal were used throughout.
Elemental analyses (C, H, N, S) were determined on a Thermo Flash
EA 1112 series elemental analyzer and were within ꢀ0.4% of the
theoretical values.
N-(4-Methoxyphenyl)acetamide
(12),
N-(3-Acetyl-4-
hydroxyphenyl)acetamide (13), 6-Acetamido-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-one (14) and 6-Amino-3,4-dihydro-
2,2-dimethyl-2H-1-benzopyran-4-one (15) were obtained accord-
ing to previously described procedures [30].
Fig. 3. Effects of KCl 80 mM on 45Ca outflow from rat aorta rings perifused throughout
in the absence (B) or presence of compound 21 (C; 30
to 5e6 individual experiments.
mM). Mean values ꢀ S.E.M refer

42
X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
5.1.1. 3,4-Dihydro-2,2-dimethyl-6-methylsulfonylamino-2H-1-
benzopyran-4-one (16a)
75.32, 44.49, 33.17, 26.34, 7.96. The product was used in the next
step without further purification.
6-Amino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-one
(15) (8 g, 41.83 mmol) was dissolved in methylene chloride (80 mL).
Pyridine (5.08 mL, 62.75 mmol) and methanesulfonyl chloride
(4.86 mL, 62.75 mmol) were added to the solution which was
stirred at 25 ꢁC. After completion of the reaction, the solvent was
evaporated under reduced pressure. The crude product was tritu-
rated with a hot sodium hydroxide solution (10% NaOH w/w). The
insoluble material was collected by filtration and concentrated
hydrochloric acid was added to the filtrate until pH 1. The resulting
precipitate was collected by filtration, washed with water and dried
5.1.5. R/S-4-Amino-3,4-dihydro-2,2-dimethyl-6-
methylsulfonylamino-2H-1-benzopyran (18a)
Raney-Nickel (2 g) was added to a stirred solution of 17a (1.7 g,
5.98 mmol) in methanol (80 mL). The solution was stirred in a
sealed hydrogenator under a hydrogen pressure of 5 bars. When the
reaction was completed (3e4 h), the catalyst was removed by
filtration and the filtrate was evaporated under reduced pressure.
The crude product was dissolved in methanol and water was added.
The title compound was collected by filtration, washed with water
(10.92 g, 97%): mp: 195e197 ꢁC; IR (KBr)
1682 (C]O), 3446e3246 (NeH) cmꢂ1
500 MHz): : 1.39 (s, 6H, CH3), 2.79 (s, 2H, CH2), 2.93 (s, 3H, e
NHSO2CH3), 6.99 (d, 1H, 8-H), 7.40 (dd, 1H, 7-H), 7.56 (d, 1H, 5-H),
9.60 (s, 1H, eNHSO2CH3); 13C NMR (DMSO-d6, 125 MHz)
: 191.83,
y
.
: 1155 and 1336 (S]O),
and dried (1.36 g, 84%): mp: 159e161 ꢁC; IR (KBr)
y
: 1153 and 1315
1H NMR (DMSO-d6,
(S]O), 3340 (NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
d: 1.19 (s,
d
3H, CH3), 1.34 (s, 3H, CH3), 1.52 (t, 1H, CH2), 2.00 (m, 1H, CH2), 2.88
(s, 3H, eNHSO2CH3), 3.80 (m, 1H, CHeNH2), 6.63 (d, 1H, 8-H), 6.93
d
(dd, 1H, 7-H), 7.43 (d, 1H, 5-H); 13C NMR (DMSO-d6, 125 MHz)
d:
156.45, 131.23, 130.02, 119.81, 119.24, 117.57, 79.41, 47.78, 38.81,
25.96. The product was used in the next step without further
purification.
150.46, 129.85, 127.92, 122.60, 122.04, 116.61, 75.14, 43.84, 43.62,
38.64, 29.68, 24.62. The product was used in the next step without
further purification.
5.1.6. R/S-4-Amino-3,4-dihydro-2,2-dimethyl-6-
ethylsulfonylamino-2H-1-benzopyran (18b)
5.1.2. 3,4-Dihydro-2,2-dimethyl-6-ethylsulfonylamino-2H-1-
benzopyran-4-one (16b)
Following the same procedure than that described for com-
pound 18a, the catalytic hydrogenation of compound 17b gener-
Compound 16b was prepared following the same procedure as
that described for compound 16a, starting from compound 15 (8 g,
41.83 mmol) and ethanesulfonyl chloride (5.95 mL, 62.75 mmol)
ated product 18b (1.44 g, 89%): mp: 148e150 ꢁC; IR (KBr)
y
: 1143
and 1320 (S]O), 3360 (NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
: 1.20 (m, 6H, eNHSO2CH2CH3 and CH3), 1.33 (s, 3H, CH3), 1.52 (t,
(11.38 g, 96%): mp: 151e153 ꢁC; IR (KBr)
y
: 1147 and 1335 (S]O),
1687 (C]O), 3253 (NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
d:
d
1H, CH2), 2.00 (m, 1H, CH2), 2.97 (m, 2H, eNHSO2CH2CH3), 3.80 (m,
1H, CHeNH2), 6.62 (d, 1H, 8-H), 6.93 (dd, 1H, 7-H), 7.42 (d, 1H, 5-H);
1.19 (t, 3H, eNHSO2CH2CH3), 1.38 (s, 6H, CH3), 2.79 (s, 2H, CH2), 3.02
(q, 2H, eNHSO2CH2CH3), 6.99 (d, 1H, 8-H), 7.40 (dd, 1H, 7-H), 7.56
(d, 1H, 5-H), 9.69 (s, 1H, eNHSO2CH2CH3); 13C NMR (DMSO-d6,
125 MHz) d: 191.83, 156.23, 131.25, 129.54, 119.81, 119.27, 117.03,
79.38, 47.79, 44.79, 25.97, 7.96. The product was used in the next
13C NMR (DMSO-d6, 125 MHz)
d: 150.23, 129.82, 127.90, 122.08,
121.49, 116.61, 75.11, 44.41, 43.88, 43.61, 29.68, 24.62, 7.99. Anal.
(C13H20N2O3S) theoretical: C, 54.92; H, 7.09; N, 9.85; S, 11.28.
Found: C, 54.92; H, 7.11; N, 9.80; S, 10.92.
step without further purification.
5.1.7. General procedure for the preparation of compounds 19e34
5.1.3. 3,4-Dihydro-2,2-dimethyl-6-methylsulfonylamino-2H-1-
benzopyran-4-hydroxyimine (17a)
The appropriate phenyl isothiocyanate or phenyl isocyanate was
added to
dimethyl-6-methylsulfonylamino-2H-1-benzopyran (18a) (0.5 g,
1.85 mmol) or R/S-4-amino-3,4-dihydro-2,2-dimethyl-6-
a boiling solution of R/S-4-amino-3,4-dihydro-2,2-
Potassium carbonate (7.18 g, 51.98 mmol) and hydroxylamine
hydrochloride (3.61 g, 51.98 mmol) were added to a stirring solu-
tion of 16a (7 g, 25.99 mmol) in ethanol (65 mL). The suspension
was refluxed for 3 h. The reaction was then poured onto ice and,
after complete melting of ice, water was added to obtain a final
volume of 195 mL. The title compound was collected by filtration,
washed with water and dried (6.95 g, 94%): mp: 221e223 ꢁC; IR
ethylsulfonylamino-2H-1-benzopyran (18b) (0.5 g, 1.76 mmol) in
methylene chloride (20 mL). The solution was refluxed for 30 min.
Thereafter, the mixture was cooled to 0 ꢁC. The resulting precipitate
was collected by filtration, washed with cold methylene chloride
and dried.
(KBr)
d6, 500 MHz):
y
: 1154 and 1322 (S]O), 3241 (NeH) cmꢂ1. 1H NMR (DMSO-
: 1.29 (s, 6H, CH3), 2.75 (s, 2H, CH2), 2.91 (s, 3H, e
d
5 . 1. 7 . 1. R / S - N - 3 - C y a n o p h e n y l - N ʹ - ( 3 , 4 - d i h y d r o - 6 -
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
(19). Was obtained from 18a (0.5 g, 1.85 mmol) and 3-cyanophenyl
isothiocyanate (356 mg, 2.22 mmol): yield: 0.772 g, 97%; mp: 198e
NHSO2CH3), 6.82 (d, 1H, 8-H), 7.11 (dd, 1H, 7-H), 7.66 (d, 1H, 5-H),
9.45 (s,1H, eNHSO2CH3),11.32 (s,1H, ¼NeOH); 13C NMR (DMSO-d6,
125 MHz) d: 150.89, 146.75, 130.95, 124.31, 118.48, 118.37, 115.51,
75.35, 38.60, 33.16, 26.35. The product was used in the next step
without further purification.
200 ꢁC; IR (KBr)
y
: 1156 and 1320 (S]O), 1519 (C]S), 2231 (C^N),
3313 (NeH) cmꢂ1
.
1H NMR (DMSO-d6, 500 MHz):
d: 1.27 (s, 3H,
CH3), 1.38 (s, 3H, CH3), 1.80 (t, 1H, CH2), 2.16 (m, 1H, CH2), 2.91 (s, 3H,
eNHSO2CH3), 5.81 (m, 1H, CH), 6.75 (d, 1H, 8-H), 7.04 (dd, 1H, 7-H),
7.18 (d, 1H, 5-H), 7.51e7.58 (m, 2H, 40-H and 50-H), 7.73 (d, 1H, 60-H),
7.98 (s, 1H, 2ʹ-H), 8.34 (d, 1H, CHeNHeCSeNHe), 9.36 (s, 1H, NH),
5.1.4. 3,4-Dihydro-2,2-dimethyl-6-ethylsulfonylamino-2H-1-
benzopyran-4-hydroxyimine (17b)
Compound 17b was prepared following the same procedure as
that described for compound 17a, starting from compound 16b (7 g,
24.71 mmol), potassium carbonate (6.83 g, 49.42 mmol) and hy-
droxylamine hydrochloride (3.43 g, 49.42 mmol) (7.22 g, 98%): mp:
9.85 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz)
d:180.80, 150.50,
139.94,130.43,129.99,128.23,127.81,126.36,122.60,122.48,120.70,
118.65, 117.41, 111.30, 75.38, 47.23, 38.67, 37.77, 29.22, 24.17. Anal.
(C20H22N4O3S2) theoretical: C, 55.79; H, 5.15; N, 13.01; S, 14.89.
Found: C, 55.57; H, 5.08; N, 13.20; S, 14.64.
207e209 ꢁC; IR (KBr)
NMR (DMSO-d6, 500 MHz):
6H, CH3), 2.75 (s, 2H, CH2), 2.99 (q, 2H, eNHSO2CH2CH3), 6.81 (d,1H,
y
: 1153 and 1321 (S]O), 3251 (NeH) cmꢂ1. 1H
: 1.20 (t, 3H, eNHSO2CH2CH3), 1.29 (s,
d
8-H), 7.11 (dd, 1H, 7-H), 7.66 (d, 1H, 5-H), 9.53 (s, 1H,
e
5 . 1. 7 . 2 . R / S - N - 4 - C y a n o p h e n y l - N ʹ - ( 3 , 4 - d i h y d r o - 6 -
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
(20). Was obtained from 18a (0.5 g, 1.85 mmol) and 4-cyanophenyl
NHSO2CH2CH3), 11.32 (s, 1H, ¼NeOH); 13C NMR (DMSO-d6,
125 MHz) d: 150.70, 146.75, 130.95, 123.99, 118.50, 118.36, 115.09,

X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
43
isothiocyanate (356 mg, 2.22 mmol): yield: 0.677 g, 85%; mp: 154e
156 ꢁC; IR (KBr)
: 1149 and 1317 (S]O), 1537 (C]S), 2228 (C^N),
3254 (NeH) cmꢂ1 1H NMR (DMSO-d6, 500 MHz):
: 1.27 (s, 3H,
CH3),1.39 (s, 3H, CH3),1.79 (t,1H, CH2), 2.20 (m,1H, CH2), 2.92 (s, 3H,
eNHSO2CH3), 5.78 (m, 1H, CH), 6.75 (d, 1H, 8-H), 7.05 (dd, 1H, 7-H),
7.18 (d, 1H, 5-H), 7.75 (m, 4H, 20-H, 60-H, 30-H and 5ʹ-H), 8.49 (d, 1H,
CHeNHeCSeNHe), 9.35 (s, 1H, NH), 10.02 (s, 1H, NH); 13C NMR
(s, 3H, CH3), 1.76 (t, 1H, CH2), 2.10 (m, 1H, CH2), 2.88 (s, 3H, e
NHSO2CH3), 4.96 (m, 1H, CH), 6.73 (d, 1H, 8-H), 6.75 (d, 1H, CHe
NHCONH-), 7.03 (dd, 1H, 7-H), 7.14 (d, 1H, 5-H), 7.63 (d, 2H, 30-H and
50-H), 7.70 (d, 2H, 20-H and 6ʹ-H), 9.08 (s, 1H, NH), 9.31 (s, 1H, NH);
y
.
d
13C NMR (DMSO-d6, 125 MHz)
d: 154.63, 150.54, 144.76, 133.22,
130.40, 123.62, 122.44, 120.91, 119.41, 117.67, 117.35, 102.61, 75.24,
42.30, 40.23, 38.65, 29.06, 24.43. Anal. (C20H22N4O3S2) theoretical:
C, 57.96; H, 5.35; N, 13.52; S, 7.73. Found: C, 58.16; H, 5.35; N, 13.59;
S, 7.33.
(DMSO-d6, 125 MHz)
d: 180.35, 150.58, 143.76, 132.87, 130.38,
122.71, 122.35, 121.96, 120.82, 119.07, 117.47, 105.08, 75.36, 47.12,
38.71, 37.71, 29.17, 24.19. Anal. (C20H22N4O3S2) theoretical: C, 55.79;
H, 5.15; N, 13.01; S, 14.89. Found: C, 55.82; H, 5.11; N, 12.99; S, 15.22.
5 .1. 7 . 7 . R / S - N - 3 - C h l o r o p h e n y l - N ʹ - ( 3 , 4 - d i h y d r o - 6 -
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (25).
Was obtained from 18a (0.5 g, 1.85 mmol) and 3-chlorophenyl
5 .1. 7 . 3 . R / S - N - 3 - C h l o r o p h e n y l - N ʹ - ( 3 , 4 - d i h y d r o - 6 -
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
(21). Was obtained from 18a (0.5 g, 1.85 mmol) and 3-chlorophenyl
isocyanate (371
189e191 ꢁC; IR (KBr)
(NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
d
m
l, 341 mg, 2.22 mmol): yield: 0.753 g, 96%; mp:
: 1152 and 1313 (S]O), 1680 (C]O), 3381
: 1.27 (s, 3H, CH3), 1.38
y
isothiocyanate (291
184e185 ꢁC; IR (KBr)
(NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
m
l, 377 mg, 2.22 mmol): yield: 0.618 g, 76%; mp:
: 1148 and 1317 (S]O), 1532 (C]S), 3252
: 1.26 (s, 3H, CH3), 1.38
y
(s, 3H, CH3), 1.75 (t, 1H, CH2), 2.10 (m, 1H, CH2), 2.88 (s, 3H, e
NHSO2CH3), 4.95 (m, 1H, CH), 6.61 (d, 1H, CHeNHCONH-), 6.72 (d,
1H, 8-H), 6.97 (d, 1H, 40-H), 7.03 (dd, 1H, 7-H), 7.15 (d, 1H, 5-H),
7.22e7.28 (m, 2H, 50-H and 60-H), 7.74 (s, 1H, 2ʹ-H), 8.74 (s, 1H, NH),
d
(s, 3H, CH3), 1.80 (t, 1H, CH2), 2.16 (m, 1H, CH2), 2.91 (s, 3H, e
NHSO2CH3), 5.80 (m, 1H, CH), 6.74 (d, 1H, 8-H), 7.04 (dd, 1H, 7-H),
7.17 (s, 2H, 5-H and 40-H), 7.34 (d, 2H, 50-H and 60-H), 7.67 (s, 1H, 2ʹ-
H), 8.22 (d, 1H, eCH-NH-CS-NH-), 9.35 (s, 1H, NH), 9.73 (s, 1H, NH);
9.32 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz)
d: 154.94, 150.55,
141.85, 133.11, 130.37, 130.27, 123.83, 122.42, 120.94, 120.85, 117.31,
117.22, 116.21, 75.26, 42.24, 40.32, 38.64, 29.09, 24.42. Anal.
(C19H22ClN3O4S) theoretical: C, 53.83; H, 5.23; N, 9.91; S, 7.56.
Found: C, 53.91; H, 5.24; N, 10.08; S, 7.21.
13C NMR (DMSO-d6, 125 MHz)
d: 180.63, 150.53, 140.51, 132.69,
130.37, 130.22, 124.02, 122.87, 122.71, 122.51, 121.85, 120.71, 117.38,
75.38, 47.16, 38.68, 37.79, 29.22, 24.19. Anal. (C19H22ClN3O3S2)
theoretical: C, 51.87; H, 5.04; N, 9.55; S, 14.57. Found: C, 51.88; H,
4.99; N, 9.72; S, 14.44.
5 .1. 7 . 8 . R / S - N - 4 - C h l o r o p h e n y l - N ʹ - ( 3 , 4 - d i h y d r o - 6 -
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (26).
Was obtained from 18a (0.5 g, 1.85 mmol) and 4-chlorophenyl
5 .1. 7 . 4 . R / S - N - 4 - C h l o r o p h e n y l - N ʹ - ( 3 , 4 - d i h y d r o - 6 -
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
(22). Was obtained from 18a (0.5 g, 1.85 mmol) and 4-chlorophenyl
isothiocyanate (377 mg, 2.22 mmol): yield: 0.740 g, 91%; mp: 165e
isocyanate (284
248e250 ꢁC; IR (KBr)
(NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
m
l, 341 mg, 2.22 mmol): yield: 0.768 g, 98%; mp:
: 1148 and 1318 (S]O), 1678 (C]O), 3382
: 1.27 (s, 3H, CH3), 1.38
y
d
167 ꢁC; IR (KBr)
y
: 1147 and 1317 (S]O), 1539 (C]S), 3254 (NeH)
(s, 3H, CH3), 1.74 (t, 1H, CH2), 2.09 (m, 1H, CH2), 2.88 (s, 3H, e
NHSO2CH3), 4.95 (m, 1H, CH), 6.55 (d, 1H, CHeNHCONH-), 6.72 (d,
1H, 8-H), 7.03 (d, 1H, 7-H), 7.15 (s, 1H, 5-H), 7.29 (d, 2H, 30-H and 50-
H), 7.48 (d, 2H, 20-H and 6ʹ-H), 8.66 (s, 1H, NH), 9.31 (s, 1H, NH); 13C
cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
d
: 1.26 (s, 3H, CH3), 1.37 (s, 3H,
CH3), 1.78 (t, 1H, CH2), 2.14 (m, 1H, CH2), 2.91 (s, 3H, eNHSO2CH3),
5.80 (m, 1H, CH), 6.73 (d, 1H, 8-H), 7.03 (dd, 1H, 7-H), 7.17 (d, 1H, 5-
H), 7.37 (d, 2H, 30-H and 50-H), 7.46 (d, 2H, 20-H and 6ʹ-H), 8.12 (d,1H,
eCHeNHeCSeNHe), 9.34 (s, 1H, NH), 9.69 (s, 1H, NH); 13C NMR
NMR (DMSO-d6, 125 MHz) d: 155.01, 150.56, 139.30, 130.36, 128.50,
124.70, 123.91, 122.45, 121.00, 119.33, 117.30, 75.26, 42.20, 40.44,
38.64, 29.08, 24.45. Anal. (C19H22ClN3O3S) theoretical: C, 53.83; H,
5.23; N, 9.91; S, 7.56. Found: C, 53.71; H, 5.20; N, 9.95; S, 7.52.
(DMSO-d6, 125 MHz)
d: 180.67, 150.54, 137.81, 130.36, 128.54,
128.34, 125.34, 122.82, 122.58, 120.76, 117.35, 75.39, 47.23, 38.65,
37.81, 29.26, 24.15. Anal. (C19H22ClN3O3S2) theoretical: C, 51.87; H,
5.04; N, 9.55; S, 14.57. Found: C, 51.53; H, 4.97; N, 9.62; S, 14.26.
5.1.7.9. R/S-N-3-Cyanophenyl-Nʹ-(6-ethylsulfonylamino-3,4-dihydro-
2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (27). Was obtained
from 18b (0.5 g, 1.76 mmol) and 3-cyanophenyl isothiocyanate
(338 mg, 2.11 mmol): yield: 0.594 g, 76%; mp: 115e117 ꢁC; IR (KBr)
5 .1. 7 . 5 . R / S - N - 3 - C y a n o p h e n y l - N ʹ - ( 3 , 4 - d i h y d r o - 6 -
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (23).
was obtained from 18a (0.5 g, 1.85 mmol) and 3-cyanophenyl iso-
cyanate (320 mg, 2.22 mmol): yield: 0.728 g, 95%; mp: 209e211 ꢁC;
y
: 1142 and 1314 (S]O), 1531 (C]S), 2232 (C^N), 3306 (NeH)
cmꢂ1 1H NMR (DMSO-d6, 500 MHz):
.
d:
1.22 (t, 3H, e
IR (KBr)
y
: 1154 and 1311 (S]O), 1683 (C]O), 2233 (C^N), 3350
NHSO2CH2CH3), 1.26 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.81 (t, 1H, CH2),
2.15 (m, 1H, CH2), 3.00 (q, 2H, eNHSO2CH2CH3), 5.80 (m, 1H, CH),
6.73 (d, 1H, 8-H), 7.03 (dd, 1H, 7-H), 7.18 (d, 1H, 5-H), 7.55 (m, 2H, 40-
H and 50-H), 7.73 (d, 1H, 60-H), 7.97 (s, 1H, 2ʹ-H), 8.32 (d, 1H, CHe
NHCSNH-), 9.44 (s, 1H, NH), 9.84 (s, 1H, NH); 13C NMR (DMSO-d6,
(NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
d: 1.27 (s, 3H, CH3), 1.38
(s, 3H, CH3), 1.77 (t, 1H, CH2), 2.09 (m, 1H, CH2), 2.88 (s, 3H, e
NHSO2CH3), 4.96 (m, 1H, CH), 6.73 (d, 2H, 8-H and CHe NHCONH-),
7.03 (dd, 1H, 7-H), 7.15 (d, 1H, 5-H), 7.37 (d, 1H, 40-H), 7.46 (t, 1H, 50-
H), 7.65 (d, 1H, 60-H), 8.00 (s, 1H, 2ʹ-H), 8.90 (s, 1H, NH), 9.31 (s, 1H,
125 MHz) d: 180.87, 150.27, 139.94, 130.35, 129.99, 128.26, 127.83,
NH); 13C NMR (DMSO-d6, 125 MHz)
d
: 155.46, 151.02, 141.70, 130.91,
126.40, 122.64, 122.12, 120.31, 118.63, 117.39, 111.29, 75.36, 47.21,
44.51, 37.74, 29.23, 24.18, 7.98. Anal. (C21H24N4O3S2) theoretical: C,
56.74; H, 5.44; N, 12.60; S, 14.42. Found: C, 56.48; H, 5.52; N, 12.65;
S, 14.03.
130.58, 125.23, 124.25, 122.97, 122.89, 121.40, 120.88, 119.45, 117.81,
111.95, 75.74, 42.79, 39.77, 39.12, 29.60, 24.89. Anal. (C20H22N4O4S2)
theoretical: C, 57.96; H, 5.35; N, 13.52; S, 7.73. Found: C, 57.76; H,
5.31; N, 13.51; S, 7.63.
5.1.7.10. R/S-N-4-Cyanophenyl-Nʹ-(6-ethylsulfonylamino-3,4-
5 .1. 7 . 6 . R / S - N - 4 - C y a n o p h e n y l - N ʹ - ( 3 , 4 - d i h y d r o - 6 -
methylsulfonylamino-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (24).
Was obtained from 18a (0.5 g, 1.85 mmol) and 4-cyanophenyl iso-
cyanate (320 mg, 2.22 mmol): yield: 0.759 g, 99%; mp: 253e255 ꢁC;
dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
(28).
Was obtained from 18b (0.5 g, 1.76 mmol) and 4-cyanophenyl iso-
thiocyanate (338 mg, 2.11 mmol): yield: 0.571 g, 73%; mp: 193e
195 ꢁC; IR (KBr)
y
: 1140 and 1318 (S]O), 1546 (C]S), 2233 (C^N),
3300 (NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
: 1.21 (t, 3H, e
NHSO2CH2CH3), 1.27 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.79 (t, 1H, CH2),
IR (KBr)
y
: 1151 and 1323 (S]O), 1685 (C]O), 3332 (C^N), 3375
d
(NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
d: 1.27 (s, 3H, CH3), 1.38

44
X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
2.19 (m, 1H, CH2), 3.00 (q, 2H, eNHSO2CH2CH3), 5.78 (m, 1H, CH),
6.74 (d, 1H, 8-H), 7.03 (dd, 1H, 7-H), 7.18 (d, 1H, 5-H), 7.75 (m, 4H, 20-
H, 30-H, 50-H and 6ʹ-H), 8.47 (d, 1H, CHeNHCSNH-), 9.43 (s, 1H, NH),
2.09 (m, 1H, CH2), 2.96 (q, 2H, eNHSO2CH2CH3), 4.95 (m, 1H, CH),
6.71 (d,1H, 8-H), 6.75 (d,1H, CHeNHCONH-), 7.02 (dd,1H, 7-H), 7.15
(d, 1H, 5-H), 7.63 (d, 2H, 30-H and 50-H), 7.70 (d, 2H, 20-H and 6ʹ-H),
10.02 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz)
d: 180.40, 150.35,
9.08 (s, 1H, NH), 9.38 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz)
d:
143.76, 132.87, 130.33, 122.39, 122.32, 121.98, 120.42, 119.07, 117.46,
105.10, 75.33, 47.10, 44.53, 37.70, 29.17, 24.19, 7.99. Anal.
(C21H24N4O3S2) theoretical: C, 56.74; H, 5.44; N, 12.60; S, 14.42.
Found: C, 57.13; H, 5.55; N, 12.76; S, 14.23.
154.65, 150.30, 144.77, 133.21, 130.38, 123.68, 122.10, 120.52, 119.41,
117.67, 117.33, 102.61, 75.21, 44.50, 42.32, 39.18, 29.07, 24.41, 7.94.
Anal. (C21H24N4O4S) theoretical: C, 58.86; H, 5.65; N, 13.07; S, 7.48.
Found: C, 58.50; H, 5.59; N, 13.15; S, 7.23.
5.1.7.11. R/S-N-3-Chlorophenyl-Nʹ-(6-ethylsulfonylamino-3,4-
5.1.7.15. R/S-N-3-Chlorophenyl-Nʹ-(6-ethylsulfonylamino-3,4-
dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (33). Was ob-
tained from 18b (0.5 g, 1.76 mmol) and 3-chlorophenyl isocyanate
dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
Was obtained from 18b (0.5 g, 1.76 mmol) and 3-chlorophenyl
isothiocyanate (277 l, 358 mg, 2.11 mmol): yield: 0.575 g, 72%;
mp: 177e179 ꢁC; IR (KBr)
: 1140 and 1315 (S]O), 1533 (C]O),
3293 (NeH) cmꢂ1. 1H NMR (DMSO-d6, 500 MHz):
: 1.20 (t, 3H, e
(29).
m
(257
IR (KBr)
NMR (DMSO-d6, 500 MHz):
m
l, 324 mg, 2.11 mmol): yield: 0.554 g, 72%; mp: 214e216 ꢁC;
y
y
: 1143 and 1318 (S]O), 1686 (C]O), 3381 (NeH) cmꢂ1. 1H
: 1.17 (t. 3H, eNHSO2CH2CH3), 1.26 (s,
d
d
NHSO2CH2CH3), 1.26 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.80 (t, 1H, CH2),
2.14 (m, 1H, CH2), 3.00 (q, 2H, eNHSO2CH2CH3), 5.80 (m, 1H, CH),
6.72 (d, 1H, 8-H), 7.02 (dd, 1H, 7-H), 7.17 (s, 2H, 5-H and 40-H), 7.34
(d, 2H, 50-H and 60-H), 7.67 (s, 1H, 2ʹ-H), 8.21 (d, 1H, CHeNHCSNHe),
3H, CH3), 1.37 (s, 3H, CH3), 1.75 (t, 1H, CH2), 2.08 (m, 1H, CH2), 2.97
(q, 2H, eNHSO2CH2CH3), 4.94 (m, 1H, CH), 6.60 (d, 1H, CHe
NHCONH-), 6.70 (d, 1H, 8-H), 6.97 (d, 1H, 40-H), 7.01 (dd, 1H, 7-H),
7.15 (d, 1H, 5-H), 7.25 (m, 2H, 50-H and 60-H), 7.74 (s, 1H, 2ʹ-H), 8.75
9.43 (s, 1H, NH), 9.73 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz)
d:
(s, 1H, NH), 9.38 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz)
d:
180.68,150.29,140.50,132.70,130.32,130.22,124.04,122.91,122.75,
122.11, 121.87, 120.31, 117.36, 75.36, 47.14, 44.51, 37.77, 29.23, 24.18,
7.99. Anal. (C20H24ClN3O3S2) theoretical: C, 52.91; H, 5.33; N, 9.26;
S, 14.12. Found: C, 52.96; H, 5.30; N, 9.30; S, 13.79.
154.97, 150.33, 141.85, 133.11, 130.32, 130.26, 123.90, 122.11, 120.85,
120.57, 117.30,117.23,116.22, 75.23, 44.49, 42.26, 39.37, 29.10, 24.40,
7.93. Anal. (C20H24ClN3O4S) theoretical: C, 54.85; H, 5.52; N, 9.59; S,
7.32. Found: C, 55.06; H, 5.58; N, 9.84; S, 7.02.
5.1.7.12. R/S-N-4-Chlorophenyl-Nʹ-(6-ethylsulfonylamino-3,4-
5.1.7.16. R/S-N-4-Chlorophenyl-Nʹ-(6-ethylsulfonylamino-3,4-
dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (34). Was ob-
tained from 18b (0.5 g, 1.76 mmol) and 4-chlorophenyl isocyanate
dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
(30).
Was obtained from 18b (0.5 g, 1.76 mmol) and 4-chlorophenyl
isothiocyanate (358 mg, 2.11 mmol): yield: 0.79 g, 99%; mp: 166e
(270
IR (KBr)
NMR (DMSO-d6, 500 MHz): d
m
l, 324 mg, 2.11 mmol): yield: 0.762 g, 99%; mp: 234e236 ꢁC;
168 ꢁC; IR (KBr)
y
: 1130 and 1310 (S]O), 1531 (C]O), 3309 (NeH)
y
: 1142 and 1312 (S]O), 1679 (C]O), 3379 (NeH) cmꢂ1. 1H
: RMN 1H (DMSO-d6, 500 MHz): 1.18
cmꢂ1
.
1H NMR (DMSO-d6, 500 MHz):
d
:
1.20 (t, 3H,
e
NHSO2CH2CH3), 1.25 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.78 (t, 1H, CH2),
2.14 (m, 1H, CH2), 3.00 (q, 2H, eNHSO2CH2CH3), 5.79 (m, 1H, CH),
6.72 (d, 1H, 8-H), 7.02 (dd, 1H, 7-H), 7.17 (d, 1H, 5-H), 7.37 (d, 2H, 30-
H and 50-H), 7.46 (d, 2H, 20-H and 6ʹ-H), 8.10 (d, 1H, CHeNHCSNH-),
(t, 3H, eNHSO2CH2CH3), 1.26 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.74 (t,
1H, CH2), 2.08 (m, 1H, CH2), 2.96 (q, 2H, eNHSO2CH2CH3), 4.94 (m,
1H, CH), 6.54 (d, 1H, CHeNHCONH-), 6.70 (d, 1H, 8-H), 7.01 (dd, 1H,
7-H), 7.15 (d, 1H, 5-H), 7.29 (d, 2H, 30-H and 50-H), 7.47 (d, 2H, 20-H
and 6ʹ-H), 8.66 (s, 1H, NH), 9.38 (s, 1H, NH); 13C NMR (DMSO-d6,
9.43 (s, 1H, NH), 9.68 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz)
d:
180.71, 150.32, 137.79, 130.29, 128.54, 128.37, 125.38, 122.86, 122.14,
120.33, 117.34, 75.37, 47.23, 44.46, 37.79, 29.27, 24.15, 8.00. Anal.
(C20H24ClN3O3S2) theoretical: C, 52.91; H, 5.33; N, 9.26; S, 14.12.
Found: C, 53.10; H, 5.55; N, 9.00; S, 13.74.
125 MHz) d: 155.03, 150.33, 139.31, 130.34, 128.49, 124.70, 123.96,
122.11, 120.62, 119.33, 117.28, 75.23, 44.49, 42.21, 39.44, 29.09,
24.44, 7.94. Anal. (C20H24ClN3O4S) theoretical: C, 54.85; H, 5.52; N,
9.59; S, 7.32. Found: C, 55.08; H, 5.61; N, 9.81; S, 7.04.
5.1.7.13. R/S-N-3-Cyanophenyl-Nʹ-(6-ethylsulfonylamino-3,4-
dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (31). Was ob-
tained from 18b (0.5 g, 1.76 mmol) and 3-cyanophenyl isocyanate
(304 mg, 2.11 mmol): yield: 0.663 g, 88%; mp: 161e163 ꢁC; IR (KBr)
5.2. Biological assays
(ꢀ)-Cromakalim (Tocris, UK), diazoxide (Sigma Chemical Co,
USA) and verapamil (Sigma Chemical Co, USA) were used as
reference compounds. All experiments were performed with aortae
or pancreatic islets isolated from adult fed Wistar Rats (Charles
River Laboratories, Belgium). The laboratory animal care was
approved by the local ethic’s committee of the Université Libre de
Bruxelles.
y
: 1142 and 1316 (S]O), 1689 (C]O), 2241 (C^N), 3358 (NeH)
cmꢂ1 1H NMR (DMSO-d6, 500 MHz):
.
d:
1.18 (t, 3H, e
NHSO2CH2CH3), 1.26 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.77 (t, 1H, CH2),
2.08 (m, 1H, CH2), 2.97 (q, 2H, eNHSO2CH2CH3), 4.95 (m, 1H, CH),
6.70e6.73 (m, 2H, 8-H and CHeNHCONH-), 7.01 (dd, 1H, 7-H), 7.16
(d, 1H, 5-H), 7.37 (d, 1H, 40-H), 7.46 (t, 1H, 50-H), 7.65 (d, 1H, 60-H),
8.00 (s, 1H, 2ʹ-H), 8.90 (s, 1H, NH), 9.38 (s,1H, NH); 13C NMR (DMSO-
5.2.1. Measurement of insulin secretion from incubated rat
pancreatic islets
d6, 125 MHz) d: 154.99,150.32,141.21,130.32,130.08,124.74,123.83,
122.48, 122.07, 120.53, 120.41, 118.96, 117.31, 111.46, 75.23, 44.49,
42.32, 39.22, 29.11, 24.38, 7.93. Anal. (C21H24N4O4S) theoretical: C,
58.86; H, 5.65; N, 13.07; S, 7.48. Found: C, 58.58; H, 5.62; N, 13.14; S,
7.12.
Pancreatic islets were isolated by the collagenase method and
freshly isolated islets were used for measurements of insulin
secretion [27,34].
Groups of 10 islets, each derived from the same batch of islets,
were pre-incubated for 30 min at 37 ꢁC in 1 mL of a physiological
salt medium (in mM: NaCl 115, KCl 5, CaCl2 2.5, MgCl2 1, NaHCO3
24) supplemented with 2.8 mM glucose, 0.5% (w/v) albumin (RIA
grade) and equilibrated against a mixture of O2 (95%) and CO2 (5%).
The islets were then incubated at 37 ꢁC for a further 90 min in 1 mL
of the same medium containing 16.7 mM glucose and, in addition,
either the reference compounds or the required chroman deriva-
tive. The release of insulin was measured radioimmunologically
5.1.7.14. R/S-N-4-Cyanophenyl-Nʹ-(6-ethylsulfonylamino-3,4-
dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (32). Was ob-
tained from 18b (0.5 g, 1.76 mmol) and 4-cyanophenyl isocyanate
(304 mg, 2.11 mmol): yield: 0.746 g, 99%; mp: 239e241 ꢁC; IR (KBr)
y
: 1148 and 1318 (S]O), 1682 (C]O), 2226 (C^N), 3376 (NeH)
cmꢂ1 1H NMR (DMSO-d6, 500 MHz):
1.18 (t, 3H,
NHSO2CH2CH3), 1.26 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.77 (t, 1H, CH2),
.
d:
e

X. Florence et al. / European Journal of Medicinal Chemistry 80 (2014) 36e46
45
using rat insulin as a standard. Residual insulin secretion was
Acknowledgments
expressed as a percentage of the value recorded in control exper-
iments (100%); that is in the absence of drug and presence of
16.7 mM glucose [27,34].
This study was supported in part by grants from the “Formation
to Research in Industry and Agriculture” (F.R.I.A., Belgium) and from
the “National Fund for Scientific Research” (F.N.R.S., Belgium) from
which P. de Tullio is a Senior Research Associate and P. Lebrun is a
Research Director. The authors gratefully acknowledge the tech-
nical assistance of Y. Abrassart, S. Counerotte, P. Fraikin, F. Leleux,
A.-M. Vanbellinghen and A. Van Praet.
5.2.2. Measurement of myorelaxant activity on rat aorta rings
The rat thoracic aorta was removed, cut into transverse rings
(3e4 mm), and adhering fat and connective tissue was detached
[34,37,38]. After removal of the endothelium by rubbing the intimal
surface with a cotton wad, the segments were suspended under
1.5 g tension in an organ bath containing 20 mL of a buffered
physiological solution (in mM: NaCl 118, KCl 4.7, CaCl2 2.5, NaHCO3
25, KH2PO4 1.2, MgSO4 1.2, glucose 5). The solution was maintained
at 37 ꢁC and continuously oxygenated with a mixture of 95% O2 and
5% CO2. After equilibration for 60 min, isometric contractions were
measured with a force-displacement transducer. Contractile activ-
ity was induced by increasing the extracellular concentration of Kþ
(30 or 80 mM KCl). When the tension was stabilized, drugs were
added cumulatively until maximal relaxation or until a maximum
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.024.
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