New R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans as KATP channel openers: Modulation of the 4-position

Article abstract of DOI:10.1016/j.bmc.2009.09.041

The present work aimed at exploring a series of diversely 4-arylthiourea-substituted R/S-3,4-dihydro-2,2-dimethyl-6-halo-2H-1-benzopyrans structurally related to (±)-cromakalim. These new compounds were examined in vitro as putative potassium channel openers (PCOs) on rat pancreatic islets (inhibition of insulin release) as well as on rat aorta rings (relaxation of aorta ring) and their activity was compared to that of the reference K_ATP channel activators (±)-cromakalim, (±)-pinacidil, diazoxide and of previously reported cromakalim analogues. Structure-activity relationships indicated that the most pronounced inhibitory activity on the insulin secretory process was obtained with molecules bearing a strong meta- or para-electron-withdrawing group (CN or NO₂) on the phenyl ring of the arylthiourea moiety at the 4-position of the benzopyran nucleus (compounds 12-23). Among those, R/S-6-chloro-4-(4-cyanophenylaminothiocarbonylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (16) was found to be the most potent benzopyran-type inhibitor of insulin release ever described. Most of these original benzopyran derivatives show increased selectivity for pancreatic versus vascular tissue. Radioisotopic investigations indicated that these new compounds activated pancreatic K_ATP channels.

Full text of DOI:10.1016/j.bmc.2009.09.041

Bioorganic & Medicinal Chemistry 17 (2009) 7723–7731
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans as KATP channel openers:
Modulation of the 4-position
Xavier Florence a, , , Sophie Sebille a, , Pascal de Tullio a, Philippe Lebrun b,à, Bernard Pirotte a,à
*
a 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
b Laboratoire de Pharmacodynamie et de Thérapeutique, Université Libre de Bruxelles, Faculté de Médecine, 808, Route de Lennik, B-1070 Bruxelles, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2009
Revised 15 September 2009
Accepted 21 September 2009
Available online 25 September 2009
The present work aimed at exploring a series of diversely 4-arylthiourea-substituted R/S-3,4-dihydro-2,2-
dimethyl-6-halo-2H-1-benzopyrans structurally related to ( )-cromakalim. These new compounds were
examined in vitro as putative potassium channel openers (PCOs) on rat pancreatic islets (inhibition of
insulin release) as well as on rat aorta rings (relaxation of aorta ring) and their activity was compared
to that of the reference KATP channel activators ( )-cromakalim, ( )-pinacidil, diazoxide and of previously
reported cromakalim analogues. Structure–activity relationships indicated that the most pronounced
inhibitory activity on the insulin secretory process was obtained with molecules bearing a strong meta-
or para-electron-withdrawing group (CN or NO2) on the phenyl ring of the arylthiourea moiety at the 4-
position of the benzopyran nucleus (compounds 12–23). Among those, R/S-6-chloro-4-(4-cyanophe-
nylaminothiocarbonylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (16) was found to be the most
potent benzopyran-type inhibitor of insulin release ever described. Most of these original benzopyran
derivatives show increased selectivity for pancreatic versus vascular tissue. Radioisotopic investigations
indicated that these new compounds activated pancreatic KATP channels.
Keywords:
Cromakalim analogues
Benzopyran derivatives
KATP channels
Potassium channel openers
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and SUR2B assemble to form the KATP channel detected in vascular
muscle cells whilst, in other smooth muscle types, the Kir6.2 and
ATP-sensitive potassium (KATP) channels belong to the wide
class of potassium channels. The activity of KATP channels is mainly
regulated by changes in the intracellular concentration of adeno-
sine triphosphate, implying that these channels couple cell metab-
olism to membrane excitability.1–3 Such channels are distributed
in many tissues where they play a large variety of physiological
roles. Indeed, KATP channels are involved in the insulin releasing
process,4–6 in cardioprotection,7 in the control of the vascular
tone,8 in neuroprotection and in the control of neurotransmitter
release.9,10
The KATP channel is an octameric complex of four inwardly rec-
tifying subunits (Kir6.x) forming the channel pore, and four sulfo-
nylurea receptor subunits (SURx) that regulate the channel
gating.11–14 According to their tissue localization, different iso-
forms of the KATP channel have been described. In pancreatic b
(insulin-secreting)-cells, the KATP channels consist of an association
of Kir6.2 and SUR115,16 whereas, in cardiac and skeletal muscle tis-
sue, the channel is composed of Kir6.2 and SUR2A subunits. Kir6.1
SUR2B subunits combine to shape the channel.17
Due to the variety of their physiological functions, targeting the
KATP channels could be an attractive mean to develop new thera-
peutic agents. Such an objective can theoretically be reached by
the synthesis of compounds exhibiting a marked specificity and a
high selectivity for a single KATP channel subtype.
By promoting KATP channel activity, potassium channel openers
(PCOs) reduce the membrane excitability and preserve metabolic
expenditure. Thus, and according to their tissue selectivity, PCOs
have been proposed to be used as antihypertensive agents,18 car-
dioprotectants,7,18 bronchodilators,18,19 bladder relaxants18,20 or
hair growth promoters.21 Moreover, by targeting the pancreatic
KATP channels, PCOs are expected to become new therapeutic
agents for the treatment (or management) of endocrine disorders
such as type 1/type 2 diabetes, obesity and hyperinsulinism.22–26
( )-Cromakalim (1), diazoxide (2) and ( )-pinacidil (3) are exam-
ples of chemically diverse PCOs (Fig. 1). ( )-Cromakalim (1), the pro-
totype benzopyran PCO, has been found to exert a marked
vasorelaxant activity27 but is also known, in contrast to diazoxide
(2), to be only slightly active as an inhibitor of insulin secretion.28,29
Recent works aiming at identifying new pancreatic b-cell selective
PCOs allowed us to synthesize a series of R/S-3,4-dihydro-2,2-di-
methyl-6-halo-4-(phenylaminothiocarbonylamino)-2H-1-benzopyrans
* Corresponding author. Tel.: +32 4 366 43 81; fax: +32 4 366 43 62.
E-mail address: xavier.florence@ulg.ac.be (X. Florence).
These authors equally contributed.
à
These authors assumed an equal supervision.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.041

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X. Florence et al. / Bioorg. Med. Chem. 17 (2009) 7723–7731
dimethyl-6-halo-4-(pyridylaminothiocarbonylamino)-2H-1-ben-
zopyrans (24–26) were obtained from the reaction of amines 6a–c
with the appropriate isothiocyanate (R–N@C@S).
Compounds bearing a thienylaminothiocarbonylamino group
(27–28) were obtained by a different synthetic pathway (Scheme 2).
Access to compounds 27 and 28 required the synthesis of thien-
3-yl isothiocyanate as intermediate for reacting with 6b–c. This
intermediate was synthesized from 3-aminothiophene-2-carbox-
ylic acid methyl ester (7) in four steps (Scheme 2).
The ester function of 3-aminothiophene-2-carboxylic acid
methyl ester (7) was first hydrolyzed by sodium hydroxide and
3-aminothiophene-2-carboxylic acid (8) was then decarboxylated
by oxalic acid to obtain 3-aminothiophene oxalate (9). Compound
9 was treated with an aqueous solution of ammonia to give 3-ami-
nothiophene (10). Thien-3-yl isothiocyanate (11) was isolated after
the reaction of 3-aminothiophene (10) with 1,10-thiocarbonyldi-
imidazole. Finally, this intermediate was converted to the final
R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(3-thienylaminothiocar-
bonylamino)-2H-1-benzopyrans 27 and 28 by reaction with the
appropriate R/S-4-amino-3,4-dihydro-2,2-dimethyl-6-halo-2H-1-
benzopyran (6b–c).
Figure 1. Chemical structure of ( )-cromakalim (1), diazoxide (2), ( )-pinacidil (3),
compound 4 and 5.
Compounds 29 and 30 were obtained by treatment of the N-(6-
bromo-2,2-dimethyl-3,4-dihydro-2H-chromen-4-yl)-N0-phenylthiou-
reas 14 and 5 with iodomethane (Scheme 3). Lastly, compounds 31 and
32 were synthesized by reaction between, respectively, compound 29
or 30 with cyanamide (Scheme 3).
All these derivatives (12–32) were crystallized from appropriate
solvents and characterized by IR, 1H NMR and elemental analyses
to obtain the final materials with the chemical purity required
prior to pharmacological evaluations.
structurally related to cromakalim.25 Several of these original
compounds were found to be less effective as vasorelaxants and much
more potent as inhibitors of insulin release than the reference molecules
diazoxide and cromakalim.25 Structure–activity relationships further
indicated that the nature of the substituent at the 4-position of the
benzopyran nucleus played a crucial role in the expression of an
inhibitory effect on insulin release. Indeed, molecules bearing a C-4 phe-
nylthiourea moiety substituted on the phenyl ring by a meta- or a para-
electron-withdrawing group (a chlorine atom) (Fig 1, compounds 4 and
5) were found to be very potent and selective for the pancreatic
b-cells.25
3. Results and discussion
According to structure–activity relationships deduced from our
previous investigations, the present work aims at developing more
potent and more insulin-secreting cell-selective dimethylchroman
derivatives. We tried to achieve this goal by preparing and testing
additional meta- or para-electron-withdrawing groups, by replac-
ing the phenyl ring with a bioisosteric heteroaromatic ring or by
replacing the thiourea function with a bioisosteric cyanoguanidine
function.
The ability of the newly synthesized cromakalim analogues
(Table 1, compounds 12–32) 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).
Moreover, the vasorelaxant activity of the compounds was deter-
mined on 30 mM K+-depolarized rat aorta (endothelium-free)
rings.
( )-Cromakalim (1), diazoxide (2), ( )-pinacidil (3), compound 4
and 5 were used as reference PCOs (Fig. 1).
On the pancreatic model, ( )-cromakalim (1) and ( )-pinacidil
(3) have previously been shown to be barely active at a 10 lM con-
centration. Diazoxide (2), however, as well as compounds 4 and 5,
clearly reduced the glucose-induced insulin release (Table 1). Com-
pounds 4 and 5 were much more potent than diazoxide at inhibi-
ting the secretory process.
The new compounds, including the S-methylisothiourea inter-
mediates, were examined as putative potassium channel openers
on rat pancreatic islets as well as on rat aorta rings. Radioisotopic
investigations were performed to confirm the mechanism of action
of these novel compounds.
2. Chemistry
Our secretory data further revealed that the new benzopyran
derivatives (12–32) were more potent on pancreatic b-cells than
the reference compounds ( )-cromakalim, ( )-pinacidil and diaz-
oxide (Table 1).
The key intermediates (6a–c) giving access to the cromakalim
analogues (12–28) were synthesized in six steps, according to the
literature,30,31 starting from the appropriate p-halophenols
(Scheme 1).
R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(phenylaminothiocar-
bonylamino)-2H-1-benzopyrans (12–23) and R/S-3,4-dihydro-2,2-
Several compounds (12–23), tested at a 10 lM concentration,
provoked a 85–90% inhibition of the glucose-induced insulin re-
lease; a result which can be considered as a near to maximal effect
considering the glucose-insensitive secretory process.
Our previous work25 indicated that an electron-withdrawing
group, such as a chlorine atom, in the meta or para position of the
phenyl ring of R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(phenylami-
nothiocarbonylamino)-2H-1-benzopyrans enhanced the inhibitory
activity on the pancreatic tissue. As expected, compounds 12–23,
bearing a strong electron-withdrawing group in the meta or para-
phenyl position (CN, NO2), exhibited a marked inhibitory effect on
the glucose-induced insulin secretion (Table 1). Compared with
the reference molecules, compounds 12–23 were found to have an
Scheme 1. Synthesis of cromakalim analogues 12–26. Reagents: (i) RNCS, CH2Cl2.

X. Florence et al. / Bioorg. Med. Chem. 17 (2009) 7723–7731
7725
Scheme 2. Synthetic pathway to R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(3-thienylaminothiocarbonylamino)-2H-1-benzopyrans 27 and 28. Reagents: (i) NaOH 2 M; (ii)
oxalic acid; (iii) NH3, H2O; (iv) 1,10-thiocarbonyldiimidazole.
Scheme 3. Synthetic pathway to R/S-N-(6-bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)-N0-(3- or 4-substituted phenyl)-S-methylisothioureas 29 and 30 and
to R/S-N-(6-bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)-N0-(3- or 4-substituted phenyl)-N00-cyanoguanidines (31–32). Reagents: (i) CH3I; (ii) H2NCN, 1,4-
diazabicyclo[2.2.2]octane.
identical activity at 10
activity than compound 4 (p <0.05). At 1
l
M (p >0.05) than compound 5 but a stronger
were found to be less potent on b-cells than their corresponding
cyano- or nitro-substituted phenyl analogues 12–23 (Table 1).
The nature of the halogen atom at the 6-position of the benzopyran
nucleus affected the activity on the pancreatic tissue (p <0.05)
(except: 24 vs 25 and 26, p >0.05), the 6-fluoro compound 24 being
the less potent and the 6-bromo compound 26 the most potent.
Compounds bearing a thienyl moiety on the thiourea chain (27–
28) were found to exert, on the pancreatic b-cells, a more pro-
nounced inhibitory activity than ( )-cromakalim, ( )-pinacidil
and diazoxide (p <0.05), and were less effective than compounds
4 and 5 (p <0.05). The nature of the halogen atom at the 6-position
of the benzopyran nucleus did not significantly affect the activity
on the pancreatic tissue (p >0.05).
Finally, the 3-pyridinyl- (24–26) and the 3-thienyl-substituted
(27–28) compounds appeared to exert close to similar inhibitory
effects on the insulin releasing process and the pyridine or the thio-
phene ring can be considered as less appropriate than the benzene
ring substituted with a meta- or para-electron-withdrawing group.
Molecules bearing a phenylisothiourea group (29–30) were also
more potent than ( )-cromakalim, ( )-pinacidil and diazoxide (p
<0.05), and less potent than the reference compounds 4 and 5 (p
<0.05). As a result, and compared to the non S-methylated deriva-
tives 14 and 5, the alkylation of the sulfur atom of the thiourea
function appeared to be responsible for a loss of activity on pancre-
atic b-cells (p <0.05).
lM, these compounds (12–
23) displayed a similar activity than compounds 4 and 5, except for
16 which was found to express a stronger inhibitory activity than
references 4 and 5 (p <0.05) and for 17 which was more potent than
the reference compound 5 (p <0.05). The nature of the halogen atom
at the 6-position of the benzopyran nucleus did not markedly affect
the activity of compounds 12–23 on the pancreatic tissue. At 1 lM,
however, compounds 13 (6-Cl) and 14 (6-Br) were found to be some-
what more potent than compound 12 (6-F) (p <0.05), 16 (6-Cl) ex-
pressed a stronger inhibitory activity than 15 (6-F) (p <0.05), as
observed for compounds 19 (6-Cl) and 20 (6-Br) versus 18 (6-F) (p
<0.05) and 23 (6-Br) versus 21 (6-F) (p <0.05). The position of the
electron withdrawing moiety on the C-4 phenyl ring (compounds
12–23) had no marked influence on the pancreatic activity, except
for compound 13 versus 16 (p <0.05 at 10 lM and 1 lM). According
to these data, compound 16 can be considered as the most potent
molecule of this series.
Compounds resulting from the introduction of a pyridinyl moi-
ety on the thiourea chain (24–26) were also more potent than ( )-
cromakalim, ( )-pinacidil and diazoxide at inhibiting insulin secre-
tion but less potent than the reference compounds 4 and 5. When
comparing the pyridinyl compounds 24–26 with their correspond-
ing unsubstituted phenyl analogues previously described,25 the
isosteric replacement of the phenyl ring with the pyridin-3-yl nu-
cleus gave compounds expressing essentially the same activity on
pancreatic b-cells. The pyridin-3-yl derivatives 24–26, however,
Compounds 31 and 32, bearing a cyanoguanidine group, were
found to be slightly less potent at reducing the glucose-dependent

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X. Florence et al. / Bioorg. Med. Chem. 17 (2009) 7723–7731
Table 1
Residual insulin secretion and myorelaxant activity of original 4-substituted R/S-3,4-dihydro-2,2-dimethyl-6-halo-2H-1-benzopyrans compared to ( )-cromakalim, ( )-pinacidil,
diazoxide, compound 4 and 5
Compounds
X
Y
R3
R4
% Residual insulin secretiona
Myorelaxant activity
b
EC50
(lM)
10
lM
1 lM
12
13
14
15
16
17
18
19
20
21
22
23
F
S
S
S
S
S
S
S
S
S
S
S
S
CN
CN
CN
16.1 1.5 (21)
15.8 0.9 (18)
14.2 1.3 (21)
12.0 0.8 (15)
10.6 0.7 (24)
11.7 0.7 (24)
11.5 0.9 (15)
14.4 0.6 (22)
13.5 0.7 (23)
10.7 1.1 (13)
10.3 0.7 (13)
13.5 1.0 (28)
80.0 4.4 (30)
66.2 2.5 (31)
65.5 3.6 (30)
64.7 4.0 (19)
47.8 2.8 (29)
57.5 3.0 (29)
76.1 4.3 (15)
64.3 3.4 (23)
64.3 2.2 (24)
74.3 3.8 (34)
67.3 3.6 (19)
61.3 3.4 (31)
2.6 0.5 (4)
0.60 0.06 (4)
0.64 0.05 (5)
>3.0 (5)
>10.0 (4)
>10.0 (6)
>3.0 (4)
>3.0 (4)
>10.0 (5)
>3.0 (4)
Cl
Br
F
Cl
Br
F
Cl
Br
F
Cl
Br
CN
CN
CN
NO2
NO2
NO2
NO2
NO2
NO2
>10.0 (4)
>10.0 (4)
24
25
26
F
Cl
Br
S
S
S
62.1 3.6 (15)
50.2 2.7 (24)
44.3 2.8 (23)
—
—
—
6.6 2.0 (4)
2.8 0.4 (6)
3.7 1.0 (4)
27
28
Cl
Br
S
S
51.9 2.8 (32)
44.0 2.9 (35)
—
—
>3.0 (4)
>10.0 (4)
29
30
Br
Br
S–CH3
S–CH3
CN
CN
37.7 2.7 (16)
40.8 2.9 (13)
93.7 5.5 (15)
89.4 6.0 (15)
>30.0 (4)
>10.0 (4)
Cl
Cl
31
32
Br
Br
N–CN
N–CN
45.3 1.7 (15)
42.7 2.7 (14)
90.5 5.6 (13)
95.8 5.3 (14)
5.2 0.6 (5)
11.4 3.5 (3)
( )-Cromakalim
94.4 4.1 (32)c
92.1 3.9 (13)d
73.9 4.4 (16)d
23.0 2.4 (31)e
12.2 1.2 (20)e
95.3 3.8 (31)c
97.7 6.7 (19)d
87.5 5.0 (15)d
74.6 4.0 (21)e
75.7 3.2 (24)e
0.13 0.01 (7)c
0.35 0.02 (11)d
22.4 2.1 (11)d
>10.0 (5)e
(
)-Pinacidil
Diazoxide
4
5
Br
Br
S
S
Cl
Cl
>10.0 (4)e
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. 33.
Published results: Ref. 32.
Published results: Ref. 25.
d
e
secretory rate than the corresponding isothiourea compounds 29
and 30 (p >0.05) and less potent than the corresponding non
S-alkylated compounds 14 and 5 (p <0.05). However, these com-
pounds (31–32) remained more potent than ( )-cromakalim, ( )-
pinacidil and diazoxide (p <0.05). Thus, the isosteric replacement
of the thiourea function of 14 or 5 by a cyanoguanidine group ap-
peared to worsen the activity on the pancreatic tissue.
meta-position of the electron-withdrawing group was clearly more
favorable than the para-position for myorelaxant activity (compare
compounds 12, 13, 14 with 15, 16, 17).
Unfortunately, and as previously observed with 4 and 5, most
compounds precipitated in the bathing medium before reaching
their maximal activity, making it difficult to accurately determine
their respective EC50 values. However, it can be concluded that
these compounds were less potent on the vascular model than
( )-cromakalim and ( )-pinacidil.
On the vascular tissue, and as previously reported, diazoxide
exhibited a moderate myorelaxant activity (EC50 = 22.4 2.1
while ( )-cromakalim and ( )-pinacidil exhibited marked vasore-
l
M)
M,
Due to its profile, additional radioisotopic experiments were
performed with 23 in order to determine if this compound was a
pancreatic ATP-sensitive potassium channel opener.
laxant properties (EC50 = 0.13 0.01
lM
and 0.35 0.02 l
respectively).25,32,33 Our two reference compounds 4 and 5 were
less potent than cromakalim and pinacidil.
Figure 2 illustrates the effects of compound 23 (10 l
M) on 86Rb
Compounds 12, 13, 14, 24, 25, 26 and 31 were more potent on
the vascular tissue than compounds 4, 5 and diazoxide (p <0.05).
The myorelaxant activity of 13 and 14 was in the same range,
although less potent (p <0.05), than ( )-pinacidil. It should be
noted that, for the cyano-substituted compounds 12–17, the
fractional outflow rate (FOR) from prelabeled and perifused rat
pancreatic islets exposed throughout to 5.6 mM glucose and extra-
cellular Ca2+. In the absence of glibenclamide in the perifusing
medium (s), 23 provoked a rapid, marked and reversible increase
in the rate of 86Rb outflow. The presence in the perifusate of the

X. Florence et al. / Bioorg. Med. Chem. 17 (2009) 7723–7731
7727
firmed that the inhibition of insulin release was mediated by the
activation of pancreatic b-cell ATP-sensitive potassium channels.
Additional pharmacomodulations, and more specifically the
substitution of the halogen atom at the 6-position by other groups,
are planned in order to discriminate more potent and more selec-
tive dimethylchroman derivatives inhibiting the insulin releasing
process.
23
6.0
5.0
4.0
3.0
2.0
1.0
5. Experimental section
5.1. Chemistry
Melting points were determined on a Stuart SMP3 capillary
apparatus and are uncorrected. IR spectra were recorded as KBr
pellets on a Perkin–Elmer 1000 FTIR spectrophotometer. The 1H
NMR spectra were recorded on a Bruker Avance (500 MHz) instru-
ment using DMSO-d6 as the solvent with TMS as an internal stan-
dard; chemical shifts are reported in d values (ppm) relative to that
of internal TMS. The abbreviations s = singlet, d = doublet, t = trip-
let, q = quadruplet, m = multiplet, and br = broad are used through-
out. Elemental analyses (C, H, N, S) were realized on a Thermo
Scientific FlashEA 1112-elemental analyzer and were within
0.4% of the theoretical values. All reactions were routinely
30
40
50
60
70
80
90
TIME (min)
checked by TLC on silica gel Merck 60 F254
.
Figure 2. Effect of 23 (10 l
M) on 86Rb outflow from rat pancreatic islets perifused
throughout in the absence (s) or presence (d) of glibenclamide (10 lM). Basal
media contained 5.6 mM glucose and extracellular Ca2+. Mean values ( SEM) refer
to 5–6 individual experiments.
5.1.1. R/S-4-Amino-3,4-dihydro-2,2-dimethyl-6-halo-2H-1-
benzopyrans (6a–c)
These compounds were prepared from the appropriate parahal-
ophenols (6a–c), according to previously described synthetic
procedures.30,31
hypoglycemic sulfonylurea glibenclamide (10 lM, d), a pharmaco-
logical tool known to block the KATP channels,34,35 abolished the
stimulatory effect of compound 23 on 86Rb outflow.
5.1.2. R/S-4-(3-Cyanophenylaminothiocarbonylamino)-3,4-
dihydro-2,2-dimethyl-6-fluoro-2H-1-benzopyran (12)
Taken as a whole, these radioisotopic data suggest that, in insu-
lin-secreting cells, compound 23 activated KATP channels.29,36–38
3-Cyanophenyl isothiocyanate (0.38 g, 2.46 mmol) was added
to a solution of 6a30 (0.4 g, 2.05 mmol) in methylene chloride
(5 mL). After 30 min, the solvent was removed under vacuum,
and the crude product was triturated with ethyl acetate. The insolu-
ble material was collected by filtration, and petroleum ether was
added to the filtrate. The resulting precipitate was collected by fil-
tration, washed with petroleum ether, and dried. The product was
then crystallized in a mixture of methanol/water. The final precip-
itate was collected by filtration, washed with water, and dried
4. Conclusion
In the present study, we have synthesized and characterized the
biological activity of several novel R/S-4-arylthiourea-substituted
3,4-dihydro-2,2-dimethyl-6-halo-2H-1-benzopyrans structurally
related to the potassium channel opener ( )-cromakalim. Some
of these new dimethylchroman derivatives included, at the 4-posi-
tion, a phenylthiourea moiety substituted on the phenyl ring by a
meta- or para-electron-withdrawing group such as NO2 or CN.
Compounds bearing an arylthiourea group with either a 3-pyridi-
nyl or a 3-thienyl moiety replacing the phenyl ring were also syn-
thesized. Moreover, the thiourea function was further replaced by
a S-methylisothiourea group or a bioisosteric cyanoguanidine func-
tion. The biological activity of these new compounds was com-
pared with that of ( )-cromakalim, ( )-pinacidil, diazoxide and
compounds 4 and 5, used as reference PCOs.
All these new dimethylchromans (12–32) were, however, more
potent than the reference molecules ( )-cromakalim, ( )-pinacidil
and diazoxide at inhibiting the insulin releasing process. Vascular
data further revealed that the new compounds were less potent
than ( )-cromakalim and ( )-pinacidil as vasorelaxant agents. Bio-
logical results also indicated that compounds bearing a strong
meta- or para-electron-withdrawing group (12–23) on the C-4 phe-
nyl ring of the thiourea moiety were potent inhibitors of insulin re-
lease. Compound 16 was found to be the most potent benzopyran
derivative tested on the present pancreatic b-cell ‘in vitro’ model.
Among these newly synthesized dimethylchromans, molecules
15–23 can be regarded as the most promising compounds due to
their marked inhibitory effect on the insulin releasing process and
their weak myorelaxant activity. Radioisotopic experiments con-
(0.20 g, 28%): mp: 168–169 °C; IR (KBr)
H aromatic), 2980, 2927 (C–H aliphatic), 2236 (C„N), 1199
(C@S) cmꢀ1 1H NMR (DMSO-d6, 500 MHz): d: 1.27 (s, 3H, CH3),
m: 3227 (N–H), 3041 (C–
;
1.40 (s, 3H, CH3), 1.80 (m, 1H, HA of CH2), 2.22 (m, 1H, HB of
CH2), 5.78 (m, 1H, CH), 6.78 (dd, J = 4.75, 8.83 Hz, 1H, 8-H), 7.01
(m, 1H, 7-H), 7.07 (d, J = 8.40 Hz, 1H, 5-H), 7.51–7.57 (m, 2H, 40-
H, 50-H), 7.76 (d, J = 7.60 Hz, 1H, 60-H), 8.05 (s, 1H, 20-H), 8.37 (d,
J = 7.55 Hz, 1H, NH(CH)), 9.82 (s, 1H, NH(C6H4)). Anal.
(C19H18N3OSF) theoretical: C, 64.21; H, 5.10; N, 11.82; S, 9.02.
Found: C, 63.85; H, 5.36; N, 11.66; S, 8.75.
5.1.3. R/S-6-Chloro-4-(3-cyanophenylaminothiocarbonyl-
amino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (13)
The title compound was obtained as described for 12, starting
from 6b31 (0.4 g, 1.89 mmol) and 3-cyanophenyl isothiocyanate
(363 mg, 2.27 mmol) (0.44 g, 62%): mp: 185–186 °C; IR (KBr):
m
: 3338, 3275, 3212 (N–H), 3056 (C–H aromatic), 2976, 2932 (C–
H aliphatic), 2237 (C„N), 1202 (C@S) cmꢀ1 1H NMR (DMSO-d6,
;
500 MHz): d: 1.28 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.81 (m, 1H, HA
of CH2), 2.23 (m, 1H, HB of CH2), 5.80 (m, 1H, CH), 6.79 (d,
J = 8.70 Hz, 1H, 8-H), 7.19 (d, J = 8.65 Hz, 1H, 7-H), 7.27 (s, 1H, 5-
H), 7.52–7.57 (m, 2H, 40-H, 50-H), 7.77 (d, J = 7.65 Hz, 1H, 60-H),
8.05 (s, 1H, 20-H), 8.40 (br d, 1H, NH(CH)), 9.84 (s, 1H, NH(C6H4)).

7728
X. Florence et al. / Bioorg. Med. Chem. 17 (2009) 7723–7731
Anal. (C19H18N3OSCl) theoretical: C, 61.36; H, 4.88; N, 11.30; S,
8.62. Found: C, 61.16; H, 5.07; N, 11.32; S, 8.95.
(443 mg, 2.46 mmol) (0.36 g, 47%): mp: 175–176 °C; IR (KBr):
3244 (N–H), 3098, 3035 (C–H aromatic), 2976, 2930 (C–H ali-
phatic), 1528, 1350 (N@O), 1197 (C@S) cmꢀ1 1H NMR (DMSO-d6,
m:
;
5.1.4. R/S-6-Bromo-4-(3-cyanophenylaminothiocarbonyl-
amino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (14)
The title compound was obtained as described for 12, starting
from 6c30 (0.4 g, 1.56 mmol) and 3-cyanophenyl isothiocyanate
(300 mg, 1.87 mmol) (0.34 g, 53%): mp: 181–182 °C; IR (KBr):
500 MHz): d: 1.28 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.80 (m, 1H, HA
of CH2), 2.24 (m, 1H, HB of CH2), 5.79 (m, 1H, CH), 6.78 (dd,
J = 4.80, 8.90 Hz, 1H, 8-H), 7.01 (m, 1H, 7-H), 7.08 (d, J = 8.95 Hz,
1H, 5-H), 7.60 (m, 1H, 50-H), 7.86 (d, J = 7.95 Hz, 1H, 40-H), 7.94
(d, J = 8.05 Hz, 1H, 60-H), 8.44–8.60 (m, 2H, 20-H, NH(CH)), 9.97
(br s, 1H, NH(C6H4)). Anal. (C18H18N3O3SF) theoretical: C, 57.59;
H, 4.83; N, 11.19; S, 8.54. Found: C, 57.24; H, 4.86; N, 11.16; S, 8.14.
m
: 3329, 3162 (N–H), 3006 (C–H aromatic), 2984, 2930 (C–H ali-
phatic), 2230 (C„N), 1200 (C@S) cmꢀ1 1H NMR (DMSO-d6,
;
500 MHz): d: 1.28 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.81 (m, 1H, HA
of CH2), 2.23 (m, 1H, HB of CH2), 5.80 (m, 1H, CH), 6.74 (d,
J = 8.70 Hz, 1H, 8-H), 7.31 (d, J = 8.65 Hz, 1H, 7-H), 7.39 (s, 1H, 5-
H), 7.52–7.57 (m, 2H, 40-H, 50-H), 7.77 (d, J = 7.70 Hz, 1H, 60-H),
8.04 (s, 1H, 20-H), 8.40 (br d, 1H, NH(CH)), 9.84 (s, 1H, NH(C6H4)).
Anal. (C19H18N3OSBr) theoretical: C, 54.81; H, 4.36; N, 10.09; S,
7.70. Found: C, 54.57; H, 4.25; N, 10.13; S, 8.13.
5.1.9. R/S-6-Chloro-3,4-dihydro-2,2-dimethyl-4-(3-nitrophenyl-
aminothiocarbonylamino)-2H-1-benzopyran (19)
The title compound was obtained as described for 12, starting
from 6b31 (0.4 g, 1.89 mmol) and 3-nitrophenyl isothiocyanate
(408 mg, 2.27 mmol) (0.60 g, 81%): mp: 188–189 °C; IR (KBr):
3183 (N–H), 3097, 3024 (C–H aromatic), 2980, 2928, 2950 (C–H
aliphatic), 1530, 1345 (N@O), 1198 (C@S) cmꢀ1 1H NMR (DMSO-
m:
;
5.1.5. R/S-4-(4-Cyanophenylaminothiocarbonylamino)-3,4-
dihydro-2,2-dimethyl-6-fluoro-2H-1-benzopyran (15)
d6, 500 MHz): d: 1.29 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.84 (m, 1H,
HA of CH2), 2.25 (m, 1H, HB of CH2), 5.80 (m, 1H, CH), 6.80 (d,
J = 8.70 Hz, 1H, 8-H), 7.20 (d, J = 8.65 Hz, 1H, 7-H), 7.29 (s, 1H, 5-
H), 7.60 (m, 1H, 50-H), 7.87 (d, J = 7.80 Hz, 1H, 40-H), 7.92 (d,
J = 7.85 Hz, 1H, 60-H), 8.45–8.61 (m, 2H, 20-H, NH(CH)), 9.95 (br s,
1H, NH(C6H4)). Anal. (C18H18N3O3SCl) theoretical: C, 55.17; H,
4.63; N, 10.72; S, 8.18. Found: C, 54.98; H, 4.83; N, 10.78; S, 7.92.
The title compound was obtained as described for 12, starting
from 6a30 (0.4 g, 2.05 mmol) and 4-cyanophenyl isothiocyanate
(394 mg, 2.46 mmol) (0.34 g, 47%): mp: 202–203 °C; IR (KBr):
3248 (N–H), 3096, 3055 (C–H aromatic), 2989, 2974, 2950, 2933
(C–H aliphatic), 2227 (C„N), 1197 (C@S) cmꢀ1 1H NMR (DMSO-
m:
;
d6, 500 MHz): d: 1.27 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.79 (m, 1H,
HA of CH2), 2.25 (m, 1H, HB of CH2), 5.77 (m, 1H, CH), 6.78 (dd,
J = 4.75, 8.80 Hz, 1H, 8-H), 7.01 (m, 1H, 7-H), 7.07 (d, J = 8.80 Hz,
1H, 5-H), 7.76 (d, J = 8.60 Hz, 2H, 20-H, 60-H), 7.80 (d, J = 8.55 Hz,
2H, 30-H, 50-H), 8.51 (br d, 1H, NH(CH)), 10.02 (s, 1H, NH(C6H4)).
Anal. (C19H18N2OSF) theoretical: C, 64.21; H, 5.10; N, 11.82; S,
9.02. Found: C, 63.86; H, 5.51; N, 11.52; S, 8.81.
5.1.10. R/S-6-Bromo-3,4-dihydro-2,2-dimethyl-4-(3-nitrophe-
nylaminothiocarbonylamino)-2H-1-benzopyran (20)
The title compound was obtained as described for 12, starting
from 6c30 (0.4 g, 1.56 mmol) and 3-nitrophenyl isothiocyanate
(338 mg, 1.87 mmol) (0.64 g, 94%): mp: 186–187 °C; IR (KBr):
3227 (N–H), 3092, 3026 (C–H aromatic), 2981, 2926, 2949 (C–H
aliphatic), 1530, 1349 (N@O), 1198 (C@S) cmꢀ1 1H NMR (DMSO-
m:
;
5.1.6. R/S-6-Chloro-4-(4-cyanophenylaminothiocarbonyl-
amino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (16)
The title compound was obtained as described for 12, starting
from 6b31 (0.4 g, 1.89 mmol) and 4-cyanophenyl isothiocyanate
d6, 500 MHz): d: 1.29 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.81 (m, 1H,
HA of CH2), 2.25 (m, 1H, HB of CH2), 5.81 (m, 1H, CH), 6.75 (d,
J = 8.70 Hz, 1H, 8-H), 7.32 (d, J = 8.68 Hz, 1H, 7-H), 7.41 (s, 1H, 5-
H), 7.60 (m, 1H, 50-H), 7.87 (d, J = 7.85 Hz, 1H, 40-H), 7.94 (d,
J = 7.95 Hz, 1H, 60-H), 8.47–8.61 (m, 2H, 20-H, NH(CH)), 9.97 (br s,
1H, NH(C6H4)). Anal. (C18H18N3O3SBr) theoretical: C, 49.55; H,
4.16; N, 9.63; S, 7.35. Found: C, 49.28; H, 4.36; N, 9.69; S, 7.09.
(363 mg, 2.27 mmol) (0.44 g, 63%): mp: 216–217 °C; IR (KBr):
3243 (N–H), 3092, 3039 (C–H aromatic), 2989, 2974, 2954, 2933
(C–H aliphatic), 2226 (C„N), 1197 (C@S) cmꢀ1 1H NMR (DMSO-
m:
;
d6, 500 MHz): d: 1.28 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.80 (m, 1H,
HA of CH2), 2.25 (m, 1H, HB of CH2), 5.78 (m, 1H, CH), 6.80 (d,
J = 8.75 Hz, 1H, 8-H), 7.20 (d, J = 8.68 Hz, 1H, 7-H), 7.27 (s, 1H, 5-
H), 7.76 (d, J = 8.65 Hz, 2H, 20-H, 60-H), 7.80 (d, J = 8.55 Hz, 2H, 30-
H, 50-H), 8.51 (br d, 1H, NH(CH)), 10.01 (s, 1H, NH(C6H4)). Anal.
(C19H18N3OSCl) theoretical: C, 61.36; H, 4.88; N, 11.30; S, 8.62.
Found: C, 61.39; H, 4.73; N, 11.28; S, 9.00.
5.1.11. R/S-3,4-Dihydro-2,2-dimethyl-6-fluoro-4-(4-nitro-
phenylaminothiocarbonylamino)-2H-1-benzopyran (21)
The title compound was obtained as described for 12, starting
from 6a30 (0.4 g, 2.05 mmol) and 4-nitrophenyl isothiocyanate
(443 mg, 2.46 mmol) (0.56 g, 73%): mp: 192–194 °C; IR (KBr):
3221 (N–H), 3040 (C–H aromatic), 2980, 2933 (C–H aliphatic),
1522, 1346 (N@O), 1196 (C@S) cmꢀ1 1H NMR (DMSO-d6,
m:
;
5.1.7. R/S-6-Bromo-4-(4-cyanophenylaminothiocarbonyl-
amino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (17)
The title compound was obtained as described for 12, starting
from 6c30 (0.4 g, 1.56 mmol) and 4-cyanophenyl isothiocyanate
500 MHz): d: 1.28 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.80 (m, 1H, HA
of CH2), 2.26 (m, 1H, HB of CH2), 5.76 (m, 1H, CH), 6.79 (br s, 1H,
8-H), 7.02–7.09 (m, 2H, 5-H, 7-H), 7.88 (d, J = 7.00 Hz, 2H, 20-H,
60-H), 8.20 (d, J = 7.25 Hz, 2H, 30-H, 50-H), 8.63 (br s, 1H, NH(CH)),
10.20 (br s, 1H, NH(C6H4)). Anal. (C18H18N3O3SF) theoretical: C,
57.59; H, 4.83; N, 11.19; S, 8.54. Found: C, 57.37; H, 4.82; N,
11.21; S, 8.17.
(300 mg, 1.87 mmol) (0.50 g, 77%): mp: 207–208 °C; IR (KBr):
3248 (N–H), 3035 (C–H aromatic), 2988, 2934 (C–H aliphatic),
2226 (C„N), 1197 (C@S) cmꢀ1 1H NMR (DMSO-d6, 500 MHz): d:
m:
;
1.28 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.80 (m, 1H, HA of CH2), 2.25
(m, 1H, HB of CH2), 5.78 (m, 1H, CH), 6.75 (d, J = 8.60 Hz, 1H, 8-
H), 7.32 (d, J = 8.55 Hz, 1H, 7-H), 7.40 (s, 1H, 5-H), 7.76–7.81 (m,
4H, 20-H, 30-H, 50-H, 60-H), 8.58 (br s, 1H, NH(CH)), 10.06 (br s,
1H, NH(C6H4)). Anal. (C19H18N3OSBr) theoretical: C, 54.81; H,
4.36; N, 10.09; S, 7.70. Found: C, 55.09; H, 4.62; N, 10.27; S, 7.73.
5.1.12. R/S-6-Chloro-3,4-dihydro-2,2-dimethyl-4-(4-nitro-
phenylaminothiocarbonylamino)-2H-1-benzopyran (22)
The title compound was obtained as described for 12, starting
from 6b31 (0.4 g, 1.89 mmol) and 4-nitrophenyl isothiocyanate
(408 mg, 2.27 mmol) (0.62 g, 84%): mp: 194–196 °C; IR (KBr):
3226 (N–H), 3059 (C–H aromatic), 2986, 2932 (C–H aliphatic),
1518, 1335 (N@O), 1198 (C@S) cmꢀ1 1H NMR (DMSO-d6,
m:
5.1.8. R/S-3,4-Dihydro-2,2-dimethyl-6-fluoro-4-(3-nitrophenyl-
aminothiocarbonylamino)-2H-1-benzopyran (18)
;
500 MHz): d: 1.29 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.81 (m, 1H, HA
of CH2), 2.27 (m, 1H, HB of CH2), 5.78 (m, 1H, CH), 6.81 (d,
J = 8.75 Hz, 1H, 8-H), 7.21 (d, J = 8.70 Hz, 1H, 7-H), 7.29 (s, 1H,
The title compound was obtained as described for 12, starting
from 6a30 (0.4 g, 2.05 mmol) and 3-nitrophenyl isothiocyanate

X. Florence et al. / Bioorg. Med. Chem. 17 (2009) 7723–7731
7729
5-H), 7.89 (d, J = 8.95 Hz, 2H, 20-H, 60-H), 8.20 (d, J = 9.05 Hz, 2H, 30-
H, 50-H), 8.64 (br s, 1H, NH(CH)), 10.20 (br s, 1H, NH(C6H4)). Anal.
(C18H18N3O3SCl) theoretical: C, 55.17; H, 4.63; N, 10.72; S, 8.18.
Found: C, 55.30; H, 4.79; N, 10.81; S, 8.45.
5.1.17. 3-Aminothiophene oxalate salt (9)
Methyl 3-amino-2-thiophenecarboxylate (15 g, 95.4 mmol) in
2 M aqueous sodium hydroxide was refluxed for 30 min. The mix-
ture was then cooled to 0 °C and the solution was adjusted to pH 1
with concentrated hydrochloric acid. The resulting precipitate was
collected by filtration. The solution of the product in acetone
(150 mL) was dried over magnesium sulfate, filtered and evapo-
rated under reduced pressure. Oxalic acid dihydrate (13.2 g,
104.7 mmol) was added to the solution of the crude product in iso-
propanol (60 mL). The solution was stirred at 38 °C for one hour
and was then cooled to 0 °C. Diethyl ether (180 mL) was added
to the solution. The final precipitate was collected by filtration,
washed with diethyl ether, and dried (13.2 g, 56%): mp: 139–
142 °C.
5.1.13. R/S-6-Bromo-3,4-dihydro-2,2-dimethyl-4-(4-nitro-
phenylaminothiocarbonylamino)-2H-1-benzopyran (23)
The title compound was obtained as described for 12, starting
from 6c30 (0.4 g, 1.56 mmol) and 4-nitrophenyl isothiocyanate
(338 mg, 1.87 mmol) (0.54 g, 80%): mp: 201–202 °C; IR (KBr):
3246 (N–H), 3050 (C–H aromatic), 2986, 2932 (C–H aliphatic),
1518, 1334 (N@O), 1199 (C@S) cmꢀ1 1H NMR (DMSO-d6,
m:
;
500 MHz): d: 1.29 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.81 (m, 1H, HA
of CH2), 2.27 (m, 1H, HB of CH2), 5.78 (m, 1H, CH), 6.75 (d,
J = 8.70 Hz, 1H, 8-H), 7.32 (d, J = 8.65 Hz, 1H, 7-H), 7.41 (s, 1H, 5-
H), 7.88 (d, J = 8.85 Hz, 2H, 20-H, 60-H), 8.20 (d, J = 9.10 Hz, 2H, 30-
H, 50-H), 8.64 (br s, 1H, NH(CH)), 10.19 (br s, 1H, NH(C6H4)). Anal.
(C18H18N3O3SBr) theoretical: C, 49.55; H, 4.16; N, 9.63; S, 7.35.
Found: C, 49.75; H, 4.20; N, 9.71; S, 7.32.
5.1.18. 3-Aminothiophene (10)
Ammonium hydroxide solution was added to a suspension of 9
(1 g, 5.29 mmol) in water (35 mL) until the solution was adjusted
to pH 14. The product was extracted with methylene chloride.
The organic layer was dried over magnesium sulfate, filtered and
evaporated under reduced pressure. The resulting oil (0.52 g,
100%) was used directly in the next step without further
purification.
5.1.14. R/S-3,4-Dihydro-2,2-dimethyl-6-fluoro-4-(3-pyridyl-
aminothiocarbonylamino)-2H-1-benzopyran (24)
3-Pyridyl isothiocyanate (274
lL, 2.46 mmol) was added to a
solution of 6a30 (0.4 g, 2.05 mmol) in methylene chloride (5 mL).
After 30 min, the solvent was removed under vacuum and the
crude product was triturated with methanol. The insoluble mate-
rial was collected by filtration, and water was added to the filtrate.
The resulting precipitate was collected by filtration, washed with
5.1.19. 3-Thienyl isothiocyanate (11)
1,10-Thiocarbonyldiimidazole (1.01 g, 5.67 mmol) was added to
a solution of 10 (0.55 g, 5.55 mmol) in dry 1,4-dioxane (15 mL). The
mixture was refluxed under nitrogen. After 20 min, the solvent was
removed under vacuum and diethyl ether was added to the oily
residue. The organic layer was washed with 0.1 M hydrochloric
acid, and evaporated under reduced pressure. The resulting oil
(0.51 g, 79%) was used directly in the next step without further
purification.
water, and dried (0.38 g, 56%): mp: 161–162 °C; IR (KBr):
m: 3174
(N–H), 2989 (C–H aliphatic), 1197 (C@S) cmꢀ1 1H NMR (DMSO-
;
d6, 500 MHz): d: 1.27 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.80 (m, 1H,
HA of CH2), 2.20 (m, 1H, HB of CH2), 5.78 (m, 1H, CH), 6.78 (dd,
J = 4.80, 8.80 Hz, 1H, 8-H), 7.00 (m, 1H, 7-H), 7.07 (d, J = 8.05 Hz,
1H, 5-H), 7.36 (m, 1H, 50-H), 7.96 (d, J = 7.75 Hz, 1H, 40-H), 8.32
(m, 2H, 60-H, NH(CH)), 8.60 (s, 1H, 20-H), 9.68 (s, 1H, NH(C5H4N)).
Anal. (C17H18N3OSF) theoretical: C, 61.61; H, 5.47; N, 12.68; S,
9.67. Found: C, 61.41; H, 5.54; N, 11.77; S, 9.36.
5.1.20. R/S-6-Chloro-3,4-dihydro-2,2-dimethyl-4-(3-thienyl-
aminothiocarbonylamino)-2H-1-benzopyran (27)
3-Thienyl isothiocyanate (11) (0.16 g, 1.13 mmol) was added to
a solution of R/S-4-amino-6-chloro-3,4-dihydro-2,2-dimethyl-2H-
1-benzopyran (6b31) (0.2 g, 0.95 mmol) in methylene chloride
(5 mL). After 30 min, the resulting precipitate was collected by fil-
tration, washed with methylene chloride, and dried (0.24 g, 69%):
5.1.15. R/S-6-Chloro-3,4-dihydro-2,2-dimethyl-4-(3-pyridyl-
aminothiocarbonylamino)-2H-1-benzopyran (25)
The title compound was obtained as described for 24, starting
from 6b31 (0.4 g, 1.89 mmol) and 3-pyridyl isothiocyanate
mp: 196–197 °C; IR (KBr):
m
: 3340, 3160 (N–H), 3087 (C–H aro-
(253
m
l
L, 2.27 mmol) (0.50 g, 76%): mp: 144–145 °C; IR (KBr):
1H NMR
matic), 2978, 2933 (C–H aliphatic), 1199 (C@S) cmꢀ1
;
1H NMR
: 3166 (N–H), 2986 (C–H aliphatic), 1198 (C@S) cmꢀ1
;
(DMSO-d6, 500 MHz): d: 1.27 (s, 3H, CH3), 1.39 (s, 3H, CH3), 1.81
(m, 1H, HA of CH2), 2.18 (m, 1H, HB of CH2), 5.77 (m, 1H, CH),
6.78 (d, J = 8.70 Hz, 1H, 8-H), 7.11 (d, J = 4.75 Hz, 1H, 40-H), 7.18
(d, J = 8.63 Hz, 1H, 7-H), 7.24 (s, 1H, 5-H), 7.46 (m, 1H, 50-H), 7.64
(s, 1H, 20-H), 8.02 (br s, 1H, NH(CH)), 9.79 (br s, 1H, NH(C4H3S)).
Anal. (C16H17N2OS2Cl) theoretical: C, 54.45; H, 4.86; N, 7.94; S,
18.17. Found: C, 54.48; H, 5.01; N, 7.74; S, 18.43.
(DMSO-d6, 500 MHz): d: 1.28 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.81
(m, 1H, HA of CH2), 2.22 (m, 1H, HB of CH2), 5.80 (m, 1H, CH),
6.79 (d, J = 8.70 Hz, 1H, 8-H), 7.19 (d, J = 8.65 Hz, 1H, 7-H), 7.27
(s, 1H, 5-H), 7.36 (m, 1H, 50-H), 7.97 (d, J = 7.90 Hz, 1H, 40-H),
8.31–8.35 (m, 2H, 60-H, NH(CH)), 8.60 (s, 1H, 20-H), 9.70 (s, 1H,
NH(C5H4N)). Anal. (C17H18N3OSCl) theoretical: C, 58.70; H 5.22;
N, 12.08; S, 9.22. Found: C, 58.59; H, 5.17; N, 11.82; S, 9.57.
5.1.21. R/S-6-Bromo-3,4-dihydro-2,2-dimethyl-4-(3-thienyl-
aminothiocarbonylamino)-2H-1-benzopyran (28)
5.1.16. R/S-6-Bromo-3,4-dihydro-2,2-dimethyl-4-(3-pyridyl-
aminothiocarbonylamino)-2H-1-benzopyran (26)
3-Thienyl isothiocyanate (11) (0.07 g, 0.5 mmol) was added to a
solution of R/S-4-amino-6-bromo-3,4-dihydro-2,2-dimethyl-2H-1-
benzopyran (6c30) (0.1 g, 0.39 mmol) in methylene chloride (5 mL).
After 30 min, the solvent was removed under vacuum, the crude
product was solubilised with ethyl acetate and n-hexane was
added to the solution. The resulting precipitate was collected by
filtration, washed with n-hexane, and dried (0.10 g, 65%): mp:
The title compound was obtained as described for 24, starting
from 6c30 (0.4 g, 1.56 mmol) and 4-nitrophenyl isothiocyanate
(255
: 3183 (N–H), 3033 (C–H aromatic), 2975, 2923 (C–H aliphatic),
1198 (C@S) cmꢀ1 1H NMR (DMSO-d6, 500 MHz): d: 1.27 (s, 3H,
lL, 1.87 mmol) (0.44 g, 72%): mp: 146–148 °C; IR (KBr):
m
;
CH3), 1.40 (s, 3H, CH3), 1.80 (m, 1H, HA of CH2), 2.21 (m, 1H, HB
of CH2), 5.80 (m, 1H, CH), 6.74 (d, J = 8.65 Hz, 1H, 8-H), 7.32 (d,
J = 8.30 Hz, 1H, 7-H), 7.36–7.40 (m, 2H, 5-H, 50-H), 7.96 (d,
J = 7.35 Hz, 1H, 40-H), 8.32–8.40 (m, 2H, 60-H, NH(CH)), 8.60 (s,
1H, 20-H), 9.75 (s, 1H, NH(C5H4N)). Anal. (C17H18N3OSBr) theoreti-
cal: C, 52.05; H, 4.62; N, 10.71; S, 8.17. Found: C, 51.92; H, 4.61;
N, 10.46; S, 8.46.
181–182 °C; IR (KBr):
m
: 3331, 3161 (N–H), 2971, 2930 (C–H ali-
;
phatic), 1198 (C@S) cmꢀ1
1H NMR (DMSO-d6, 500 MHz): d: 1.27
(s, 3H, CH3), 1.39 (s, 3H, CH3), 1.81 (m, 1H, HA of CH2), 2.18 (m,
1H, HB of CH2), 5.79 (m, 1H, CH), 6.73 (d, J = 8.65 Hz, 1H, 8-H),
7.11 (d, J = 4.65 Hz, 1H, 40-H), 7.29 (d, J = 8.62 Hz, 1H, 7-H), 7.36
(s, 1H, 5-H), 7.46 (m, 1H, 50-H), 7.64 (s, 1H, 20-H), 7.99 (br d, 1H,

7730
X. Florence et al. / Bioorg. Med. Chem. 17 (2009) 7723–7731
NH(CH)), 9.78 (s, 1H, NH(C4H3S)). Anal. (C16H17N2OS2Br) theoreti-
cal: C, 48.36; H, 4.31; N, 7.05; S, 16.14. Found: C, 48.19; H, 4.14;
N, 7.79; S, 15.96.
aliphatic), 2161 (C„N), 1568 (C@N) cmꢀ1 1H NMR (DMSO-d6,
;
500 MHz): d: 1.26 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.80 (m, 1H,
CH2), 2.18 (m, 1H, CH2), 5.13 (m, 1H, CH), 6.71 (d, J = 8.70 Hz, 1H,
8-H), 7.30 (d, J = 8.65 Hz, 1H, 7-H), 7.40 (s, 1H, 5-H), 7.53–7.60
(m, 3H, 40-H, 50-H, 60-H), 7.70 (s, 1H, 20-H), 7.84 (d, J = 8.35 Hz,
1H, NH(CH)), 9.54 (s, 1H, NH(C6H4)). Anal. (C20H18N5OBr) theoreti-
cal: C, 56.61; H, 4.28; N, 16.51. Found: C, 56.35; H, 4.32; N, 16.12.
5.1.22. R/S-N-(6-Bromo-3,4-dihydro-2,2-dimethyl-2H-1-ben-
zopyran-4-yl)-N0-(3-cyanophenyl)-S-methylisothiourea (29)
Iodomethane (179 lL, 2.88 mmol) was added to a solution of de
R/S-6-bromo-4-(3-cyanophenylaminothiocarbonylamino)-3,4-
dihydro-2,2-dimethyl-2H-1-benzopyran (14) (0.4 g, 0.96 mmol) in
acetone (1.5 mL) and the solution was stirred overnight at rt. The
next day, R/S-N-(6-bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzo-
pyran-4-yl)-N0-(3-cyanophenyl)-S-methylisothiouronium hydroio-
dide was collected by filtration, washed with n-hexane, and dried
5.1.25. R/S-N-(6-Bromo-3,4-dihydro-2,2-dimethyl-2H-1-ben-
zopyran-4-yl)-N0-(4-chlorophenyl)-N00-cyanoguanidine (32)
The title compound was obtained as described for 31, starting
from 30 (0.4 g, 0.71 mmol), cyanamide (0.089 g, 2.1 mmol) and
1,4-diazabicyclo[2.2.2]octane (0.0112 g, 0.1 mmol) (46 mg, 15%):
(0.36 g, 67%): mp: 128–130 °C; IR (KBr):
m
: 3435 (N–H), 2975,
mp: 187–189 °C; IR (KBr):
m
: 3373 (N–H), 3059 (C–H aromatic),
2928 (C–H aliph.), 2233 (C„N), 1578 (C@N) cmꢀ1
.
Anal.
2977, 2942 (C–H aliphatic), 2186 (C„N), 1577 (C@N) cmꢀ1
;
1H
(C20H21BrN3OSI) theoretical: C, 43.03; H, 3.79; N, 7.53; S, 5.74.
Found: C, 42.95; H, 4.00; N, 7.29; S, 5.99.
After purification by column chromatography (ethyl acetate–
petroleum 1:4) on silica gel, the title compound was isolated as
the corresponding base (0.041 g, 15%): mp: 155–156 °C; IR (KBr):
NMR (DMSO-d6, 500 MHz): d: 1.25 (s, 3H, CH3), 1.37 (s, 3H, CH3),
1.82 (m, 1H, CH2), 2.14 (m, 1H, CH2), 5.13 (m, 1H, CH), 6.70 (d,
J = 8.68 Hz, 1H, 8-H), 7.26–7.31 (m, 3H, 7-H, 30-H, 50-H), 7.35 (s,
1H, 5-H), 7.40 (d, J = 8.80 Hz, 2H, 20-H, 60-H), 7.64 (d, J = 8.28 Hz,
1H, NH(CH)), 9.37 (s, 1H, NH(C6H4)). Anal. (C17H18N3OSBr) theoret-
ical: C, 52.61; H, 4.18; N, 12.92. Found: C, 52.74; H, 4.32; N, 12.81.
m
: 3375 (N–H), 2972, 2930 (C–H aliphatic), 2226 (C„N), 1586
(C@N) cmꢀ1 1H NMR (DMSO-d6, 500 MHz): d: 1.25 (s, 3H, CH3),
;
1.41 (s, 3H, CH3), 1.89 (m, 1H, HA of CH2), 2.20 (m, 1H, HB of
CH2), 2.38 (s, 3H, S(CH3)), 5.32 (m, 1H, CH), 6.73 (d, J = 8.65 Hz,
1H, 8-H), 7.13–7.20 (m, 3H, 20-H, 60-H, NH(CH)), 7.30 (d,
J = 8.63 Hz, 1H, 7-H), 7.39–7.46 (m, 2H, 40-H, 50-H), 7.51 (s, 1H, 5-
H). Anal. (C20H20N3OSBr) theoretical: C, 55.82; H, 4.68; N, 9.76; S,
7.45. Found: C, 55.88; H, 4.37; N, 9.69; S, 7.77.
5.2. Biological assays
( )-Cromakalim (Tocris, UK), diazoxide (Sigma Chemical, USA)
and ( )-pinacidil (Laboratory of Medicinal Chemistry, ULg, Bel-
gium) were tested as reference compounds.
5.3. Measurement of insulin release from incubated rat
pancreatic islets
5.1.23. R/S-N-(6-Bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzo-
pyran-4-yl)-N0-(4-chlorophenyl)-S-methylisothiourea (30)
The title compound, as the hydroiodide, salt was obtained as de-
scribed for 29, starting from 5 (0.4 g, 0.94 mmol) and iodomethane
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
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 radioimmunologi-
cally using rat insulin as a standard.28 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.
(175
base.
lL, 2.82 mmol) and further converted to the corresponding
R/S-N-(6-bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-
4-yl)-N0-(3-cyanophenyl)-S-methylisothiouronium hydroiodide:
Yield: 0.44 g, 83%; IR (KBr):
1584 (C@N) cmꢀ1
m: 3396 (N–H), 2976, 2928 (C–H aliph.),
.
R/S-N-(6-Bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-
4-yl)-N0-(3-cyanophenyl)-S-methylisothiourea: Yield: 0.096 g,
28%; mp: 113 °C; IR (KBr):
phatic), 1584 (C@N) cmꢀ1
m
: 3396 (N–H), 2976, 2928 (C–H ali-
;
1H NMR (DMSO-d6, 500 MHz): d: 1.24
(s, 3H, CH3), 1.40 (s, 3H, CH3), 1.89 (m, 1H, HA of CH2), 2.18 (m,
1H, HB of CH2), 2.35 (s, 3H, S(CH3)), 5.31 (m, 1H, CH), 6.72 (d,
J = 8.70 Hz, 1H, 8-H), 6.79 (d, J = 8.50 Hz, 2H, 30-H, 50-H), 6.97 (d,
J = 8.30 Hz, 1H, NH(CH)), 7.26–7.30 (m, 3H, 7-H, 20-H, 60-H), 7.50
(d, 1H, 5-H). Anal. (C19H20N2OSBrCl) theoretical: C, 51.89; H,
4.58; N, 6.37; S, 7.29. Found: C, 51.75; H, 4.62; N, 6.53; S, 7.45.
5.4. Measurement of tension in rat aorta rings
Experiments were performed with aortas removed from adult
fed Wistar rats (Charles River Laboratories, Belgium).
5.1.24. R/S-N-(6-Bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzo-
pyran-4-yl)-N0-(3-cyanophenyl)-N00-cyanoguanidine (31)
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-
Cyanamide (0.06 g, 1.4 mmol) and 1,4-diazabicyclo[2.2.2]oc-
tane (0.0071 g, 0.063 mmol) were added to a solution of R/S-N-
(6-bromo-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)-N0-
(3-cyanophenyl)-S-methylisothiouronium hydriodide (0.25 g,
0.45 mmol) in butanol (1.7 mL) and the solution was refluxed for
5 h. The solvent was removed under vacuum. Water was added
to the residue and the product was extracted with methylene chlo-
ride. The organic layer was evaporated under reduced pressure.
The crude product was solubilised in methanol and 3 volumes of
water were added. The resulting precipitate was collected by filtra-
tion, washed with water and dried. The final product was purified
on silica gel eluted with ethyl acetate/petroleum ether (1:1 v/v) to
give the title compound (28 mg, 15%): mp: 218–219 °C; IR (KBr):
ing concentrations until maximal relaxation (or until 300 lM). The
relaxation response was expressed as the percentage of the
m: 3352, 3240 (N–H), 3153 (C–H aromatic), 2978, 2934 (C–H
contractile response to KCl. The EC50 values (concentration evoking

X. Florence et al. / Bioorg. Med. Chem. 17 (2009) 7723–7731
7731
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50% inhibition of the plateau phase induced by KCl) were assessed
from dose–response curves using Datanalyst software (EMKA
Technologies, France).39
5.5. Measurements of 86Rb outflow from perifused rat
pancreatic islets
8. Standen, N. B.; Quayle, J. M.; Davies, N. W.; Brayden, J. E.; Huang, Y.; Nelson, M.
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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.28,29,38 Groups of 100 islets were incubated for 60 min at
37 °C in a bicarbonate-buffered medium (in mM: NaCl 115, KCl 5,
CaCl2 2.56, MgCl2 1, NaHCO3 24) supplemented with 0.5% (w/v)
dialyzed albumin (fraction V, Sigma) and containing 16.7 mM glu-
cose and 86Rb (0.15–0.25 mmol/L:50
lCi/mL). The incubation med-
ium was gassed with O2 (95%) and CO2 (5%). After incubation, the
islets were washed three times with a nonradioactive medium
and then placed in a perifusion chamber. The perifusate [bicarbon-
ate-buffered medium supplemented with 0.5% (w/v) dialyzed albu-
min (fraction V, Sigma) and gassed with O2 (95%)/CO2 (5%)] was
delivered at a constant 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 counting. At the end of the perifusion, the radioac-
tive content of the islets was also determined. The outflow of 86Rb
(cpm/min) was expressed as a fractional outflow rate (FOR, % of
instantaneous islet content per min).28,29,38
23. Bjork, E.; Berne, C.; Kampe, O.; Wibell, L.; Oskarsson, P.; Karlsson, F. A. Diabetes
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24. Rasmussen, S. B.; Sorensen, T. S.; Hansen, J. B.; Mandrup-Poulsen, T.; Hornum,
L.; Markholst, H. Horm. Metab. Res. 2000, 32, 294.
25. Sebille, S.; de Tullio, P.; Florence, X.; Becker, B.; Antoine, M. H.; Michaux, C.;
Wouters, J.; Pirotte, B.; Lebrun, P. Bioorg. Med. Chem. 2008, 16, 5704.
26. Zdravkovic, M.; Kruse, M.; Rost, K. L.; Moss, J.; Kecskes, A. Exp. Clin. Endocrinol.
Diabetes 2007, 115, 405.
27. Hamilton, T. C.; Weir, S. W.; Weston, A. H. Br. J. Pharmacol. 1986, 88, 103.
28. Lebrun, P.; Antoine, M. H.; Devreux, V.; Hermann, M.; Herchuelz, A. J.
Pharmacol. Exp. Ther. 1990, 255, 948.
29. Lebrun, P.; Arkhammar, P.; Antoine, M. H.; Nguyen, Q. A.; Hansen, J. B.; Pirotte,
B. Diabetologia 2000, 43, 723.
30. Khelili, S.; de Tullio, P.; Lebrun, P.; Fillet, M.; Antoine, M. H.; Ouedraogo, R.;
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Med. Chem. 1999, 7, 1513.
5.6. Statistical calculation
The statistical significance of difference between mean data was
assessed by using the Student0s t-test or by an analysis of variance
followed for multiple comparisons by a Bonferroni test procedure.
The biological results were considered as statistically different
when p was <0.05.
Acknowledgements
31. Sebille, S.; de Tullio, P.; Becker, B.; Antoine, M. H.; Boverie, S.; Pirotte, B.;
Lebrun, P. J. Med. Chem. 2005, 48, 614.
32. de Tullio, P.; Becker, B.; Boverie, S.; Dabrowski, M.; Wahl, P.; Antoine, M. H.;
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Chem. 2003, 46, 3342.
33. Sebille, S.; Gall, D.; de Tullio, P.; Florence, X.; Lebrun, P.; Pirotte, B. J. Med. Chem.
2006, 49, 4690.
34. Ashcroft, F. M.; Rorsman, P. Prog. Biophys. Mol. Biol. 1989, 54, 87.
35. Malaisse, W. J.; Lebrun, P. Diabetes Care 1990, 13, 9.
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37. Lebrun, P.; Antoine, M. H.; Ouedraogo, R.; Kane, C.; Dunne, M.; Hermann, M.;
Herchuelz, A.; Masereel, B.; Delarge, J.; de Tullio, P.; Pirotte, B. J. Pharmacol. Exp.
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This study was supported by grants from the Formation to Re-
search 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.
References and notes
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