Modulation of the 6-position of benzopyran derivatives and inhibitory effects on the insulin releasing process

Article abstract of DOI:10.1016/j.bmc.2011.05.040

The synthesis of different series of 4- and 6-substituted R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans is described. All of these new benzopyran derivatives were bearing, at the 4-position, a phenylthiourea moiety substituted on the phenyl ring by a meta or a para-electron-withdrawing group such as Cl or CN. The study aimed at exploring the influence of the nature of the substituent at the 6-position in order to develop new benzopyran-type K _ATP channel activators exhibiting an improved selectivity towards the insulin secreting cells. The original compounds were examined in vitro on rat pancreatic islets (inhibition of insulin release) as well as on rat aorta rings (vasorelaxant effect) and their activity was compared to that of the reference K_ATP channel activators (±)-cromakalim, (±)-pinacidil, diazoxide and to previously synthesized cromakalim analogues. Structure-activity relationships indicated that the inhibitory effect on the insulin secreting cells was related to the lipophilicity of the molecules and to the size of the substituent located at the 6-position. A marked inhibitory activity on the insulin secretory process was obtained with molecules bearing a bulky tert-butyloxycarbonylamino group at the 6-position (20-23). The latter compounds were found to have the same efficacy on the pancreatic endocrine tissue than some previously described molecules. Lastly, radioisotopic experiments further identified R/S-N-4-chlorophenyl-N′-(6-tert-butyloxycarbonylamino-3,4- dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (23) as a K_ATP channel opener.

Full text of DOI:10.1016/j.bmc.2011.05.040

Bioorganic & Medicinal Chemistry 19 (2011) 3919–3928
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Modulation of the 6-position of benzopyran derivatives and inhibitory
effects on the insulin releasing process
Xavier Florence a, , Sébastien Dilly b, Pascal de Tullio b, Bernard Pirotte b, , Philippe Lebrun a,
⇑
a Laboratoire de Pharmacodynamie et de Thérapeutique, Université Libre de Bruxelles, CP617, Faculté de Médecine, 808, Route de Lennik, B-1070 Brussels, Belgium
b Laboratoire de Chimie Pharmaceutique, Centre Interfacultaire de Recherche du Médicament (Drug Research Center), Université de Liège, C.H.U., 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:
The synthesis of different series of 4- and 6-substituted R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans
is described. All of these new benzopyran derivatives were bearing, at the 4-position, a phenylthiourea
moiety substituted on the phenyl ring by a meta or a para-electron-withdrawing group such as Cl or
CN. The study aimed at exploring the influence of the nature of the substituent at the 6-position in order
to develop new benzopyran-type KATP channel activators exhibiting an improved selectivity towards the
insulin secreting cells. The original compounds were examined in vitro on rat pancreatic islets (inhibition
of insulin release) as well as on rat aorta rings (vasorelaxant effect) and their activity was compared to
that of the reference KATP channel activators ( )-cromakalim, ( )-pinacidil, diazoxide and to previously
synthesized cromakalim analogues. Structure–activity relationships indicated that the inhibitory effect
on the insulin secreting cells was related to the lipophilicity of the molecules and to the size of the sub-
stituent located at the 6-position. A marked inhibitory activity on the insulin secretory process was
obtained with molecules bearing a bulky tert-butyloxycarbonylamino group at the 6-position (20–23).
The latter compounds were found to have the same efficacy on the pancreatic endocrine tissue than some
previously described molecules. Lastly, radioisotopic experiments further identified R/S-N-4-chloro-
phenyl-N0-(6-tert-butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea
(23) as a KATP channel opener.
Received 24 January 2011
Revised 26 April 2011
Accepted 19 May 2011
Available online 24 May 2011
Keywords:
Benzopyran derivatives
ATP-sensitive potassium channels
Potassium channel openers
Cromakalim analogues
Insulin secretion
Smooth muscle contractile activity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
channels. For instance, the assembly of Kir6.2 and SUR1 generates
pancreatic B-cell KATP channels10 whereas the combination of
ATP-sensitive potassium (KATP) channels are transmembrane
proteins which have been initially described, more than 25 years
ago, by Noma1 in cardiac cells. Since then, KATP channels have been
identified in many other excitable tissues into which they play a
large variety of physiological roles.2 Such ionic channels have been
depicted in pancreatic B-cells3 and have been shown to be tightly
involved in the control of the insulin secretory process.4,5 In vascu-
lar smooth muscle cells, KATP channels participate in the control of
muscle tone.6
By coupling their activity to the intracellular concentration of
adenosine triphosphate, KATP channels have been shown to link cell
metabolism to membrane excitability.7
KATP channels consist of an octameric complex of two mem-
brane proteins: the inwardly rectifying potassium channel (Kir6.1,
Kir6.2) forming the pore of the channel and the sulfonylurea recep-
tor (SUR1, SUR2A, SUR2B) which regulates channel activity.2,8,9 The
combination of the different subunits leads to specific tissue KATP
Kir6.1 and SUR2B has been found to be expressed in vascular
smooth muscles.11
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 synthe-
sis of compounds exhibiting a marked specificity and a high selec-
tivity for a single KATP channel subtype.
The potential and recognized therapeutic indications for
potassium channel openers (PCOs) include, among others, the
treatment of arterial hypertension,12,13 angina pectoris,12,14 cardiac
arrhythmias,12,15 bronchial asthma,12,16 urinary incontinence12,17
and androgenic alopecia.12,18 PCOs have also been proposed for
the prevention and/or management of type I, type II diabetes, obes-
ity,12,13,19,20 nesidioblastosis,12 insulinomas12 and polycystic ovary
syndrome.19,21
PCOs such as diazoxide (1), ( )-pinacidil (2) and ( )-cromakalim
(3) exemplify the chemical diversity of this pharmacological family
(Fig. 1). ( )-Cromakalim (3), leader of the benzopyran derivatives,
has been found to exert a marked myorelaxant activity22,23
although the drug has also been reported to be slightly active
as an inhibitor of the insulin secretory process.24 By contrast,
⇑
Corresponding author. Tel.: +32 2 555 60 91; fax: +32 2 555 63 70.
E-mail address: xavier.florence@ulb.ac.be (X. Florence).
These authors assumed an equal supervision.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.040

3920
X. Florence et al. / Bioorg. Med. Chem. 19 (2011) 3919–3928
oxy group located at the para-position of the acetamido moiety.
Treatment of compound 10 with acetone, in the presence of pyrrol-
idine led to chromanone intermediate 11.
The previously described way of synthesis of the 4-amino-
substituted benzopyran key intermediates by the Ritter reaction28
did not give easy access to the target compounds 15a and 15b, due
to hydrolysis sensitivity of the molecules. Thus, we have developed
an original scheme of synthesis that allowed us to obtain, more
rapidly and with an improved yield, the desired compounds.
The acetamido group of intermediate 11 was hydrolyzed in an
alcoholic solution of diluted hydrochloric acid. The amine function
of the resulting intermediate (12) was protected by a tert-butyl-
oxycarbonyl group (t-BOC) after treatment of compound 12 with
di-tert-butyl dicarbonate (t-BOC anhydride or BOC2O), providing
compound 13.
In order to obtain the key intermediates 15a–b, the two ketonic
compounds 11 or 13 were treated with hydroxylamine hydrochlo-
ride and potassium carbonate to give the oxime intermediates
14a–b which were further hydrogenated in the presence of Raney-
NickelÒ.
Diazoxide (1)
( )-Pinacidil (2)
4
6
( )-Cromakalim (3)
4 : R3 = CN, R4 = H
5 : R3 = H, R4 = CN
6 : R3 = Cl, R4 = H
7 : R3 = H, R4 = Cl
Figure 1. Chemical structure of diazoxide (1), ( )-pinacidil (2), ( )-cromakalim (3)
and compounds 4–7.
R/S-N-(m/p-substituted)phenyl-N0-(6-acetamido-3,4-dihydro-2,
2-dimethyl-2H-1-benzopyran-4-yl)thioureas (16–19) and R/S-N-
(m/p-substituted)phenyl-N0-(6-tert-butyloxycarbonylamino-3,4-di
hydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thioureas (20–23) were
obtained from the reaction of the amines 15a–b with the appropri-
ate phenyl isothiocyanate (R–N@C@S) (Scheme 2).
To prepare compounds 24–27, the t-BOC protective group of
compounds 20–23 was removed by treatment with diluted hydro-
chloric acid in ethanol.
diazoxide (1) has been reported to be equipotent on the vascular
smooth muscle and the insulin secreting-cells.24,25
Recently, by exploring a series of R/S-3,4-dihydro-2,2-dimethyl-
6-halo-2H-1-benzopyrans structurally related to cromakalim, we
have identified four derivatives (4–7) exhibiting a modified tissue
selectivity profile.26,27 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 were found
to be less effective as vasorelaxants but more potent as inhibitors
of the insulin secretory process than the reference molecules diaz-
oxide (1) and ( )-cromakalim (3).27 Structure–activity relation-
ships further indicated that the nature of the substituent located
at the 4-position on the benzopyran nucleus played a crucial role
in the expression of an inhibitory effect on insulin release.
Molecules bearing, at the 4-position, a phenylthiourea moiety
substituted on the phenyl ring by a meta- or a para- electron-with-
Finally, the reaction of compounds 24–27 with acetic formic
anhydride, generated in situ by the reaction of formic acid with
acetic anhydride, provided compounds 28–31.
3. Results and discussion
The ability of the original compounds (Table 1, compounds
16–31) to inhibit the insulin releasing process was evaluated on
isolated rat pancreatic islets incubated in the presence of an insu-
linotropic glucose concentration (16.7 mM). The vasorelaxant
activity of the compounds was determined on 30 mM K+-depolar-
ized rat aorta (endothelium-free) rings.
drawing group (a cyano group or
a chlorine atom) (Fig 1,
compounds 4–7) were found to be much more potent and selective
for the pancreatic B-cells than the reference molecule ( )-cromaka-
lim (3).27
( )-Cromakalim (1), diazoxide (2) and ( )-pinacidil (3) were
used as reference PCOs (Fig. 1). The activity of the original chroman
derivatives was also compared to that of previously described mol-
ecules (4–7)26,27 (Fig 1).
Biological results obtained with classical KATP channel openers
indicated that ( )-cromakalim and ( )-pinacidil, at a 10 lM con-
centration, were roughly inactive on the pancreatic endocrine tis-
sue (percentage of residual insulin secretion: 94.4 4.1% and
92.1 3.9%, respectively) (Table 1). Diazoxide (10 lM), however,
In the light of such data, and keeping the same substitutions at
the 4-position, the present work aimed at exploring the influence
of the nature of the substituent at the 6-position in order to
develop new 4,6-disubstituted R/S-2,2-dimethylchromans exhibiting
an improved selectivity towards insulin secreting cells. The newly
synthesized compounds bearing a 6-amino, a 6-formamido, a 6-
acetamido or
a
6-tert-butyloxycarbonylamino group were
examined in vitro as putative potassium channel openers on rat
pancreatic islets as well as on rat aorta rings.
provoked a 25% reduction in the insulin secretory rate (Table 1).
By contrast, previously synthesized benzopyrans (4–7) were found
to be much more potent than diazoxide at inhibiting the glucose-
induced insulin release (Table 1).
Our previous investigations indicated that an electron-with-
drawing group, such as a chlorine atom or a cyano group at the
meta or para position of the phenyl ring of R/S-N-phenyl-N0-(6-bro-
mo-3,4-dihydro-2,2-dimethyl-4-2H-1-benzopyran-4-yl)thioureas,
enhanced the inhibitory activity on the insulin secretory rate.26,27
Therefore, we decided to keep such an electron-withdrawing group
at the 3- or 4-position and further explored new moieties located
at the 6-position.
2. Chemistry
The key intermediates (15a–b) giving access to the target
compounds (16–31), R/S-6-acetamido-4-amino-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran (15a) and R/S-4-amino-6-tert-butyl-
oxy carbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran
(15b) were, respectively, synthesized in five and seven steps, start-
ing from 4-methoxyaniline (Scheme 1).
The synthesis of the common intermediate, 6-acetamido-3,4-
dihydro-2,2-dimethyl-2H-1-benzopyran-4-one (11), was achieved
in three steps. First, 4-methoxyaniline (8) was acetylated by acetic
anhydride to provide N-(4-methoxyphenyl)acetamide (9). This
The current secretory data revealed that the new benzopyran
derivatives (16–31, except 24–27) were more active on pancreatic
B-cells than the reference compounds ( )-cromakalim and
( )-pinacidil (Table 1). Numerous cromakalim analogues were even
reaction was followed by
N-(3-acetyl-4-hydroxyphenyl)acetamide (10). It should be noted
that the reaction conditions led to the demethylation of the meth-
a Friedel–Crafts acylation to give

X. Florence et al. / Bioorg. Med. Chem. 19 (2011) 3919–3928
3921
Scheme 1. Synthesis of key intermediates 15a–b. Reagents: (i) Ac2O, HOAc, NaOAc; (ii) AcCl, AlCl3; (iii) acetone, pyrrolidine; (iv) NH2OHꢀHCl, K2CO3, ethanol; (v) H2, Raney-
NickelÒ, ethanol; (vi) HCl 5 N in ethanol; (vii) BOC2O, K2CO3.
16-23
15 a-b
R = CH3, OC(CH3)3
20-23
24-27
28-31
16, 20, 24, 28 : R3 = CN, R4 = H
17, 21, 25, 29 : R3 = H, R4 = CN
18, 22, 26, 30 : R3 = Cl, R4 = H
19, 23, 27, 31 : R3 = H, R4 = Cl
Scheme 2. Synthesis of cromakalim analogues 16–31. Reagents: (i) 3- or 4-chloro/3- or 4-cyanophenyl isothiocyanate (R–N@C@S), CH2Cl2; (ii) HCl 5 N in ethanol; (iii)
HCOOH, Ac2O, THF.
more potent than diazoxide at reducing the glucose-induced insulin
response (Table 1).
Pharmacological results obtained on pancreatic endocrine tis-
sue further indicated that drugs bearing an amine group (24–27)
at the 6 position barely affected the insulin secretory process. At
The nature and the position of the electron-withdrawing group
on the phenyl ring of the phenylthiourea chain did not markedly
affect the activity of compounds 24–27 on the pancreatic tissue
(p >0.05).
Drugs bearing a formamide group (–NHCHO) (28–31) at the 6
position exhibited a more pronounced inhibitory activity than
( )-cromakalim (p <0.05) and ( )-pinacidil (p <0.05) on the glu-
cose-induced insulin release. The inhibitory activity of compounds
28–31 was comparable to that of diazoxide (p >0.05). The efficacy
of such compounds was also more marked than that of molecules
a 10 lM concentration, their activity was equivalent to that of
( )-cromakalim (p >0.05) and ( )-pinacidil (p >0.05). Compared
to diazoxide, compound 24, 25 and 27 were less effective
(p <0.05) whilst compound 26 was equipotent (p >0.05) at reducing
the insulin secretory rate.

3922
X. Florence et al. / Bioorg. Med. Chem. 19 (2011) 3919–3928
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
R3
R4
AC log P
Residual insulin secretiona (%)
Myorelaxant activity
b
10
lM
1
lM
EC50
(lM)
24
25
26
27
CN
Cl
3.21
3.21
4.01
4.01
96.9 3.4 (23)
87.0 4.1 (24)
84.6 4.1 (22)
85.0 3.2 (23)
—
—
—
—
>30.0 (4)
>10.0 (6)
24.6 6.7 (5)
15.6 0.2 (3)
CN
Cl
28
29
30
31
CN
Cl
3.27
3.27
4.07
4.07
70.6 3.1 (30)
71.3 3.3 (31)
68.9 3.1 (23)
69.6 2.9 (30)
—
—
—
—
>30.0 (4)
>30.0 (4)
>30.0 (3)
>30.0 (4)
CN
Cl
16
17
18
19
CN
Cl
3.54
3.54
4.34
4.34
60.9 3.3 (21)
55.8 3.4 (21)
63.1 2.9 (21)
60.4 3.3 (21)
—
—
—
—
11.9 1.4 (4)
>10.0 (4)
>10.0 (6)
CN
Cl
8.3 1.7 (4)
20
21
22
23
CN
Cl
4.79
4.79
5.59
5.59
13.3 1.8 (19)
12.9 1.7 (19)
17.1 1.5 (23)
15.5 1.4 (18)
72.2 4.3 (24)
51.6 2.9 (21)
69.0 2.5 (22)
76.4 3.3 (23)
8.5 1.0 (7)
>3.0 (4)
>3.0 (4)
CN
Cl
>30.0 (5)
( )-Cromakalim
( )-Pinacidil
Diazoxide
1.82
nd
nd
94.4 4.1 (32)c
92.1 3.9 (13)c
73.9 4.4 (16)c
95.3 3.8 (31)c
97.7 6.7 (19)c
87.5 5.0 (15)c
0.13 0.01 (7)c
0.35 0.02 (11)c
22.4 2.1 (11)c
4
5
6
7
CN
Cl
4.63
4.63
5.43
5.43
14.2 1.3 (21)c
11.7 0.7 (24)c
65.5 3.6 (30)c
57.5 3.0 (29)c
0.64 0.05 (5)c
>10.0 (6)c
CN
Cl
23.0 2.4 (31)d
12.2 1.2 (20)d
74.6 4.0 (21)d
75.7 3.2 (24)d
>10.0 (5)d
>10.0 (4)d
a
b
c
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 results: Ref. 27.
Published results: Ref. 26.
d
24–27 (p <0.05) but less pronounced than the reference 4-substi-
tuted R/S-6-bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran
derivatives (4–7).
Compounds substituted with
a
tert-butyloxycarbonylamino
bulky group (20–23) at the 6 position were found to exert a pro-
nounced inhibitory effect on the insulin secretory rate. These benz-
opyran derivatives were much more potent than ( )-cromakalim
(p <0.05), ( )-pinacidil (p <0.05) and diazoxide (p <0.05). Com-
Molecules substituted by an acetamido moiety (–NHCOCH3)
(16–19) at the 6-position provoked a marked inhibition of the
insulin secretory process. Such molecules were more active than
( )-cromakalim (p <0.05), ( )-pinacidil (p <0.05) and even diazox-
ide (p <0.05). Moreover, these original compounds were more
effective than molecules 24–27 and 28–31 (p <0.05) [except 18
vs 30 (p >0.05)]. Nevertheless, the efficacy of the benzopyran deriv-
atives 16–19 remained less pronounced than that of the previously
described reference molecules 4–7.
pounds (20–23), tested at a 10 lM concentration, provoked a
85% inhibition of the glucose-induced insulin release; an effect
which can be considered as near to maximal according to the glu-
cose-insensitive secretory rate.25,29 The inhibitory activity of com-
pounds 20–23, tested at 10 and 1 lM, was equivalent to that
exhibited by their brominated reference analogues (4–7) (p
>0.05) (Table 1).
As depicted above, the nature of the substituent on the phenyl
ring of the phenylthiourea chain did not affect the activity of com-
pounds 28–31 and 16–19 (p >0.05).
As previously reported for compounds 4–7,26,27 the most effec-
tive molecule of the 6-tert-butyloxycarbonylamino series was
bearing a 4-cyanophenylthiourea chain (21) at the 4 position.

X. Florence et al. / Bioorg. Med. Chem. 19 (2011) 3919–3928
3923
An important aspect to consider for the establishment of
structure–activity relationships is the electronic impact of the
substituent at the 6-position. Thus, the replacement of the weak
electron-withdrawing bromine atom (compounds 4–7) by an
electron-donating group such as NH2 (compounds 24–27) resulted
in a marked decrease of the biological activity on insulin secreting
cells. By acylating the amine function (formylation for compounds
28–31 and acetylation for compounds 16–19), which moderately
increased the electron-withdrawing impact of the substituent at
the 6-position, the activity on the pancreatic tissue was slightly
improved. However, only the concomitant increase in both the size
and the lipophilicity of the molecule, by the introduction of a
tert-butoxycarbonylamino moiety (compounds 20–23), was
responsible for a marked improvement of the inhibitory effect on
the insulin releasing process. The predicted log P values (expressed
as the AC log P values30) reported in Table 1 were in accordance
with the impact on lipophilicity of the substituent at the 6-position
and of the substituent on the phenyl ring. Thus, the replacement of
the cyano group by a chlorine atom on the phenyl ring at the 4-po-
sition provoked an increase of the AC log P value of about 0.8 log P
units. Regarding the nature of the substituent at the 6-position, an
increasing lipophilicity (as well as an increasing activity on pancre-
atic B-cells) was observed with the following order: –NH2
< –NHCOH < –NHCOCH3 < –NHCOOC(CH3)3. With the tert-butoxy-
carbonylamino group, the lipophilicity of the compounds reached
that of the brominated derivatives.
Unfortunately, and as previously observed with 5–7,27 most ori-
ginal dimethylchromans derivatives precipitated in the bathing
medium before reaching their maximal myorelaxant activity, mak-
ing it difficult to accurately determine their respective EC50 values.
However, and despite the poor solubility of several compounds,
all new benzopyran analogues (16–31) exhibited a weaker vasore-
laxant activity than ( )-pinacidil and ( )-cromakalim (p <0.05).
When accurately quantifiable, the EC50 values indicated that the
vasorelaxant potency of cromakalim was at least 60 times higher
than that of the newly synthesized compounds. The latter mole-
cules also appeared to be less potent on the vascular tissue than
the reference 4-substituted R/S-6-bromo-3,4-dihydro-2,2-di-
methyl-2H-1-benzopyran derivative 4.
Compound 23 was selected to perform additional radioisotopic
experiments in order to determine whether such a compound be-
haved as a pancreatic ATP-sensitive potassium channel opener.
Figure 2 illustrates the effects of compound 23, tested at a
10 l
M concentration, on the 86Rb fractional outflow rate (FOR)
from prelabeled and perifused rat pancreatic islets exposed
throughout to 5.6 mM glucose and extracellular Ca2+. The addition
of compound 23 provoked a marked, sustained and reversible in-
crease in the rate of 86Rb outflow (d). When the perifusate was en-
riched with the hypoglycemic sulfonylurea glibenclamide (s,
10 lM), a pharmacological tool known to block the KATP chan-
nels,24,25,27 the stimulatory effect of compound 23 was completely
abolished (Fig. 2).
Additional experiments were conducted on rat aorta rings in or-
der to detect and to quantify the potential vasorelaxant properties
of the newly synthesized compounds.
On the vascular tissue, diazoxide displayed a moderate myore-
laxant activity while ( )-pinacidil and ( )-cromakalim were at least
50 to 150-fold more potent at inducing a vasorelaxant effect (Table
1).27,29,31 Reference compounds 4–7 were found to be less potent
than ( )-pinacidil and ( )-cromakalim, although compound 4 was
Such radioisotopic data suggest that compound 23 activates
KATP channels in islet cells.25
4. Conclusion
In the present work, 16 original 4,6-disubstituted R/S-3,4-dihy-
dro-2,2-dimethyl-2H-1-benzopyrans (16–31) structurally related
to previously described 4-substituted R/S-6-bromo-3,4-dihydro-
2,2-dimethyl-2H-1-benzopyrans (4–7) were synthesized and their
biological activity evaluated. All of these new benzopyran deriva-
tives comprised, at the 4-position, a phenylthiourea moiety substi-
tuted on the phenyl ring by a meta or a para-electron-withdrawing
group such as Cl or CN. Such compounds (16–31) were also bear-
ing, at the 6-position, either an amino, a formamido, an acetamido
or a tert-butyloxycarbonylamino moiety instead of a bromine atom
(compounds 4–7). The biological activity of these original com-
pounds was compared to that of ( )-cromakalim, ( )-pinacidil,
diazoxide and previously reported compounds 4–7, used as refer-
ence PCOs.
Some of the new dimethylchromans were found to be barely ac-
tive (24–27) whilst the others were more active than ( )-cromaka-
lim and ( )-pinacidil at inhibiting the insulin releasing process.
Among the latter compounds, drugs bearing at the 6-position an
acetamido group (16–19) or a tert-butyloxycarbonylamino moiety
(20–23) were even more active than diazoxide.
found to exert a marked vasorelaxant activity (EC50 = 0.64
lM)
(Table 1).27
23
5.0
4.0
3.0
2.0
1.0
0.0
All these new benzopyrans were found to be less active than
( )-cromakalim and ( )-pinacidil as vasorelaxant agents.
Biological results clearly indicated that the inhibitory activity of
the new drugs on the insulin secretory process raised with increas-
ing the size of the substituent located at the 6-position and with
increasing the lipophilicity of the molecules. Indeed, molecules
bearing a bulky group such as a tert-butyloxycarbonylamino moi-
ety on the 6-position (20–23) provoked a marked inhibition of
the insulin secretory rate. These compounds 20–23 were more ac-
tive than drugs bearing an acetamido group (16–19), themselves
more efficient than drugs bearing the less bulky formamido group
(28–31). The less active compounds (24–27) were those only bear-
ing the amino function at the 6-position. Compounds 20–23 were
also found to be equipotent to compounds 4–7 at inhibiting the
insulin secretory rate but less active on the vascular tissue than
30
40
50
60
70
80
90
TIME (min)
Figure 2. Effect of 23 (10 l
M) on 86Rb outflow from rat pancreatic islets perifused
throughout in the absence (d) or presence (s) of glibenclamide (10 lM). Basal
media contained 5.6 mM glucose and extracellular Ca2+. Mean values ( SEM) refer
to 5–6 individual experiments.

3924
X. Florence et al. / Bioorg. Med. Chem. 19 (2011) 3919–3928
the reference R/S-6-bromo-4-(3-cyanophenylaminothiocarbonyla-
mino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (4). Therefore,
molecules 20–23 can be regarded as the most promising com-
pounds, being active on the insulin secretory cells and exhibiting
a pharmacological profile clearly different from that of the parent
molecule ( )-cromakalim.
(21.05 g, 90%): mp: 162–164 °C; IR (KBr)
1657 (C@O) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d: 2.02 (s, 3H,
–NHCOCH3), 2.58 (s, 3H, –COCH3), 6.90 (d, 1H, 5-H), 7.65 (dd, 1H,
6-H), 8.06 (s, 1H, 2-H), 9.89 (s, 1H, –NHCOCH3), 11.54 (s, 1H, –
OH). Anal. (C10H11NO3) theoretical: C, 62.17; H, 5.74; N, 7.25.
Found: C, 62.35; H, 5.82; N, 7.19.
m
: 3452 cmꢁ1 (O–H),
;
Lastly, radioisotopic experiments performed on prelabeled and
perifused rat pancreatic islet indicated that the inhibitory effect
on the insulin releasing process of the new benzopyran derivatives
was mediated by the activation of pancreatic B-cell ATP-sensitive
potassium channels.
Additional pharmacomodulations, and more specifically the
exploration of the requirement and the steric hindrance of the
carbamate function, are planned in order to further characterize
original benzopyran derivatives specifically activating the insulin
secreting-cell KATP channels.
5.1.3. 6-Acetamido-3,4-dihydro-2,2-dimethyl-2H-1-
benzopyran-4-one (11)
A solution of 10 (25 g, 0.129 mol), acetone (32 ml, 0.434 mol),
and pyrrolidine (15 ml, 0.182 mol) in methanol (575 ml) was stir-
red at 25 °C overnight. On the next day, the solvents were removed
under reduced pressure. Water was added to the residue and the
mixture was adjusted to pH 1 with concentrated hydrochloric acid.
The resulting precipitate was collected by filtration. The crude
product was then dissolved in methanol. The solution was treated
with charcoal, filtered and water was added to the filtrate. The title
compound was collected by filtration, washed with water and
5. Experimental section
5.1. Chemistry
dried (27.47 g, 91%): mp: 164–165 °C; IR (KBr)
m: 1664 and 1693
(C@O), 3313 (N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d: 1.38
;
(s, 6H, CH3), 2.01 (s, 3H, –NHCOCH3), 2.76 (s, 2H, CH2), 6.92
(d, 1H, 8-H), 7.66 (dd, 1H, 7-H), 7.99 (s, 1H, 5-H), 9.91 (s, 1H, –
NHCOCH3). Anal. (C13H15NO3) theoretical: C, 66.94; H, 6.48; N,
6.00. Found: C, 66.79; H, 6.59; N, 5.96.
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 with UV light (254 nm).
Melting points were determined on a Stuart SMP3 apparatus in
open capillary tubes and were uncorrected. IR spectra were re-
corded as KBr pellets on a Perkin–Elmer 1000 FTIR spectrophotome-
ter. The 1H NMR spectra were recorded on a Bruker Avance
(500 MHz) instrument using DMSO-d6 as solvent and tetramethyl-
silane (TMS) as internal standard; chemical shifts were reported in
d values (ppm) relative to internal TMS. The abbreviations s = sin-
glet, d = doublet, t = triplet, q = quadruplet, m = multiplet, and
b = 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.
5.1.4. 6-Amino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-
one (12)
Compound 11 (20 g, 85.74 mmol) was dissolved in an ethanolic
solution (250 ml) of hydrochloric acid 5 N. The solution was then
refluxed for 3 h. When the reaction was ended, the mixture was
poured in water (500 ml). The title compound was precipitated
by addition of a solution of sodium hydroxide 40% until pH 10.
The final product was collected by filtration, washed with water
and dried (16.23 g, 99%): mp: 148–149 °C; IR (KBr)
m: 1667
(C@O), 3446–3246 (N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d:
;
1.36 (s, 6H, CH3), 2.66 (s, 2H, CH2), 4.85 (s, 2H, –NH2), 6.68 (d,
1H, 8-H), 6.82 (dd, 1H, 7-H), 6.90 (d, 1H, 5-H). Anal. (C11H13NO2)
theoretical: C, 69.09; H, 6.85; N, 7.32. Found: C, 69.35; H, 6.96;
N, 7.34.
5.1.1. N-(4-Methoxyphenyl)acetamide (9)
4-Methoxyaniline (50 g, 0.406 mol) was dissolved in a solution
of sodium acetate (10.24 g, 0.125 mol) in glacial acetic acid (41 ml,
0.716 mol), the mixture was then cooled to 0 °C and acetic anhy-
dride (45.05 ml, 0.477 mol) was added dropwise. At the end of
the reaction, water (100 ml) was added to the mixture. The result-
ing precipitate was collected by filtration. The product was then
crystallized in boiling water. The final precipitate was collected
by filtration, washed with water and dried (61.70 g, 92%): mp:
5.1.5. 6-tert-Butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-
2H-1-benzopyran-4-one (13)
Compound 12 (15 g, 78.44 mmol) was dissolved in a mixture
(385 ml) of tetrahydrofuran and water (v/v 1:1). Potassium carbon-
ate (21.9 g, 0.158 mol) and di-tert-butyl dicarbonate (20.1 g,
92.10 mmol) were added to the solution. The reaction mixture
was stirred at 25 °C for 4 days. The organic layer was isolated
and the water layer was extracted three times with ethyl acetate.
Organic layers were pooled together and then dried over anhy-
drous MgSO4, filtered and evaporated under reduced pressure.
The residue was dissolved in methanol and water was added.
The title compound was collected by filtration, washed with water
126–127 °C; IR (KBr)
m
: 3068 (C–H aromatic), 2934 (C–H aliphatic),
1647 (C@O), 1245 (C–O) cmꢁ1
;
1H NMR (DMSO-d6, 500 MHz): d:
2.00 (s, 3H, –NHCOCH3), 3.75 (s, 3H, –OCH3), 6.85 (d, 2H, 3-H and
5-H), 7.47 (d, 2H, 2-H and 6-H), 9.74 (s, 1H, –NHCOCH3). Anal.
(C9H11NO2) theoretical: C, 65.44; H, 6.71; N, 8.48. Found: C,
65.68; H, 6.82; N, 8.32.
and dried (22.40 g, 98%): mp: 168–170 °C; IR (KBr)
m: 1684 and
1719 (C@O), 3360 (N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d:
;
5.1.2. N-(3-Acetyl-4-hydroxyphenyl)acetamide (10)
1.36 (s, 6H, CH3), 1.47 (s, 9H, –NHCOOC(CH3)3), 2.75 (s, 2H, CH2),
6.88 (d, 1H, 8-H), 7.50 (dd, 1H, 7-H), 7.89 (s, 1H, 5-H), 9.30 (s,
1H, –NHCOOC(CH3)3). Anal. (C16H21NO4) theoretical: C, 65.96; H,
7.27; N, 4.81. Found: C, 65.87; H, 7.16; N, 4.84.
Acetyl chloride (25 ml, 0.352 mol) was added to a stirred sus-
pension of 9 (20 g, 0.121 mol) in carbon disulfide (50 ml). Alumi-
num chloride (55 g, 0.412 mol) was added step by step. The
mixture was then heated at 80–90 °C. After 90 min, the solvent
was evaporated under vacuum. Crushed ice and water was added
cautiously to the residue. The resulting precipitate was collected
by filtration and washed with water. The crude product was then
dissolved in an aqueous solution of sodium hydroxide 5% (w/v)
and filtered through CeliteÒ. The filtrate was then acidified to pH
1 by addition of concentrated hydrochloric acid. The title com-
pound was collected by filtration, washed with water and dried
5.1.6. 6-Acetamido-3,4-dihydro-2,2-dimethyl-2H-1-
benzopyran-4-hydroxyimine (14a)
Potassium carbonate (5.93 g, 42.87 mmol) and hydroxylamine
hydrochloride (2.98 g, 42.87 mmol) were added to a stirred solu-
tion of 11 (5 g, 21.44 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

X. Florence et al. / Bioorg. Med. Chem. 19 (2011) 3919–3928
3925
volume of 195 ml. The title compound was collected by filtration,
washed with water and dried (5.06 g, 95%): mp: 203–204 °C; IR
(KBr)
: 1634 (C@O), 3229 (N–H) cmꢁ1 1H NMR (DMSO-d6,
500 MHz): d: 1.28 (s, 6H, CH3), 2.00 (s, 3H, –NHCOCH3), 2.74 (s,
2H, CH2), 6.75 (d, 1H, 8-H), 7.39 (dd, 1H, 7-H), 8.06 (s, 1H, 5-H),
9.77 (s, 1H, –NHCOCH3), 11.19 (s, 1H, @N–OH). Anal.
(C13H16N2O3) theoretical: C, 62.89; H, 6.50; N, 11.28. Found: C,
62.57; H, 6.78; N, 11.63.
5.1.11. R/S-N-4-Cyanophenyl-N0-(6-acetamido-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (17)
The title compound was obtained as described for 16, starting
from 15a (0.5 g, 2.13 mmol) and 4-cyanophenyl isothiocyanate
m
;
(410.3 mg, 2.56 mmol) (0.63 g, 75%): mp: 172–174 °C; IR (KBr)
m:
1652 (C@O), 2226 (C„N), 3323 (N–H) cmꢁ1 1H NMR (DMSO-d6,
;
500 MHz): d: 1.26 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.77 (t, 1H, 3-H),
1.98 (s, 3H, –NHCOCH3), 2.20 (m, 1H, 3-H), 5.77 (m, 1H, 4-H),
6.69 (d, 1H, 8-H), 7.43 (d, 2H, 7-H, 5-H), 7.74–7.82 (dd, 4H, 20-H,
60-H, 30-H and 50-H), 8.43 (br s, 1H, –NHCSNH–C7H4N). Anal.
(C21H22N4O2S) theoretical: C, 63.94; H, 5.92; N, 14.20; S, 8.13.
Found: C, 63.68; H, 5.77; N, 14.23; S, 8.12.
5.1.7. R/S-6-Acetamido-4-amino-3,4-dihydro-2,2-dimethyl-2H-
1-benzopyran (15a)
Raney-Nickel (3 g) was added to a stirred solution of 14a (2.5 g,
10.07 mmol) in methanol. The solution was stirred in a sealed
hydrogenator under a hydrogen pressure of 5 bars. When the reac-
tion was ended (3–4 h), the catalyst was filtered off 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 and dried (2.03 g,
5.1.12. R/S-N-3-Chlorophenyl-N0-(6-acetamido-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (18)
The title compound was obtained as described for 16, starting
from 15a (0.5 g, 2.13 mmol) and 3-chlorophenyl isothiocyanate
(336
l
l, 2.56 mmol) (0.70 g, 81%): mp: 170–172 °C; IR (KBr)
m:
1653 (C@O), 3257 (N–H) cmꢁ1
;
1H NMR (DMSO-d6, 500 MHz): d:
86%): mp: 103–104 °C; IR (KBr)
m: 1658 (C@O), 3308–3468 (N–
1.25 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.77 (t, 1H, 3-H), 1.98 (s, 3H,
–NHCOCH3), 2.16 (m, 1H, 3-H), 5.78 (br s, 1H, 4-H), 6.68 (d, 1H,
8-H), 7.14–7.44 (br m, 5H, 5-H, 7-H, 20-H, 30-H and 40-H), 7.76 (s,
1H, 60-H), 8.21 (br s, 1H, –NHCSNH–C6H4Cl), 9.69 (br s, 1H, –
NHCSNH–C6H4Cl), 9.76 (s, 1H, –NHCOCH3). Anal. (C20H22ClN3O2S)
theoretical: C, 59.47; H, 5.49; N, 10.40; S, 7.94. Found: C, 59.42;
H, 5.66; N, 10.55; S, 7.98.
H2) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d: 1.17 (s, 3H, CH3),
;
1.32 (s, 3H, CH3), 1.51 (t, 1H, 3-H), 1.79 (br s, 2H, –CH–NH2), 1.99
(m, 4H, –NHCOCH3 and 3-H), 3.79 (m, 1H, –CH–NH2), 6.57 (d, 1H,
8-H), 7.29 (d, 1H, 7-H), 7.64 (s, 1H, 5-H), 9.69 (s, 1H, –NHCOCH3).
Anal. (C13H18N2O2) theoretical: C, 66.64; H, 7.74; N, 11.96. Found:
C, 66.84; H, 7.45; N, 11.81.
5.1.13. R/S-N-4-Chlorophenyl-N0-(6-acetamido-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (19)
5.1.8. 6-tert-Butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-
2H-1-benzopyran-4-hydroxyimine (14b)
The title compound was obtained as described for 14a, starting
from 13 (20 g, 68.65 mmol), K2CO3 (18.98 g, 137.3 mmol) and
hydroxylamine hydrochloride (9.54 g, 137.3 mmol) (19.77 g,
The title compound was obtained as described for 16, starting
from 15a (0.5 g, 2.13 mmol) and 4-chlorophenyl isothiocyanate
(434.3 mg, 2.56 mmol) (0.71 g, 82%): mp: 151–154 °C; IR (KBr)
m:
1657 (C@O), 3264 (N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d:
;
94%): mp: 162–163 °C; IR (KBr)
;
m
: 1705 (C@O), 3369 (N–H) cm
ꢁ1
1H NMR (DMSO-d6, 500 MHz): d: 1.27 (s, 6H, CH3), 1.49 (s,
1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.76 (t, 1H, 3-H), 1.99 (s, 3H,
–NHCOCH3), 2.15 (m, 1H, 3-H), 5.78 (br s, 1H, 4-H), 6.67 (d, 1H,
8-H), 7.36 (d, 2H, 30-H and 50-H), 7.41 (s, 2H, 7-H, 5-H), 7.53 (d,
2H, 20-H, 60-H), 8.12 (br s, 1H, –NHCSNH–C6H4Cl), 9.64 (br s, 1H,
–NHCSNH–C6H4Cl), 9.76 (s, 1H, –NHCOCH3). Anal. (C20H22ClN3O2S)
theoretical: C, 59.47; H, 5.49; N, 10.40; S, 7.94. Found: C, 59.46; H,
5.56; N, 10.32; S, 7.98.
9H, –NHCOOC(CH3)3), 2.72 (s, 2H, CH2), 6.71 (d, 1H, 8-H), 7.23
(dd, 1H, 7-H), 7.95 (s, 1H, 5-H), 9.13 (s, 1H, –NHCOOC(CH3)3),
11.16 (s, 1H, @N–OH). Anal. (C16H22N2O4) theoretical: C, 62.73;
H, 7.24; N, 9.14. Found: C, 62.64; H, 7.05; N, 8.88.
5.1.9. R/S-4-Amino-6-tert-butyloxycarbonylamino-3,4-dihydro-
2,2-dimethyl-2H-1-benzopyran (15b)
5.1.14. R/S-N-3-Cyanophenyl-N0-(6-tert-butyloxycarbonylamino-
3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (20)
3-Cyanophenyl isothiocyanate (789 mg, 4.92 mmol) was added
to a solution of 15b (1.20 g, 4.10 mmol) in methylene chloride
(5 ml). After 30 min, the solvent was removed under vacuum and
the crude product was dissolved in ethyl acetate. The final product
was crystallized by addition of n-hexane, collected by filtration,
washed with n-hexane, and dried (1.75 g, 94%): mp: 121–124 °C;
The title compound was obtained as described for 15a, starting
from 14b (6.25 g, 20.41 mmol) (5.25 g, 88%): mp: 144–146 °C; IR
(KBr)
m ;
: 1710 (C@O), 3366 (N–H) cmꢁ1 1H NMR (DMSO-d6,
500 MHz): d: 1.17 (s, 3H, CH3), 1.31 (s, 3H, CH3), 1.45 (m, 10H, –
NHCOOC(CH3)3 and 3-H), 1.79 (s, 2H, NH2), 1.98 (dd, 1H, 3-H),
3.78 (dd, 1H, –CH–NH2), 6.54 (d, 1H, 8-H), 7.04 (d, 1H, 7-H), 7.62
(s, 1H, 5-H), 8.94 (s, 1H, –NHCOOC(CH3)3). Anal. (C16H24N2O3) the-
oretical: C, 65.73; H, 8.27; N, 9.58. Found: C, 65.94; H, 8.17; N, 9.59.
IR (KBr) m ;
: 1691 (C@O), 2231 (C„N), 3339 (N–H) cmꢁ1 1H NMR
(DMSO-d6, 500 MHz): d: 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.45
(s, 9H, –NHCOOC(CH3)3), 1.76 (t, 1H, 3-H), 2.16 (m, 1H, 3-H), 5.75
(br s, 1H, 4-H), 6.65 (d, 1H, 8-H), 7.17 (d, 1H, 7-H), 7.45–7.56 (m,
3H, 5-H and 40-H and 50-H), 7.76 (d, 1H, 60-H), 8.04 (s, 1H, 20-H),
8.30 (d, 1H, –NHCSNH–C7H4N), 9.10 (s, 1H, –NHCSNH–C7H4N),
9.78 (s, 1H, –NHCOOC(CH3)3). Anal. (C24H28N4O3S) theoretical: C,
63.69; H, 6.24; N, 12.38; S, 7.08. Found: C, 63.66; H, 5.97; N,
12.58; S, 6.86.
5.1.10. R/S-N-3-Cyanophenyl-N0-(6-acetamido-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (16)
3-Cyanophenyl isothiocyanate (410.3 mg, 2.56 mmol) was
added to a solution of 15a (0.5 g, 2.13 mmol) in methylene chloride
(10 ml). After 30 min, the resulting precipitate was collected by fil-
tration, washed with methylene chloride, and dried (0.63 g, 75%):
mp: 147–149 °C; IR (KBr)
m: 1655 (C@O), 2231 (C„N), 3335
(N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d: 1.26 (s, 3H, CH3),
;
5.1.15. R/S-N-4-Cyanophenyl-N0-(6-tert-butyloxycarbonylamino-
3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (21)
The title compound was obtained as described for 20, starting
from 15b (1.2 g, 4.10 mmol) and 4-cyanophenyl isothiocyanate
1.37 (s, 3H, CH3), 1.77 (t, 1H, 3-H), 2.02 (s, 3H, –NHCOCH3), 2.17
(m, 1H, 3-H), 5.78 (m, 1H, 4-H), 6.69 (d, 1H, 8-H), 7.41 (d, 1H, 7-
H), 7.46–7.55 (m, 3H, 5-H and 40-H and 50-H), 7.75 (d, 1H, 60-H),
8.06 (s, 1H, 20-H), 8.35 (br s, 1H, –NHCSNH–C7H4N), 9.76 (br s,
2H, –NHCOCH3 and –NHCSNH–C7H4N). Anal. (C21H22N4O2S) theo-
retical: C, 63.94; H, 5.92; N, 14.20; S, 8.13. Found: C, 63.65; H,
5.54; N, 14.27; S, 8.26.
(789 mg, 4.92 mmol) (1.71 g, 92%): mp: 188–189 °C; IR (KBr)
1703 (C@O), 2224 (C„N), 3309–3369 (N–H) cmꢁ1 1H NMR
(DMSO-d6, 500 MHz): d: 1.23 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.45
m:
;

3926
X. Florence et al. / Bioorg. Med. Chem. 19 (2011) 3919–3928
(s, 9H, –NHCOOC(CH3)3), 1.75 (t, 1H, 3-H), 2.21 (m, 1H, 3-H), 5.73
(br s, 1H, 4-H), 6.66 (d, 1H, 8-H), 7.18 (s, 1H, 7-H), 7.42 (s, 1H, 5-
H), 7.74 (d, 2H, 20-H and 60-H), 7.80 (d, 2H, 30-H and 50-H), 8.45
(br s, 1H, –NHCSNH–C7H4N), 9.11 (br s, 1H, –NHCSNH–C7H4N),
9.97 (s, 1H, –NHCOOC(CH3)3). Anal. (C24H28N4O3S) theoretical: C,
63.69; H, 6.24; N, 12.38; S, 7.08. Found: C, 63.35; H, 6.54; N,
12.38; S, 7.05.
5.1.20. R/S-N-3-Chlorophenyl-N0-(6-amino-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (26)
The title compound was obtained as described for 24, starting
from 22 (600 mg, 1.3 mmol) (418 mg, 89%): mp: 161–163 °C; IR
(KBr) m ;
: 3214–3338 (N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz):
d: 1.21 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.70 (t, 1H, 3-H), 2.13 (m,
1H, 3-H), 4.59 (s, 2H, –NH2), 5.65 (br s, 1H, 4-H), 6.41–6.51 (m,
3H, 5-H, 7-H, 8-H), 7.14 (d, 1H, 40-H), 7.31–7.37 (m, 2H, 50-H and
60-H), 7.83 (s, 1H, 20-H), 8.13 (br s, 1H, –NHCSNH–C6H4Cl), 9.62
(s, 1H, –NHCSNH–C6H4Cl). Anal. (C18H20ClN3OS) theoretical: C,
59.74; H, 5.57; N, 11.61; S, 8.86. Found: C, 59.75; H, 5.80; N,
11.74; S, 8.48.
5.1.16. R/S-N-3-Chlorophenyl-N0-(6-tert-
butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-
benzopyran-4-yl)thiourea (22)
The title compound was obtained as described for 20, starting
from 15b (1.2 g, 4.10 mmol) and 3-chlorophenyl isothiocyanate
(646
l
l, 4.92 mmol) (1.67 g, 88%): mp: 117–120 °C; IR (KBr)
m:
5.1.21. R/S-N-4-Chlorophenyl-N0-(6-amino-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (27)
1689 (C=O), 3338 (N–H) cmꢁ1
;
1H NMR (DMSO-d6, 500 MHz): d:
1.24 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.47 (s, 9H, –NHCOOC(CH3)3),
1.75 (t, 1H, 3-H), 2.16 (m, 1H, 3-H), 5.74 (br s, 1H, 4-H), 6.65 (d,
1H, 8-H), 7.16 (m, 2H, 20-H and 60-H), 7.34 (m, 2H, 30-H and 50-H),
7.44 (s, 1H, 7-H), 7.74 (s, 1H, 5-H), 8.18 (d, 1H, –NHCSNH–
C6H4Cl), 9.10 (s, 1H, –NHCSNH–C6H4Cl), 9.67 (s, 1H,
NHCOOC(CH3)3). Anal. (C23H28ClN3O3S) theoretical: C, 59.79; H,
The title compound was obtained as described for 24, starting
from 23 (600 mg, 1.3 mmol) (428 mg, 91%): mp: 173–174 °C; IR
(KBr) m ;
: 3240 (N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d: 1.21
(s, 3H, CH3), 1.32 (s, 3H, CH3), 1.69 (t, 1H, 3-H), 2.12 (m, 1H, 3-H),
4.59 (s, 2H, –NH2), 5.65 (br s, 1H, 4-H), 6.40–6.51 (m, 3H, 5-H, 7-
H, 8-H), 7.36 (d, 2H, 20-H, 60-H), 7.55 (d, 2H, 30-H, 50-H), 8.04 (br
s, 1H, –NHCSNH–C6H4Cl), 9.56 (br s, 1H, –NHCSNH–C6H4Cl). Anal.
(C18H20ClN3OS) theoretical: C, 59.74; H, 5.57; N, 11.61; S, 8.86.
Found: C, 59.75; H, 5.84; N, 11.74; S, 8.48.
–
6.11; N, 9.10; S, 6.94. Found: C, 59.67; H, 6.45; N, 9.08; S, 7.03.
5.1.17. R/S-N-4-Chlorophenyl-N0-(6-tert-
butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-
benzopyran-4-yl)thiourea (23)
The title compound was obtained as described for 20, starting
from 15b (1.2 g, 4.10 mmol) and 4-chlorophenyl isothiocyanate
5.1.22. R/S-N-3-Cyanophenyl-N0-(6-formamido-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (28)
A mixture of acetic anhydride (2 ml, 21.16 mmol) and formic
acid (1 ml, 26.50 mmol) was heated at 55 °C for 2 h. The mixture
was then cooled to 0 °C and anhydrous tetrahydrofuran (2 ml)
was added. A solution of 24 (300 mg, 0.851 mmol) in anhydrous
tetrahydrofuran was then added dropwise. At the end of the reac-
tion, water (15 ml) was added and the resulting precipitate was
collected by filtration. The crude product was then purified by col-
umn chromatography on silica gel using a chloroform/methanol
solution (20:1) as eluent. The purified product was then crystal-
lized in methanol/water mixture, the resulting precipitate col-
lected by filtration, washed with water and dried (139 mg, 43%):
(835 mg, 4.92 mmol) (1.86 g, 98%): mp: 176–178 °C; IR (KBr)
m:
1689 (C„O), 3336 (N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d:
;
1.24 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.46 (s, 9H, –NHCOOC(CH3)3),
1.73 (t, 1H, 3-H), 2.16 (m, 1H, 3-H), 5.73 (br s, 1H, 4-H), 6.64 (d,
1H, 8-H), 7.16 (br s, 1H, 7-H), 7.35 (d, 2H, 20-H and 60-H), 7.43 (s,
1H, 5-H), 7.54 (d, 2H, 30-H and 50-H), 8.09 (br s, 1H, –NHCSNH–
C6H4Cl), 9.11 (br s, 1H, –NHCSNH–C6H4Cl), 9.62 (s, 1H,
NHCOOC(CH3)3). Anal. (C23H28ClN3O3S) theoretical: C, 59.79; H,
–
6.11; N, 9.10; S, 6.94. Found: C, 60.03; H, 6.42; N, 9.18; S, 7.00.
5.1.18. R/S-N-3-Cyanophenyl-N0-(6-amino-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (24)
mp: 197–198 °C; IR (KBr)
m: 1661 (C@O), 2232 (C„N), 3314
(N–H) cmꢁ1 1H NMR (DMSO-d6, 500 MHz): d: 1.26 (s, 3H, CH3),
;
A suspension of compound 20 (600 mg, 1.3 mmol) in an etha-
nolic solution (20 ml) of hydrochloric acid 5 N was heated for
5 min. The mixture was then poured into water (40 ml). The title
compound was precipitated by addition of an aqueous solution
of sodium hydroxide 40% until pH=10. The final product was col-
lected by filtration, washed with water and dried (360 mg, 77%):
1.38 (s, 3H, CH3), 1.78 (t, 1H, 3-H), 2.18 (m, 1H, 3-H), 5.78 (br s, 1H,
4-H), 6.72 (d, 1H, 8-H), 7.42 (d, 1H, 7-H), 7.51–7.56 (m, 3H, 5-H, 40-
H and 50-H), 7.75 (d, 1H, 60-H), 8.03 (s, 1H, 20-H), 8.18 (s, 1H, –
NHCHO), 8.35 (br s, 1H, –NHCSNH–C7H4N), 9.81 (s, 1H, –NHCSN
H–C7H4N), 10.03 (m, 1H, –NHCHO). Anal. (C20H20N4O2S) theoreti-
cal: C, 63.14; H, 5.30; N, 14.73; S, 8.43. Found: C, 62.97; H, 5.41;
N, 14.75; S, 8.63.
mp: 156–158 °C; IR (KBr) m ;
: 2229 (C„N), 3373 (N–H) cmꢁ1 1H
NMR (DMSO-d6, 500 MHz): d: 1.22 (s, 3H, CH3), 1.33 (s, 3H, CH3),
1.71 (t, 1H, 3-H), 2.13 (m, 1H, 3-H), 4.67 (br s, 2H, –NH2), 5.66
(br s, 1H, 4-H), 6.41–6.52 (m, 3H, 5-H, 7-H, 8-H), 7.51 (m, 2H, 40-
H and 50-H), 7.77 (d, 1H, 60-H), 8.11 (s, 1H, 20-H), 8.29 (d, 1H, –
NHCSNH–C7H4N), 9.75 (s, 1H, –NHCSNH–C7H4N). Anal.
(C19H20N4OS) theoretical: C, 64.75; H, 5.72; N, 15.90; S, 9.10.
Found: C, 64.46; H, 5.79; N, 15.77; S, 8.73.
5.1.23. R/S-N-4-Cyanophenyl-N0-(6-formamido-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (29)
The title compound was obtained as described for 28, starting
from 25 (300 mg, 0.851 mmol) (282 mg, 87%): mp: 180–181 °C;
IR (KBr) m ;
: 1659 (C@O), 2228 (C„N), 3308 (N–H) cmꢁ1 1H NMR
(DMSO-d6, 500 MHz): d: 1.27 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.77
(t, 1H, 3-H), 2.22 (m, 1H, 3-H), 5.77 (br s, 1H, 4-H), 6.73 (d, 1H,
8-H), 7.43 (d, 1H, 7-H), 7.50 (s, 1H, 5-H), 7.75–7.80 (dd, 4H, 20-H,
30-H, 50-H and 60-H), 8.18 (s, 1H, –NHCHO), 8.47 (br s, 1H, –
NHCSNH–C7H4N), 9.98–10.01 (m, 2H, –NHCSNH–C7H4N and –
NHCHO). Anal. (C20H20N4O2S) theoretical C, 63.14; H, 5.30; N,
14.73; S, 8.43. Found: C, 62.75; H, 5.43; N, 14.65; S, 8.12.
5.1.19. R/S-N-4-Cyanophenyl-N0-(6-amino-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)thiourea (25)
The title compound was obtained as described for 24, starting
from 21 (600 mg, 1.3 mmol) (458 mg, 98%): mp: 171–174 °C; IR
(KBr) m ;
: 2229 (C„N), 3320–3386 (N–H) cmꢁ1 1H NMR (DMSO-
d6, 500 MHz): d: 1.22 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.70 (t, 1H,
3-H), 2.16 (m, 1H, 3-H), 4.59 (s, 2H, –NH2), 5.64 (br s, 1H, 4-H),
6.41–6.50 (m, 3H, 5-H, 7-H, 8-H), 7.75 (d, 2H, 20-H, 60-H), 7.84 (d,
2H, 30-H, 50-H), 8.37 (br s, 1H, –NHCSNH–C7H4N), 9.91 (br s, 1H, –
NHCSNH–C7H4N). Anal. (C19H20N4OS) theoretical: C, 64.75; H,
5.72; N, 15.90; S, 9.10. Found: C, 64.51; H, 6.06; N, 15.53; S, 8.76.
5.1.24. R/S-N-3-Chlorophenyl-N0-(6-formamido-3,4-dihydro-
2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (30)
The title compound was obtained as described for 28, starting
from 26 (300 mg, 0.829 mmol) (307 mg, 95%): mp: 171–173 °C;
IR (KBr)
m ;
: 1666 (C@O), 3233 (N–H) cmꢁ1 1H NMR (DMSO-d6,

X. Florence et al. / Bioorg. Med. Chem. 19 (2011) 3919–3928
3927
500 MHz): d: 1.26 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.78 (t, 1H, 3-H),
2.17 (m, 1H, 3-H), 5.78 (br s, 1H, 4-H), 6.71 (d, 1H, 8-H), 7.15 (d,
1H, 50-H), 7.32–7.37 (br m, 2H, 20-H0 and 40-H), 7,43 (d, 1H, 7-H),
7,49 (s, 1H, 5-H), 7,73 (s, 1H, 60-H), 8,18–8,23 (m, 2H, –NHCSNH–
C6H4Cl and –NHCHO), 9.69 (br s, 1H, –NHCSNH–C6H4Cl), 10.01 (s,
1H, –NHCHO). Anal. (C19H20ClN3O2S) theoretical C, 58.53; H,
5.17; N, 10.78; S, 8.22. Found: C, 58.90; H, 5.23; N, 10.56; S, 7.87.
from dose–response curves using Datanalyst software (EMKA
Technologies, France).32
5.5. Measurements of 86Rb outflow from perifused rat
pancreatic islets
Experiments were performed with pancreatic islets isolated
from adult fed Wistar rats (Charles River Laboratories, Belgium).
The methods used to measure 86Rb (42K substitute) outflow
from perifused rat pancreatic islets have been described previ-
ously.24,25,27 Briefly, groups of 100 rat pancreatic islets were incu-
bated for 60 min at 37 °C in a bicarbonate-buffered medium (in
mM: NaCl 115, KCl 5, CaCl2 2.56, MgCl2 1, NaHCO3 24) supple-
mented with 0.5% (w/v) albumin (fraction V, Sigma) and containing
5.1.25. R/S-N-4-Chlorophenyl-N0-(6-formamido-3,4-dihydro-
2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (31)
The title compound was obtained as described for 28, starting
from 27 (300 mg, 0.829 mmol) (268 mg, 95%): mp: 197–198 °C;
IR (KBr)
m ;
: 1663 (C@O), 3311 (N–H) cmꢁ1 1H NMR (DMSO-d6,
500 MHz): d: 1.25 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.76 (t, 1H, 3-H),
2.16 (m, 1H, 3-H), 5.76 (br s, 1H, 4-H), 6.70 (d, 1H, 8-H), 7.36 (d,
2H, 20-H, 60-H), 7.41 (d, 1H, 7-H), 7.51 (m, 3H, 5-H, 30-H and 50-
H), 8.13 (br s, 1H, –NHCSNH–C6H4Cl), 8.18 (s, 1H, –NHCHO), 9.64
(br s, 1H, –NHCSNH–C6H4Cl), 10.01 (s, 1H, –NHCHO). Anal.
(C19H20ClN3O2S) theoretical C, 58.53; H, 5.17; N, 10.78; S, 8.22.
Found: C, 58.30; H, 5.36; N, 10.70; S, 7.93.
16.7 mM glucose and 86Rb (0.15–0.25 mmol/L:50
lCi/ml). After
incubation, the islets were washed three times with a nonradioac-
tive medium and then placed in a perifusion chamber. The perifu-
sate [in mM: NaCl 115, KCl 5, CaCl2 2.56, MgCl2 1, NaHCO3 24
supplemented with 0.5% (w/v) albumin] was delivered at a con-
stant rate (1.0 ml/min). From the 31st to the 90th min, the effluent
was continuously collected over successive periods of 1 min each.
An aliquot of the effluent (0.5 ml) was used for scintillation count-
ing and, at the end of the experiment, the radioactive content of the
islets was determined. The outflow of 86Rb (cpm/min) was ex-
pressed as a fractional outflow rate (FOR, % of instantaneous islet
content per min).24,25,27
5.2. Biological assays
( )-Cromakalim (Tocris, UK), diazoxide (Sigma Chemical, USA)
and ( )-pinacidil (Sigma Chemical, USA) were tested as reference
compounds.
5.6. Statistical evaluation
5.3. Measurement of insulin release from incubated rat
pancreatic islets
The statistical significance of differences between mean data
was assessed by using the Student’s t-test or by an analysis of var-
iance followed for multiple comparisons by a Bonferroni test pro-
cedure. The biological results were considered as statistically
different when p value was <0.05.
Experiments were performed with pancreatic islets isolated
from adult fed Wistar rats (Charles River Laboratories, Belgium).
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) dialyzed albu-
min (fraction V, Sigma) 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, the reference compound or the required
chroman derivative. The release of insulin was measured radioim-
munologically using rat insulin as a standard.24,25 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.
5.7. Predicted partition coefficients
The program ALOGPS 2.1 (VCCLAB, Virtual Computational
Chemistry Laboratory, http://www.vcclab.org, 2005) was used for
the calculation of AC log P values.30 Such a program is available
at the Virtual Computational Chemistry Laboratory and used
algorithms have been previously described in the literature.30
Acknowledgments
This study was supported 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 Senior Research Associate and P. Lebrun is Re-
search Director. The authors gratefully acknowledge the technical
assistance of Y. Abrassart, S. Counerotte, P. Fraikin, F. Leleux,
A.-M. Vanbellinghen and A. Van Praet.
5.4. Measurement of tension in rat aorta rings
Experiments were performed with aortas removed from adult
fed Wistar rats (Charles River Laboratories, Belgium).
A section of the thoracic aorta was cleared of adhering fat and
connective tissue and was cut into transverse rings (3–4 mm long).
The endothelium was removed by rubbing the intimal surface with
forceps. The segments were suspended under 1.5 g tension by
means of steel hooks in an organ bath containing 20 ml of a phys-
iological solution (in mM: NaCl 118, KCl 4.7, CaCl2 2.5, NaHCO3 25,
KH2PO4 1.2, MgSO4 1.2, glucose 5). 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, the rings were exposed to KCl (30 mM). When the
tension had stabilized, the drug was added to the bath at increas-
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