4-Phenylureido/thioureido-substituted 2,2-dimethylchroman analogs of cromakalim bearing a bulky 'carbamate' moiety at the 6-position as potent inhibitors of glucose-sensitive insulin secretion

Article abstract of DOI:10.1016/j.ejmech.2016.05.042

The synthesis of 2,2-dimethylchromans bearing a 3/4-chloro/cyano-substituted phenylureido or phenylthioureido moiety at the 4-position and an alkoxycarbonylamino ('carbamate') group at the 6-position is described. These new analogs of the potassium channe

Full text of DOI:10.1016/j.ejmech.2016.05.042

Accepted Manuscript
4-Phenylureido/thioureido-substituted 2,2-dimethylchroman analogs of cromakalim
bearing a bulky ‘carbamate’ moiety at the 6-position as potent inhibitors of glucose-
sensitive insulin secretion
Bernard Pirotte, Xavier Florence, Eric Goffin, Marlen Borges Medeiros, Pascal de
Tullio, Philippe Lebrun
PII:
S0223-5234(16)30440-8
10.1016/j.ejmech.2016.05.042
EJMECH 8634
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 June 2015
Revised Date: 19 May 2016
Accepted Date: 20 May 2016
Please cite this article as: B. Pirotte, X. Florence, E. Goffin, M.B. Medeiros, P. de Tullio, P. Lebrun,
4-Phenylureido/thioureido-substituted 2,2-dimethylchroman analogs of cromakalim bearing a bulky
‘carbamate’ moiety at the 6-position as potent inhibitors of glucose-sensitive insulin secretion, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.042.
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ACCEPTED MANUSCRIPT
4-Phenylureido/thioureido-substituted 2,2-dimethylchroman analogs of cromakalim
bearing a bulky ‘carbamate’ moiety at the 6-position as potent inhibitors of
glucose-sensitive insulin secretion
Bernard Pirotte, Xavier Florence, Eric Goffin, Marlen Borges Medeiros,
Pascal de Tullio, Philippe Lebrun
Graphical abstract

ACCEPTED MANUSCRIPT
4-Phenylureido/thioureido-substituted 2,2-dimethylchroman analogs of cromakalim
bearing a bulky ‘carbamate’ moiety at the 6-position as potent inhibitors of glucose-
sensitive insulin secretion
Bernard Pirotte1*, Xavier Florence1,2, Eric Goffin1, Marlen Borges Medeiros1, Pascal de
Tullio1, Philippe Lebrun2
1 Laboratoire de Chimie Pharmaceutique, Center for Interdisciplinary Research on Medicines
(CIRM), University of Liege, Quartier Hôpital, Avenue Hippocrate 15, B-4000 Liège,
Belgium
2
Laboratoire de Physiologie et Pharmacologie, Université Libre de Bruxelles, Faculté de
Médecine, 808 Route de Lennik, B-1070 Bruxelles, Belgium
* Corresponding author
Keywords
2,2-Dimethylchromans; ATP-sensitive potassium channels; potassium channel openers;
cromakalim analogs; insulin secretion; smooth muscle contractile activity
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Abstract
The synthesis of 2,2-dimethylchromans bearing a 3/4-chloro/cyano-substituted phenylureido
or phenylthioureido moiety at the 4-position and an alkoxycarbonylamino (‘carbamate’)
group at the 6-position is described. These new analogs of the potassium channel opener (±)-
cromakalim were further tested on rat pancreatic islets as putative inhibitors of insulin release
and on rat aorta rings as putative vasorelaxants. All compounds inhibited insulin secretion and
induced
a
myorelaxant activity. Compound 14o [R/S-N-3-cyanophenyl-N'-(6-tert-
butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea; BPDZ 711]
emerged as the most potent inhibitor of the glucose-sensitive insulin releasing process (IC50 =
0.24 µM) and displayed selectivity towards the pancreatic endocrine tissue. Radioisotopic,
fluorimetric and pharmacological investigations were performed on rat pancreatic islet and rat
vascular smooth muscle cells in order to decipher its mechanism of action. Our findings
suggest that the mechanism of action of 14o is rather unspecific. The compound behaves as a
KATP channel opener, a Ca2+ entry blocker, and promotes an intracellular calcium
translocation.
Manuscript text
1. Introduction
ATP-sensitive potassium channels (KATP channels) located at the plasma membrane of a
variety of excitable cells are potassium ion channels that link cell metabolism to membrane
excitability [1]. An increase in the intracellular concentration ratio of adenosine triphosphate
to adenosine diphosphate (ATP/ADP ratio) provokes an inhibition of the channel activity with
subsequent membrane depolarization while a decrease of this ratio exerts opposite effects and
induces cell membrane hyperpolarization [1-3].
KATP channels are widely distributed throughout tissues where they are involved in multiple
physiological processes, including the control of insulin release from pancreatic β-cells and
the modulation of vascular smooth muscle tone [4-6]. The structure of those channels at the
molecular level has been elucidated as being octameric complexes of four pore-forming
inwardly rectifying K+ (Kir6.x) channels (Kir6.1 or Kir6.2) and four regulatory sulfonylurea
receptor (SURx) subunits (SUR1, SUR2A or SUR2B) [7]. Different combinations of the Kir
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and SUR subunits lead to tissue-specific KATP channels; the pancreatic β-cell KATP channel is
known to express a SUR1/Kir6.2 KATP channel subtype while the SUR2B/Kir6.1 combination
is found in vascular smooth muscle cells [8,9].
KATP channels are recognized as important therapeutic targets and several modulators
(openers and/or blockers) are currently used in clinical practice (i.e. hypoglycemic
sulfonylureas as oral antidiabetic drugs are pancreatic β-cell KATP channel blockers) [10].
New modulators of these channels are expected to become promising therapeutic agents
provided that they are able to exert high potency and selectivity for a single KATP channel
subtype. Selective openers of the SUR1/Kir6.2 channel subtype have been proposed for the
prevention and/or management of type 1 - type 2 diabetes, congenital hyperinsulinism and
insulinoma [11-13]. Diazoxide (1) was the first reported reference potassium channel opener
(PCO) displaying an activity on the pancreatic (SUR1-type) KATP channels [14,15] with
subsequent inhibition of insulin secretion, while 2,2-dimethylchroman PCOs such as (±)-
cromakalim (2) were shown to be much more effective on the smooth muscle (SUR2B-type)
KATP channels (inducing a myorelaxant effect) [16]. As a result, diazoxide is currently the
therapeutic agent used in medical practice to treat hypoglycemia due to excessive insulin
release [17,18].
Recent researches, however, led to the identification of original highly selective pancreatic β-
cell (SUR1)-type KATP channel openers, among which ring-fused 3-alkylamino-substituted
4H-1,2,4-thiadiazine 1,1-dioxides (benzo/pyrido/thienothiadiazine dioxides i.e. 3, 4 and 5; fig.
1) structurally related to diazoxide [19-21]. More recently, the identification of a xanthine
derivative, VU0071063 (6), as a selective SUR1/Kir6.2-type channel opener was also reported
[22]. By contrast, more restricted investigations have been focused on 2,2-dimethylchromans,
with marked pancreatic β-cell selectivity, structurally related to (±)-cromakalim. In this
specific field, we reported that, among 2,2-dimethylchroman structures studied as PCOs, 4-
phenylureido- and 4-phenylthioureido-substituted 2,2-dimethylchromans diversely substituted
at the 6-position exerted a potent inhibitory activity on insulin-secreting cells; an observation
attributed to the activation of the pancreatic KATP channels [23-26]. For a few drugs, such as
compounds 7 and 8 (fig. 1), a significant selectivity for the pancreatic tissue versus the
vascular smooth muscle tissue was observed [23,25]. Furthermore, a surprising marked
activity was disclosed with a series of 4-phenylthioureido-substituted 2,2-dimethylchromans
bearing a bulky tert-butoxycarbonylamino group at the 6-position of the chroman ring (i.e.
compound 9 with an IC50 value on pancreatic β-cells close to 1 µM; fig. 1) [26].
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According to these observations, we decided to further explore the pharmacological interest
of 2,2-dimethylchromans bearing, at the 4-position, the previously selected monosubstituted
(3-/4-Cl, 3-/4-CN) phenylureido/thioureido group and, at the 6-position, an
alkoxycarbonylamino group (‘carbamate’ moiety) with a variety of small-sized branched or
not branched alkyl groups (see formulae 14 and 15, scheme 1).
The new compounds were tested as putative inhibitors of insulin release and as putative
vasorelaxants. The most active drug was further investigated in radioisotopic, fluorimetric and
pharmacological experiments in order to elucidate its mechanism of action.
2. Results and discussion
2.1. Chemistry
The synthesis of the new compounds 14 and 15 is described on scheme 1. The starting
intermediate 6-amino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-one (10) was obtained as
previously described [26]. The reaction of 10 with the appropriate alkyl chloroformate (except
when R was tert-butyl, di-tert-butyl dicarbonate was used [26]) gave rise to the ‘carbamate’
intermediates 11a-d (6-alkoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-
ones). Hydroxylamine hydrochloride in the presence of potassium carbonate converted the
ketonic compounds 11a-d into the corresponding oximes 12a-d, which were further treated
with hydrogen gas in the presence of Raney-Nickel° to provide the corresponding amines
13a-d. In the last step, the reaction of 13a-d with the appropriate monosubstituted phenyl
isocyanate or isothiocyanate generated the final compounds 14a-p (N,N’-disubstituted ureas)
and 15a-l (N,N’-disubstituted thioureas).
2.2. Biological results
2.2.1. Insulin secretion from rat pancreatic islets
The newly synthesized compounds (compounds 14a-p and 15a-l; Table 1) were evaluated as
inhibitors of the insulin releasing process on isolated rat pancreatic islets incubated in the
presence of an insulinotropic glucose concentration (16.7 mM). An inhibition of the glucose-
induced insulin release might reflect the opening of pancreatic β-cells KATP channels. A
residual 5-10% insulin secretion can be considered as reflecting a close to maximal inhibitory
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effect according to the glucose-insensitive basal endocrine secretion [27]. (±)-Cromakalim
and diazoxide were used as reference PCOs (Table 1). The activity of the new 2,2-
dimethylchromans 14a-p and 15a-l was also compared to that of the previously described
compounds 9, 16, 17 and 18 (Table 1) [26].
As previously reported, a 10 µM concentration of (±)-cromakalim was roughly inactive on the
pancreatic endocrine tissue (Table 1) [25,26]. Diazoxide at the same concentration (10 µM),
however, significantly reduced the insulin secretory rate by about 20% (Table 1) [25,26].
The new 4-phenylureido/thioureido-substituted 2,2-dimethylchromans 14 and 15 exhibited a
strong inhibitory activity on the insulin releasing process (Table 1). Among these 2,2-
dimethylchromans, the most potent compound was found to be 14o (BPDZ 711) (Table 1).
The following structure-activity relationships can be deduced from the secretory data.
Concerning the substituent on the benzene ring of 4-phenylureido-substituted compounds
14a-p, the lowest inhibitory activity was systematically observed with the 4-chloro-
substituted derivatives (see 14b, 14f, 14j and 14n at 1 and 10 µM; Table 1). By contrast, the
best choice of substituent appeared to be the cyano group at the 3-position (see 14c, 14g, 14k
and 14o at 1 and 10 µM; Table 1), with compound 14o clearly emerging as the most potent
inhibitor of insulin release.
The corresponding thioureas 9, 15a-l, 16-18 also markedly inhibited the glucose-induced
insulin release, although less obvious differences were detected according to the nature and
the position of the substituent on the phenyl ring (Table 1).
Moreover, by comparing ureas and thioureas bearing similar substituents (R, R3 or R4), it was
noticed that, in most cases, the thioureas 15 were more potent than their corresponding ureas
14, except when R3 corresponded to the cyano group (compare 14c, 14g, 14k and 14o with
15c, 15g, 15k and 18; Table 1).
Two structural considerations might explain differences in the biological activity observed
between ureas and thioureas. Firstly, the carbon-sulfur interatomic distance is known to be
greater than that of carbon-oxygen [for example, the C=S bond length in N,N’-
diphenylthiourea (1.681 Å) [28] is greater than the C=O bond length in N,N’-diphenylurea
(1.233 Å) [29]], presumably causing differences in the establishment of C=S---H or C=O---H
hydrogen bonds at the level of the receptor binding site. Secondly, ureas and thioureas, in the
solid state and/or in solution, are expected to adopt multiple conformations ensuring optimal
electron delocalization in the N-C(=X)-N plane system (see Fig. 2). According to X-ray data
previously published for N,N’-disubstituted ureas and N,N’-disubstituted thioureas, the A
conformation seems to predominate for ureas while thioureas are known to exist in the A as
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well as in the B/C conformations (i.e. N,N’-diphenylurea was found in the A conformation
[29,30], while N,N’-diphenylthiourea was found in conformations A [28] and B [31] in the
solid state). The D conformation is the less probable due to steric hindrance resulting from the
proximity of the two hydrocarbon chains R and R’. It is tempting to speculate that the active
conformation assuming optimal interaction with the receptor binding site might be the A
conformation.
An additional complication results from the possible presence, in solution, of multiple
tautomeric forms corresponding to the equilibrium between ol/one or thiol/thione forms (see
conformations A, A’ and A”, Fig. 2). In the solid state, for N,N’-diarylureas/thioureas, the A
conformation seems to predominate [28-31]. Moreover, according to the NMR data obtained
with the described ureas and thioureas (see Experimental section and “Supplementary data”
section), the nitrogen atom linked to the carbon atom at the 4-position (4-C) of the chroman
ring undoubtedly bears a hydrogen atom since this proton appears as a doublet due to its
coupling with the hydrogen atom attached to the neighbor 4-C carbon atom, thus precluding
the A” conformation (see Fig. 2).
The nature of the alkyl chain linked to the ‘carbamate’ moiety also appeared to affect the
biological activity on pancreatic islets. The rank order of potency, both for the 4-
phenylureido- and the 4-phenylthioureido-substituted 2,2-dimethylchromans, could be
roughly summarized as follows: tert-butyl > isopropyl ≥ ethyl ≥ methyl. Thus, there is a
favorable impact, on the biological activity, of the increase of the steric hindrance at the 6-
position.
2.2.2. Myorelaxant activity on rat aortic rings
The vasorelaxant activity of the new 2,2-dimethylchromans 14a-p and 15a-l was determined
on 30 mM K+-depolarized rat aorta (endothelium-free) rings and was compared to that of (±)-
cromakalim, diazoxide and the previously described compounds 9, 16, 17, 18 (Table 1) [26].
As reported, (±)-cromakalim displayed a marked myorelaxant activity on the vascular tissue
while diazoxide was less potent (Table 1). Such a vasorelaxant effect of the two reference
compounds is known to result from the primary opening of the vascular smooth muscle cells
KATP channels [32,33].
The vasorelaxant activity of compounds 14 and 15 was found to be weaker than that of (±)-
cromakalim, although most new drugs were equipotent or more potent than diazoxide (Table
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1). The best choice of substituent on the phenyl ring appeared to be the cyano group at the 3-
position, as observed for the inhibitory effect on insulin release.
Taken as a whole, these biological data indicated that the new 2,2-dimethylchromans exerted
a more pronounced activity on the pancreatic endocrine tissue than on the vascular smooth
muscle tissue.
Surprisingly, compound 14o (BPDZ 711), the most potent inhibitor of insulin secretion (IC50
= 0.24 µM, see below), did not come out as the most potent vasodilator. Considering an
EC50/IC50 ratio equal to 44, a clear selectivity of 14o for the pancreatic versus the vascular
tissue can be deduced.
This compound was selected for further characterization in order to elucidate its mechanism
of action.
2.2.3. Effects of 14o (BPDZ 711) on insulin release from incubated rat pancreatic islets
The addition of increasing concentrations of 14o provoked a concentration-dependent
decrease in insulin output from rat pancreatic islets incubated in the presence of 16.7 mM
glucose (Fig. 3). After the addition of 0.1 µM, 0.5 µM, 1 µM and 10 µM 14o to the
incubation medium, the residual insulin release averaged 82.0 ± 4.6 % (n = 30); 28.6 ± 1.8 %
(n = 21), 15.6 ± 1.2 % (n = 20) and 7.4 ± 1.0 % (n = 25) of the control value, respectively.
The IC50 value (drug concentration evoking a 50 % reduction of the secretory response to
glucose) amounted to 0.24 µM.
In contrast, at the same concentrations, (±)-cromakalim did not affect insulin release from
glucose-stimulated pancreatic islets. In the presence of 1 µM and 10 µM (±)-cromakalim in
the incubation medium, the insulin output averaged 93.8 ± 4.1 % (n = 16) and 96.4 ± 6.5 % (n
= 16) of the control experiments, respectively.
2.2.4. Effects of 14o (BPDZ 711) on 86Rb, 45Ca outflow and insulin release from perifused rat
pancreatic islets
Figure 4 clearly reveals that 10 µM 14o provoked a rapid, marked and sustained increase in
the rate of 86Rb (42K substitute) outflow from prelabeled and perifused rat pancreatic islets.
Such a massive enhancing effect on 86Rb efflux has never been observed with other reference
KATP channel openers.
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The addition of 1 µM 14o to prelabeled pancreatic islets also induced a sustained, although
less pronounced, rise in 86Rb outflow (data not shown). Under the same experimental
conditions, 0.1 µM 14o did not affect the rate of 86Rb outflow (data not shown).
When the basal medium contained 10 µM of the hypoglycemic sulfonylurea glibenclamide, a
recognized KATP channel blocker [34], the stimulatory effect of 10 µM 14o was abolished
(Fig. 4).
These radioisotopic data indirectly suggest that 14o increases the membrane K+ permeability
and activates KATP channels in islets cells. Such an activation of KATP channels should
hyperpolarize the insulin-secreting cells, reduce Ca2+ inflow through voltage-sensitive Ca2+
channels, decrease the cytosolic calcium concentration and, consequently, inhibit insulin
secretion.
To corroborate this hypothesis, additional experiments have been conducted. Figure 5 (upper
panel) shows that, in islets exposed throughout to an insulinotropic glucose concentration
(16.7 mM) and extracellular Ca2+, the addition of 10 µM 14o elicited an immediate and
pronounced inhibition of 45Ca outflow from prelabeled and perifused rat pancreatic islets.
Under such experimental conditions, a decrease in the 45Ca fractional outflow rate is known to
reflect a reduction in 40Ca2+ entry into the islets cells [27,35].
Moreover, the simultaneous measurement of the glucose-sensitive secretory process further
indicated that 10 µM 14o provoked an inhibition of the insulin secretory rate displaying a
time course identical to that of the 45Ca response (Fig. 5, lower panel).
To further characterize the effects of 14o on 45Ca movements, similar experiments were
conducted in the continuous presence of 16.7 mM glucose but absence of extracellular Ca2+ in
the perifusing medium. In islets exposed to Ca2+-depleted media, the basal rate of 45Ca
outflow (min 40-44) was lower but the addition of 10 µM 14o induced a clear-cut increase in
45Ca efflux (Fig. 5, upper panel).
14o (10 µM) also provoked a sustained increase in 45Ca outflow from islets perifused in the
presence of a non-insulinotropic glucose concentration (2.8 mM), whether the physiological
medium contained or was deprived of extracellular Ca2+ (data not shown). Under such
experimental conditions, Ca2+ entry is impaired and the isotopic exchange between influent
40Ca and effluent 45Ca inhibited.
Thus, the 14o-induced increases in 45Ca outflow from islets perifused either in the absence of
extracellular Ca2+ or in the presence of extracellular Ca2+ but at a non-stimulatory glucose
concentration might result from an intracellular calcium redistribution with subsequent
changes in 45Ca outflow [36,37].
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In the last series of radioisotopic experiments, we determined the effects of 14o on the 45Ca
response elicited by a rise in the extracellular K+ concentration from islets perifused in the
presence of a non-insulinotropic glucose concentration (2.8 mM). A sudden increase in the
extracellular concentration of K+ from 5 to 50 mM provoked a rapid, sustained and reversible
enhancement in 45Ca outflow (Fig. 6).
When the same experiment was repeated in the continuous presence of 14o (10 µM) in the
perifusate, the basal rate of 45Ca outflow was higher. Such an observation, detected in islets
exposed throughout to 2.8 mM glucose, corroborates previous findings suggesting that the
drug might interfere with an intracellular target site.
Moreover, the continuous presence of 14o (10 µM) in the perifusing medium counteracted the
45Ca response evoked by 50 mM extracellular K+.
These experimental data indirectly suggest that 14o also inhibits Ca2+ entry by acting at the
level of the voltage-sensitive Ca2+ channels. Indeed, the 45Ca response to high K+
concentrations, which is mediated by the opening of voltage-sensitive Ca2+ channels, is
known to be sensitive to Ca2+ entry blockers but rather resistant to “pure” K+ channel openers
[27,35,38].
2.2.5. Effects of 14o (BPDZ 711) on the fura-2 fluorescence from single rat pancreatic islet
cells
A rise in the extracellular glucose concentration from 2.8 to 20 mM provoked, as expected, an
initial decrease rapidly followed by a biphasic and sustained rise in cytosolic Ca2+
concentration (fluorescence ratio 340:380 nm) (Fig. 7, upper panel) [27]. The addition of 14o
(10 µM) during the sustained phase induced a slow but continuous reduction in the cytosolic
Ca2+ concentration (Fig. 7, upper panel).
When the same experiment was performed with a physiological medium deprived of external
calcium, an experimental condition into which Ca2+ influx was prevented, 20 mM glucose
induced a slight reduction in cytosolic calcium with no secondary rise. Under such
experimental conditions, the subsequent addition of 14o (10 µM) provoked a slow but
sustained increase in the intracellular Ca2+ concentration (Fig. 7, lower panel).
Additional experiments indicated that, whether the physiological medium contained or was
deprived of extracellular calcium, the addition of 10 µM 14o increased the fluorescence
intensity of fura 2-loaded islets cells exposed to a non-insulinotropic glucose concentration
(2.8 mM). (data not shown).
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These calcium fluorimetry experiments indicate that, in islets cells stimulated by an
insulinotropic glucose concentration (20 mM), a reduction in the cytosolic Ca2+ concentration
as mediated by 14o evidences a decrease in Ca2+ entry.
Moreover, the fluorimetric data further support the hypothesis that 14o could promote an
intracellular calcium redistribution with subsequent changes in the cytosolic calcium
concentration.
2.2.6. Effects of 14o (BPDZ 711) on the contractile activity of rat aortic rings
In rat aortic rings, a sudden rise in the extracellular K+ concentration provoked an increase in
muscle tension, which rapidly reached a sustained plateau phase. The cumulative application
of 14o (0.1 µM to 200 µM) to aortic rings continuously exposed to 30 mM K+ induced
concentration-dependent relaxations.
The vasorelaxant potency of 14o, as calculated from the EC50 value (drug concentration
giving 50% relaxation), was roughly 50 fold lower than that of the reference compound
cromakalim (Table 2).
The concomitant presence of glibenclamide (1 µM or 10 µM) in the bathing solution failed to
affect the myorelaxant properties of 14o (Table 2). By contrast, the KATP channel blocker
glibenclamide induced a dose-dependent reduction in the vasorelaxant response to
cromakalim (Table 2).
Incubation of aortic rings with glibenclamide (1 µM or 10 µM) did not impair the
myorelaxant capacity of the Ca2+ entry blocker verapamil (Table 2).
The myorelaxant activity of 14o was further characterized on rat aortic rings precontracted by
80 mM extracellular K+. Table 2 indicates that, under the latter experimental condition, the
vasorelaxant potency of 14o was maintained. Under identical experimental conditions, the
myorelaxant effects of the KATP channel opener cromakalim were drastically blunted whilst
those of the Ca2+ entry blocker verapamil were unaffected (Table 2).
These findings indicate that, on vascular smooth muscle cells, 14o mainly behaved as a Ca2+
entry blocker rather than as a K+ channel opener.
3. Conclusions
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We reported here the synthesis of original 2,2-dimethylchromans bearing a phenylureido or a
phenylthioureido moiety at the 4-position and an alkoxycarbonylamino group at the 6-
position. The new compounds are structurally related to the reference potassium channel
opener (±)-cromakalim. Most compounds exerted a marked inhibitory activity on the insulin
releasing process and were found to be much more active than (±)-cromakalim and diazoxide.
The thioureas were commonly more potent than their corresponding ureas, except when
bearing a cyano group at the 3-position. Compared with (±)-cromakalim, the new 4-
phenylureido/thioureido-substituted 2,2-dimethylchromans displayed a weaker vasorelaxant
activity.
R/S-N-3-Cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-
2H-1-benzopyran-4-yl)urea (14o) was the most active compound on pancreatic islets and also
exhibited a pancreatic tissue selectivity. This drug was selected to be further investigated on
rat pancreatic islet cells and rat vascular smooth muscle cells in order to characterize its
mechanism of action. Altogether, our radiosiotopic, fluorimetric and pharmacological
observations suggest that the mechanism of action of 14o is rather “unspecific”. The
compound activates KATP channels, expresses Ca2+ entry blocker properties and promotes a
calcium translocation by acting at the level of intracellular organelles.
4. Experimental section
4.1. Chemistry
All commercial chemicals (Sigma-Aldrich, Belgium; Appolo Scientific, United Kingdom and
Fluorochem, United Kingdom) and solvents were reagent grade and used without further
purification. Melting points were determined on a Stuart SMP3 apparatus in open capillary
tubes and are uncorrected. NMR spectra were recorded on a Bruker Avance 500 spectrometer
(1H: 500 MHz; 13C: 125 MHz) using DMSO-d6 as solvent and tetramethylsilane (TMS) as
internal standard; chemical shifts are reported in δ values (ppm) relative to internal TMS. The
abbreviation s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet and bs = broad
signal are used throughout. Elemental analyses (C, H, N, S) were carried out on a Thermo
Flash EA 1112 series elemental analyzer and were within ± 0.4% of the theoretical values. This
analytical process ensured, for each target compound, a purity equal or greater than 95 %. All
reactions were followed by TLC (silica gel 60F254 Merck) and visualization was accomplished
with UV light (254 or 366 nm).
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The synthesis of compounds 10, 11d, 12d, 13d and 15a-d has been previously described [26].
4.1.1. General synthetic pathway to 6-alkoxycarbonylamino-3,4-dihydro-2,2-dimethyl-
2H-1-benzopyran-4-ones (11a-c)
The solution of 6-amino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-one (10) [26] (7 g, 36.6
mmol) in acetonitrile (100 mL) was supplemented with potassium carbonate (30.36 g, 219.6
mmol, 6 equiv.), then with the appropriate alkyl chloroformate (51.2 mmol, 1.4 equiv.), and
stirred for 30 min. at room temperature. The reaction medium was treated with water (200 mL)
to solubilize potassium carbonate. The resulting suspension was extracted three times with
ethyl acetate (3 x 200 mL). The organic layers were collected, dried over magnesium sulfate,
and filtered. The filtrate was concentrated to dryness under reduced pressure and the solid
residue was dissolved in the minimum volume of methanol. The addition of two volumes of
water under stirring provoked the precipitation of the title compound, which was collected by
filtration, washed with water and dried. The crude compound was used in the next step without
further purification (yields: 85-95%).
4.1.2. 3,4-Dihydro-2,2-dimethyl-6-methoxycarbonylamino-2H-1-benzopyran-4-one (11a):
IR (KBr) : υ : 3343 (N-H), 1724, 1682 (C=O) cm-1; RMN 1H (DMSO-d6, 500 MHz) : 1.37 (s,
6H, CH3), 2.76 (s, 2H, CH2), 3.66 (s, 3H, CH3), 6.93 (d, 1H, 8-H), 7.58 (dd, 1H, 7-H), 7.84 (s,
1H, 5-H), 9.57 (s, 1H, -NH).
4.1.3. 3,4-Dihydro-2,2-dimethyl-6-ethoxycarbonylamino-2H-1-benzopyran-4-one (11b):
1
IR (KBr) : υ : 3343 (N-H), 1721, 1671 (C=O) cm-1; RMN H (DMSO-d6, 500 MHz) : 1.24 (t,
3H, CH3-CH2), 1.37 (s, 6H, CH3), 2.76 (s, 2H, CH2), 4.11 (q, 2H, CH3-CH2), 6.93 (d, 1H, 8-H),
7.57 (dd, 1H, 7-H), 7.86 (s, 1H, 5-H), 9.60 (s, 1H, -NH).
4.1.4.
3,4-Dihydro-2,2-dimethyl-6-isopropoxycarbonylamino-2H-1-benzopyran-4-one
(11c) : IR (KBr) : υ : 3322 (N-H), 1726, 1682 (C=O) cm-1; RMN 1H (DMSO-d6, 500 MHz) :
1.24 (d, 6H, (CH3)2-CH), 1.37 (s, 6H, CH3), 2.76 (s, 2H, CH2), 4.88 (m, 1H, (CH3)2-CH), 6.92
(d, 1H, 8-H), 7.56 (dd, 1H, 7-H), 7.88 (s, 1H, 5-H), 9.54 (s, 1H, -NH).
12

ACCEPTED MANUSCRIPT
4.1.5. General synthetic pathway to 6-alkoxycarbonylamino-3,4-dihydro-2,2-dimethyl-
2H-1-benzopyran-4-hydroxyimines (12a-c)
The appropriate 6-alkoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-one
(11a-d) (21.44 mmol) was solubilized in ethanol (65 mL) and supplemented with potassium
carbonate (42.88 mmol, 2 equiv.) and hydroxylamine hydrochloride (42.88 mmol, 2 equiv.).
The resulting suspension was refluxed for 3 h, after which the reaction medium was poured
on ice and water. The resulting precipitate of the title compound was collected by filtration,
washed with water and dried. The crude compound was used in the next step without further
purification (yields: 80-95%).
4.1.6.
3,4-Dihydro-2,2-dimethyl-6-methoxycarbonylamino-2H-1-benzopyran-4-
hydroxyimine (12a): IR (KBr) : υ : 3427 (N-H), 1743 (C=O) cm-1; RMN 1H (DMSO-d6, 500
MHz) : 1.28 (s, 6H, CH3), 2.74 (s, 2H, CH2), 3.64 (s, 3H, CH3), 6.77 (d, 1H, 8-H), 7.32 (dd,
1H, 7-H), 7.89 (s, 1H, 5-H), 9.48 (s, 1H, -NH), 11.23 (s, 1H, =NOH).
4.1.7.
3,4-Dihydro-2,2-dimethyl-6-ethoxycarbonylamino-2H-1-benzopyran-4-
hydroxyimine (12b): IR (KBr) : υ : 3295 (N-H), 1697 (C=O) cm-1; RMN 1H (DMSO-d6, 500
MHz) : 1.24 (t, 3H, CH3-CH2), 1.28 (s, 6H, CH3), 2.73 (s, 2H, CH2), 4.10 (q, 2H, CH3-CH2),
6.75 (d, 1H, 8-H), 7.31 (dd, 1H, 7-H), 7.90 (s, 1H, 5-H), 9.45 (s, 1H, -NH), 11.22 (s, 1H, =N-
OH).
4.1.8.
3,4-Dihydro-2,2-dimethyl-6-isopropoxycarbonylamino-2H-1-benzopyran-4-
hydroxyimine (12c): IR (KBr) : υ : 3348 (N-H), 1699 (C=O) cm-1; RMN 1H (DMSO-d6, 500
MHz) : 1.24 (d, 6H, (CH3)2-CH), 1.28 (s, 6H, CH3), 2.73 (s, 2H, CH2), 4.86 (m, 1H, (CH3)2-
CH), 6.75 (d, 1H, 8-H), 7.30 (dd, 1H, 7-H), 7.92 (s, 1H, 5-H), 9.39 (s, 1H, -NH), 11.22 (s, 1H,
=N-OH).
4.1.9. General synthetic pathway to R/S-6-alkoxycarbonylamino-4-amino-3,4-dihydro-
2,2-dimethyl-2H-1-benzopyrans (13a-c)
Raney-Nickel° (3 g) was suspended in a solution of the appropriate 6-alkoxycarbonylamino-
3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-hydroxyimine (12a-d) (2.5 g, 8-9 mmol) in
13

ACCEPTED MANUSCRIPT
methanol (50 mL). The reaction mixture was stirred for 3-4 h in a sealed hydrogenator under
a hydrogen pressure of 5 bars. The catalyst was filtered off and the filtrate was evaporated to
dryness under reduced pressure. The crude product was dissolved in the minimum methanol
and the resulting solution was mixed with two volumes of water. The precipitate of the title
compound was collected by filtration, washed with water and dried. The crude compound was
used in the next step without further purification (yields: 80-95%).
4.1.10.
R/S-4-Amino-3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-2H-1-
1
benzopyran (13a): IR (KBr) : υ : 3365 (N-H), 1719 (C=O) cm-1; RMN H (DMSO-d6, 500
MHz) : 1.17 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.51 (t, 1H, CH2), 1.80 (s, 2H, NH2), 1.99 (m, 1H,
CH2), 3.62 (s, 3H, CH3), 3.79 (m, 1H, CH-NH2), 6.58 (d, 1H, 5-H), 7.12 (d, 1H, 7-H), 7.57 (s,
1H, 8-H), 9,29 (s, 3H, NH).
4.1.11. R/S-4-Amino-3,4-dihydro-2,2-dimethyl-6-ethoxycarbonylamino-2H-1-benzopyran
(13b): IR (KBr) : υ : 3352 (N-H), 1723 (C=O) cm-1; RMN 1H (DMSO-d6, 500 MHz) : 1.17 (s,
3H, CH3), 1.22 (t, 3H, CH3-CH2), 1.32 (s, 3H, CH3), 1.51 (t, 1H, CH2), 1.79 (s, 2H, NH2), 1.99
(m, 1H, CH2), 3.79 (m, 1H, CH), 4.08 (q, 2H, CH3-CH2), 6.57 (d, 1H, 8-H), 7.11 (d, 1H, 7-H),
7.59 (s, 1H, 5-H), 9.26 (s, 1H, -NH).
4.1.12.
R/S-4-Amino-3,4-dihydro-2,2-dimethyl-6-isopropoxycarbonylamino-2H-1-
1
benzopyran (13c): IR (KBr) : υ : 3361 (N-H), 1720 (C=O) cm-1; RMN H (DMSO-d6, 500
MHz) : 1.17 (s, 3H, CH3), 1.23 (d, 6H, (CH3)2CH), 1.32 (s, 3H, CH3), 1.51 (t, 1H, CH2), 1.77
(s, 2H, NH2), 1.99 (m, 1H, CH2), 3.79 (m, 1H, CH-NH2), 4.84 (m, 1H, (CH3)2CH), 6.56 (d, 1H,
5-H), 7.09 (d, 1H, 7-H), 7.61 (s, 1H, 8-H), 9.17 (s, 3H, NH).
4.1.13. General synthetic pathway to R/S-N-(3/4-monosubstituted (Cl/CN)-phenyl)-N’-(6-
alkoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)ureas (14) and
R/S-N-(3/4-monosubstituted
(Cl/CN)-phenyl)-N’-(6-alkoxycarbonylamino-3,4-dihydro-
2,2-dimethyl-2H-1-benzopyran-4-yl)thioureas (15)
The
appropriate
R/S-6-alkoxycarbonylamino-4-amino-3,4-dihydro-2,2-dimethyl-2H-1-
benzopyran (13) (0.5 g) was dissolved in methylene chloride (8 mL) and then supplemented
with the appropriate 3- or 4-monosubstituted (Cl, CN)-phenyl isocyanate (1.2 equiv.) or the
14

ACCEPTED MANUSCRIPT
appropriate 3- or 4-monosubstituted (Cl, CN)-phenyl isothiocyanate (1.2 equiv.). The reaction
mixture was stirred during 30 min., after which the resulting precipitate of the title compound
was collected by filtration, washed with methylene chloride and dried (yields: 85-95%). The
title compound can further be purified by DCVC chromatography.
4.1.14. R/S-N-3-Chlorophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-
1
2H-1-benzopyran-4-yl)urea (14a): m.p. : 217-219°C; H NMR (DMSO-d6) δ 1.25 (s, 3H,
CH3), 1.36 (s, 3H, CH3), 1.73 (dd, J=12.9 Hz/11.2 Hz, 1H, 3-H), 2.08 (dd, J=13.2 Hz/6.2 Hz,
1H, 3-H), 3.60 (s, 3H, NHCOOCH3), 4.93 (dd, J=15.8 Hz/10.0 Hz, 1H, 4-H), 6,61 (d, J=8.8
Hz, 1H, CHNHCONHAr), 6.67 (d, J=8.8 Hz, 1H, 8-H), 6.97 (ddd, J=7.7 Hz/2 Hz/1.4 Hz, 1H,
6'-H), 7.22-7.28 (m, 3H, 4'-H/5'-H/7-H), 7.35 (s, 1H, 5-H), 7.75 (t, J=2 Hz, 1H, 2'-H), 8.75 (s,
1H, CHNHCONHAr), 9.40 (s, 1H, NHCOOCH3); 13C NMR (DMSO-d6) δ 24.4 (CH3), 29.1
(CH3), 40.0 (C-3), 42.3 (C-4), 51.4 (OCH3), 74.9 (C-2), 116.1 (C-6'), 116.8 (C-8), 117.1 (C-
2'), 117.6 (C-5), 119.4 (C-7), 120.8 (C-4'), 123.1 (C-4a), 130.3 (C-5'), 131.6 (C-6), 133.1 (C-
3'), 141.9 (C-1'), 148.8 (C-8a), 154.1 (NHCOOCH3), 154.9 (CHNHCONHAr). Anal.
(C20H22ClN3O4) theoretical: C, 59.48; H, 5.49; N, 10.40. Found: C, 59.10; H, 5.54; N, 10.04.
4.1.15. R/S-N-4-Chlorophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-
1
2H-1-benzopyran-4-yl)urea (14b): m.p. : 191-193°C; H NMR (DMSO-d6) d 1.25 (s, 3H,
CH3), 1.36 (s, 3H, CH3), 1.71 (dd, J=13 Hz/11.2 Hz, 1H, 3-H), 2.08 (dd, J=13.2 Hz/6.2 Hz,
1H, 3-H), 3.60 (s, 3H, NHCOOCH3), 4.93 (dd, J=15.9 Hz/9.9 Hz, 1H, 4-H), 6.56 (d, J=8.8
Hz, 1H, CHNHCONHAr), 6,66 (d, J=8.8 Hz, 1H, 8-H), 7.23 (d, J=7.8 Hz, 1H, 7-H), 7.29 (d,
J=8.9 Hz , 2H, 3'-H/5'-H), 7.35 (s, 1H, 5-H), 7.48 (d, J=8.9 Hz, 1H, 2'-H/6'-H), 8.68 (s, 1H,
CHNHCONHAr), 9.40 (s, 1H, NHCOOCH3); 13C NMR (DMSO-d6) δ 24.4 (CH3), 29.1
(CH3), 40.2 (C-3), 42.2 (C-4), 51.4 (OCH3), 74.9 (C-2), 116.8 (C-8), 117.7 (C-5), 119.2 (C-
2'/C-6'), 119.4 (C-7), 123.2 (C-4a), 124.6 (C-4'), 128.5 (C-3'/C-5'), 131.6 (C-6), 139.4 (C-1'),
148.8 (C-8a), 154.1 (NHCOOCH3), 154.9 (CHNHCONHAr). Anal. (C20H22ClN3O4)
theoretical: C, 59.48; H, 5.49; N, 10.40. Found: C, 59.84; H, 5.43; N, 10.66.
4.1.16. R/S-N-3-Cyanophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-
1
2H-1-benzopyran-4-yl)urea (14c) : m.p. 127-129°C; H NMR (DMSO-d6) δ 1.25 (s, 3H,
CH3), 1.36 (s, 3H, CH3), 1.74 (t, J=12 Hz, 1H, 3-H), 2.08 (dd, J=13 Hz/6 Hz, 3-H), 3.60 (s,
3H, NHCOOCH3), 4.95 (dd, J=16 Hz/9.5 Hz, 1H, 4-H), 6.67 (d, J=8.8 Hz, 1H, 8-H), 6,76 (d,
15

ACCEPTED MANUSCRIPT
J=8.6 Hz, 1H, CHNHCONHAr), 7.23 (d, J=8.2 Hz, 1H, 7-H), 7.36 (s, 1H, 5-H), 7.37 (d,
J=7.3 Hz, 1H, 4'-H), 7.46 (t, J=8 Hz, 1H, 5'-H), 7.65 (d, J=8.3 Hz, 1H, 6'-H), 8.01 (s, 1H, 2'-
H), 8.94 (s, 1H, CHNHCONHAr), 9.37 (s, 1H, NHCOOCH3); 13C NMR (DMSO-d6) δ 24.4
(CH3), 29.1 (CH3), 40.0 (C-3), 42.4 (C-4), 51.4 (NHCOOCH3), 74.9 (C-2), 111.5 (C-3'),
116.8 (C-8), 117.6 (C-5), 119.0 (CN), 119.5 (C-7), 120.4 (C-2'), 122.4 (C-6'), 123.1 (C-4a),
124.6 (C-4'), 130.1 (C-5'), 131.6 (C-6), 141.3 (C-1'), 148.8 (C-8a), 154.1 (NHCOOCH3),
154.9 (CHNHCONHAr). Anal. (C21H22N4O4) theoretical: C, 63.95; H, 5.62; N, 14.20. Found:
C, 63.61; H, 5.51; N, 14.39.
4.1.17. R/S-N-4-Cyanophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-
1
2H-1-benzopyran-4-yl)urea (14d) : m.p. : 221-223°C; H NMR (DMSO-d6) δ 1.25 (s, 3H,
CH3), 1.36 (s, 3H, CH3), 1.74 (dd, J=13 Hz/11.2 Hz, 1H, 3-H), 2.09 (dd, J=13.2 Hz/6.3 Hz,
1H, 3-H), 3.59 (s, 3H, NHCOOCH3), 4.94 (dd, J=15.9 Hz/9.9 Hz, 1H, 4-H), 6.67 (d, J=8.8
Hz, 1H, 8-H), 6,76 (d, J=8.7 Hz, 1H, CHNHCONHAr), 7.24 (d, J=8 Hz, 1H, 7-H), 7.35 (s,
1H, 5-H), 7.64 (d, J=8.9 Hz , 2H, 3'-H/5'-H), 7.70 (d, J=8.9 Hz, 1H, 2'-H/6'-H), 9.08 (s, 1H,
CHNHCONHAr), 9.40 (s, 1H, NHCOOCH3); 13C NMR (DMSO-d6) δ 24.4 (CH3), 29.0
(CH3), 39.8 (C-3), 42.4 (C-4), 51.4 (OCH3), 74.9 (C-2), 102.5 (C-4'), 116.8 (C-8), 117.6 (C-
5/C-3'/C-5'), 119.4 (CN), 119.8 (C-7), 122.9 (C-4a), 131.7 (C-6), 133.2 (C-2'/C-6'), 144.8 (C-
1'), 148.8 (C-8a), 154.1 (NHCOOCH3), 154.6 (CHNHCONHAr). Anal. (C21H22N4O4)
theoretical: C, 63.95; H, 5.62; N, 14.20. Found: C, 63.90; H, 5.62; N, 13.91.
4.1.18. R/S-N-3-Chlorophenyl-N'-(6-ethoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-
1
1-benzopyran-4-yl)urea (14e): m.p. : 197-199°C; H NMR (DMSO-d6) δ 1.19 (t, J=7.1 Hz,
3H, CH2CH3), 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.72 (dd, J=12.9 Hz/11.3 Hz, 1H, 3-H),
2.08 (dd, J=13.2 Hz/6.2 Hz, 1H, 3-H), 4.06 (q, J=7.1 Hz, 2H, CH2CH3), 4.94 (dd, J=15.9
Hz/9.9 Hz, 1H, 4-H), 6,61 (d, J=8.7 Hz, 1H, CHNHCONHAr), 6.66 (d, J=8.8 Hz, 1H, 8-H),
6.96 (ddd, J=7.7 Hz/2 Hz/1.4 Hz, 1H, 6'-H), 7.22-7.28 (m, 3H, 4'-H/5'-H/7-H), 7.36 (s, 1H, 5-
H), 7.75 (t, J=2 Hz, 1H, 2'-H), 8.75 (s, 1H, CHNHCONHAr), 9.37 (s, 1H, NHCOOCH2CH3);
13C NMR (DMSO-d6) δ 14.5 (CH2CH3), 24.4 (CH3), 29.1 (CH3), 40.0 (C-3), 42.3 (C-4), 59.9
(CH2CH3), 74.9 (C-2), 116.1 (C-6'), 116.7 (C-8), 117.2 (C-2'), 117.6 (C-5), 119.5 (C-7), 120.8
(C-4'), 123.1 (C-4a), 130.3 (C-5'), 131.7 (C-6), 133.1 (C-3'), 141.9 (C-1'), 148.7 (C-8a), 153.7
(NHCOOCH2CH3), 154.9 (CHNHCONHAr). Anal. (C21H24ClN3O4) theoretical: C, 60.36; H,
5.79; N, 10.06. Found: C, 60.25; H, 5.81; N, 9.78.
16

ACCEPTED MANUSCRIPT
4.1.19. R/S-N-4-Chlorophenyl-N'-(6-ethoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-
1
1-benzopyran-4-yl)urea (14f): m.p. : 211-213°C; H NMR (DMSO-d6) δ 1.19 (t, J=7.1 Hz,
3H, CH2CH3), 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.71 (dd, J=12.9 Hz/11.2 Hz, 1H, 3-H),
2.08 (dd, J=13.2 Hz/6.2 Hz, 1H, 3-H), 4.05 (q, J=7.1 Hz, 2H, CH2CH3), 4.93 (dd, J=15.9
Hz/9.8 Hz, 1H, 4-H), 6.53 (d, J=8.8 Hz, 1H, CHNHCONHAr), 6,66 (d, J=8.8 Hz, 1H, 8-H),
7.21 (d, J=7.7 Hz, 1H, 7-H), 7.29 (d, J=8.9 Hz, 2H, 3'-H/5'-H), 7.38 (s, 1H, 5-H), 7.48 (d,
13
J=8.9 Hz, 2H, 2'-H/6'-H), 8.66 (s, 1H, CHNHCONHAr), 9.36 (s, 1H, NHCOOCH2CH3); C
NMR (DMSO-d6) δ 14.5 (CH2CH3), 24.4 (CH3), 29.1 (CH3), 40.2 (C-3), 42.3 (C-4), 59.9
(CH2CH3), 74.9 (C-2), 116.7 (C-8), 117.7 (C-5), 119.2 (C-2'/C-6'), 119.3 (C-7), 123.2 (C-4a),
124.6 (C-4'), 128.5 (C-3'/C-5'), 131.7 (C-6), 139.4 (C-1'), 148.8 (C-8a), 153.7
(NHCOOCH2CH3), 154.9 (CHNHCONHAr). Anal. (C21H24ClN3O4) theoretical: C, 60.36; H,
5.79; N, 10.06. Found: C, 60.55; H, 5.83; N, 9.88.
4.1.20. R/S-N-3-Cyanophenyl-N'-(6-ethoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-
1-benzopyran-4-yl)urea (14g): m.p. 167-169°C; 1H NMR (DMSO-d6) δ 1.19 (t, J=7 Hz, 3H,
NHCOOCH2CH3), 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.74 (t, J=12 Hz, 1H, 3-H), 2.08 (dd,
J=13 Hz/6 Hz, 3-H), 4.06 (q, J=7 Hz, 2H, NHCOOCH2CH3), 4.94 (dd, J=16 Hz/9.5 Hz, 1H,
4-H), 6.66 (d, J=8.8 Hz, 1H, 8-H), 6,75 (d, J=8.7 Hz, 1H, CHNHCONHAr), 7.21 (d, J=7.8
Hz, 1H, 7-H), 7.36-7.39 (m, 2H, 5-H/4'-H), 7.46 (t, J=8 Hz, 1H, 5'-H), 7.65 (d, J=8.3 Hz, 1H,
13
6'-H), 8.01 (s, 1H, 2'-H), 8.93 (s, 1H, CHNHCONHAr), 9.34 (s, 1H, NHCOOCH2CH3); C
NMR (DMSO-d6) δ
NHCOOCH2CH3 , 24.9 (CH3), 29.6 (CH3), 40.5 (C-3), 42.9
(C-4), 60.3 (NHCOOCH2CH3), 75.3 (C-2), 112.0 (C-3'), 117.2 (C-8), 118.1 (C-5), 119.5
(CN), 120.0 (C-7), 120.9 (C-2'), 122.9 (C-6'), 123.5 (C-4a), 125.1 (C-4'), 130.5 (C-5'), 132.2
(C-6), 141.8 (C-1'), 149.2 (C-8a), 154.1 (NHCOOCH2CH3), 155.4 (CHNHCONHAr). Anal.
(C22H24N4O4) theoretical: C, 64.69; H, 5.92; N, 13.72. Found: C, 64.70; H, 6.04; N, 13.63.
4.1.21. R/S-N-4-Cyanophenyl-N'-(6-ethoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-
1
1-benzopyran-4-yl)urea (14h): m.p. : 208-210°C; H NMR (DMSO-d6) δ 1.19 (t, J=7.1 Hz,
3H, CH2CH3), 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.74 (dd, J=12.9 Hz/11.3 Hz, 1H, 3-H),
2.08 (dd, J=13.2 Hz/6.2 Hz, 1H, 3-H), 4.05 (q, J=7.1 Hz, 2H, CH2CH3), 4.94 (dd, J=15.9
Hz/9.8 Hz, 1H, 4-H), 6.66 (d, J=8.8 Hz, 1H, 8-H), 6,77 (d, J=8.7 Hz, 1H, CHNHCONHAr),
7.22 (d, J=7.6 Hz, 1H, 7-H), 7.37 (s, 1H, 5-H), 7.64 (d, J=8.8 Hz, 2H, 3'-H/5'-H), 7.70 (d,
17

ACCEPTED MANUSCRIPT
13
J=8.8 Hz, 2H, 2'-H/6'-H), 9.09 (s, 1H, CHNHCONHAr), 9.36 (s, 1H, NHCOOCH2CH3); C
NMR (DMSO-d6) δ 14.5 (CH2CH3), 24.4 (CH3), 29.0 (CH3), 39.8 (C-3), 42.4 (C-4), 59.9
(CH2CH3), 74.9 (C-2), 102.5 (C-4'), 116.8 (C-8), 117.6 (C-5/C-3'/C-5'), 119.4 (CN), 119.6 (C-
7), 122.9 (C-4a), 131.7 (C-6), 133.2 (C-2'/C-6'), 144.8 (C-1'), 148.7 (C-8a), 153.7
(NHCOOCH2CH3), 154.6 (CHNHCONHAr). Anal. (C22H24N4O4) theoretical: C, 64.69; H,
5.92; N, 13.72. Found: C, 64.70; H, 5.94; N, 13.45.
4.1.22.
R/S-N-3-Chlorophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-
1
isopropoxycarbonylamino-2H-1-benzopyran-4-yl)urea (14i): m.p. : 105-107°C; H NMR
(DMSO-d6) δ 1.20 (dd, J=6.2 Hz/1 Hz, 6H, CH(CH3)2), 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3),
1.72 (dd, J=12.9 Hz/11.2 Hz, 1H, 3-H), 2.08 (dd, J=13.2 Hz/6.2 Hz, 1H, 3-H), 4.82 (hept,
J=6.2 Hz, 1H, CH(CH3)2), 4.92 (dd, J=15.8 Hz/9.9 Hz, 1H, 4-H), 6,59 (d, J=8.7 Hz, 1H,
CHNHCONHAr), 6.65 (d, J=8.8 Hz, 1H, 8-H), 6.96 (ddd, J=7.7 Hz/2 Hz/1.3 Hz, 1H, 6'-H),
7.23-7.26 (m, 3H, 4'-H/5'-H/7-H), 7.40 (s, 1H, 5-H), 7.76 (t, J=2 Hz, 1H, 2'-H), 8.74 (s, 1H,
CHNHCONHAr), 9.31 (s, 1H, NHCOOCH(CH3)2); 13C NMR (DMSO-d6) δ 21.2
(CH(CH3)2), 24.4 (CH3), 29.1 (CH3), 40.0 (C-3), 42.3 (C-4), 67.1 (CH(CH3)2), 74.8 (C-2),
116.1 (C-6'), 116.7 (C-8), 117.2 (C-2'), 117.6 (C-5), 119.5 (C-7), 120.8 (C-4'), 123.0 (C-4a),
130.3 (C-5'), 131.8 (C-6), 133.1 (C-3'), 141.9 (C-1'), 148.7 (C-8a), 153.3
(NHCOOCH(CH3)2), 154.9 (CHNHCONHAr). Anal. (C22H26ClN3O4) theoretical: C, 61.18;
H, 6.07; N, 9.73. Found: C, 60.91; H, 6.23; N, 9.62.
4.1.23.
R/S-N-4-Chlorophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-
1
isopropoxycarbonylamino-2H-1-benzopyran-4-yl)urea (14j): m.p. : 195-197°C; H NMR
(DMSO-d6) δ 1.20 (d, J=6.2 Hz, 6H, CH(CH3)2), 1.24 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.71
(dd, J=13 Hz/11.2 Hz, 1H, 3-H), 2.08 (dd, J=13.2 Hz/6.2 Hz, 1H, 3-H), 4.82 (hept, J=6.3 Hz,
1H, CH(CH3)2), 4.92 (dd, J=15.8 Hz/9.8 Hz, 1H, 4-H), 6.53 (d, J=8.8 Hz, 1H,
CHNHCONHAr), 6,65 (d, J=8.8 Hz, 1H, 8-H), 7.19 (d, J=6.6 Hz, 1H, 7-H), 7.29 (d, J=8.9
Hz, 2H, 3'-H/5'-H), 7.40 (s, 1H, 5-H), 7.48 (d, J=8.9 Hz, 2H, 2'-H/6'-H), 8.66 (s, 1H,
CHNHCONHAr), 9.30 (s, 1H, NHCOOCH(CH3)2); 13C NMR (DMSO-d6) δ 21.2
(CH(CH3)2), 24.4 (CH3), 29.1 (CH3), 40.2 (C-3), 42.3 (C-4), 67.0 (CH(CH3)2), 74.8 (C-2),
116.7 (C-8), 117.6 (C-5), 119.2 (C-2'/C-6'), 119.5 (C-7), 123.1 (C-4a), 124.6 (C-4'), 128.5 (C-
3'/C-5'), 131.8 (C-6), 139.4 (C-1'), 148.7 (C-8a), 153.3 (NHCOOCH(CH3)2), 154.9
18

ACCEPTED MANUSCRIPT
(CHNHCONHAr). Anal. (C22H26ClN3O4) theoretical: C, 61.18; H, 6.07; N, 9.73. Found: C,
61.11; H, 6.08; N, 9.50.
4.1.24. R/S-N-3-Cyanophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-isopropoxycarbonylamino-
1
2H-1-benzopyran-4-yl)urea (14k): m.p. : 105-107°C; H NMR (DMSO-d6) 1.20 (t, J=7
Hz, 6H, NHCOOCH(CH3)2), 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.74 (t, J=12 Hz, 1H, 3-H),
2.08 (dd, J=13 Hz/6 Hz, 3-H), 4.82 (dt, J=12 Hz/6 Hz, 1H, NHCOOCH(CH3)2), 4.94 (dd,
J=16.2 Hz/9.5 Hz, 1H, 4-H), 6.65 (d, J=8.8 Hz, 1H, 8-H), 6,74 (d, J=8.7 Hz, 1H,
CHNHCONHAr), 7.20 (d, J=7.2 Hz, 1H, 7-H), 7.37 (d, J=7.5 Hz, 1H, 4'-H), 7.40 (m, 2H, 5-
H), 7.46 (t, J=7.9 Hz, 1H, 5'-H), 7.65 (d, J=8.3 Hz, 1H, 6'-H), 8.01 (s, 1H, 2'-H), 8.93 (s, 1H,
CHNHCONHAr), 9.27 (s, 1H, NHCOOCH(CH3)2); 13C NMR (DMSO-d6)
NHCOOCH(CH3 , 24.9 (CH3), 29.6 (CH3), 40.2 (C-3), 42.9 (C-4), 67.6
δ
(NHCOOCH(CH3)2), 75.3 (C-2), 112.0 (C-3'), 117.2 (C-8), 118.1 (C-5), 119.5 (CN), 120.0
(C-7), 120.9 (C-2'), 122.9 (C-6'), 123.5 (C-4a), 125.1 (C-4'), 130.5 (C-5'), 132.3 (C-6), 141.8
(C-1'), 149.2 (C-8a), 153.8 (NHCOOCH(CH3)2), 155.4 (CHNHCONHAr). Anal.
(C23H26N4O4) theoretical: C, 65.39; H, 6.20; N, 13.26. Found: C, 65.23; H, 6.07; N, 13.36.
4.1.25. R/S-N-4-Cyanophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-isopropoxycarbonylamino-
1
2H-1-benzopyran-4-yl)urea (14l): m.p. : 210-212°C; H NMR (DMSO-d6) δ 1.20 (d, J=6.2
Hz, 6H, CH(CH3)2), 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.73 (dd, J=12.9 Hz/11.2 Hz, 1H,
3-H), 2.08 (dd, J=13.2 Hz/6.3 Hz, 1H, 3-H), 4.82 (hept, J=6.2 Hz, 1H, CH(CH3)2), 4.94 (dd,
J=15.9 Hz/9.8 Hz, 1H, 4-H), 6.66 (d, J=8.8 Hz, 1H, 8-H), 6,73 (d, J=8.7 Hz, 1H,
CHNHCONHAr), 7.20 (d, J=6.9 Hz, 1H, 7-H), 7.39 (s, 1H, 5-H), 7.64 (d, J=8.9 Hz , 2H, 3'-
H/5'-H), 7.70 (d, J=8.9 Hz, 2H, 2'-H/6'-H), 9.06 (s, 1H, CHNHCONHAr), 9.30 (s, 1H,
13
NHCOOCH(CH3)2); C NMR (DMSO-d6) δ 22.0 (CH(CH3)2), 24.4 (CH3), 29.0 (CH3), 39.8
(C-3), 42.4 (C-4), 67.1 (CH(CH3)2), 74.8 (C-2), 102.5 (C-4'), 116.8 (C-8), 117.6 (C-3'/C-5'),
118.3 (C-5), 119.4 (CN), 119.6 (C-7), 122.8 (C-4a), 131.8 (C-6), 133.2 (C-2'/C-6'), 144.8 (C-
1'), 148.7 (C-8a), 153.3 (NHCOOCH(CH3)2), 154.5 (CHNHCONHAr). Anal. (C23H26N4O4)
theoretical: C, 65.39; H, 6.20; N, 13.26. Found: C, 65.46; H, 6.15; N, 13.22.
4.1.26.
R/S-N-3-Chlorophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-
dimethyl-2H-1-benzopyran-4-yl)urea (14m): m.p. 113°C (dec); 1H NMR (DMSO-d6)
1.24 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.42 (s, 9H, NHCOOC(CH3)3), 1.71 (dd, J=13 Hz/11
19

ACCEPTED MANUSCRIPT
Hz, 1H, 3-H), 2.09 (dd, J=13 Hz/6 Hz, 3-H), 4.92 (dd, J=16 Hz/10 Hz, 1H, 4-H), 6.55 (d,
J=8.7 Hz, 1H, CHNHCONHAr), 6,63 (d, J=8.8 Hz, 1H, 8-H), 6.96 (ddd, J=1.2 Hz/2 Hz/8.7
Hz, 1H, 4'-H), 7.17 (d, J=7.1 Hz, 1H, 7-H), 7.22 (d, J=8.6 Hz, 1H, 6'-H), 7.26 (t, J=8 Hz, 1H,
5'-H), 7.40 (s, 1H, 5-H), 7.75 (t, J=2 Hz, 1H, 2'-H), 8.70 (s, 1H, CHNHCONHAr), 9.08 (s,
1H, NHCOOC(CH3)3); 13C NMR (DMSO-d6) δ 24.4 (CH3), 28.1 (C(CH3)3), 29.0 (CH3), 40.1
(C-3), 42.4 (C-4), 74.8 (C-2), 78.6 (C(CH3)3), 116.2 (C-6'), 116.7 (C-8), 117.2 (C-2'), 117.6
(C-5), 119.6 (C-7), 120.8 (C-4'), 122.9 (C-4a), 130.2 (C-5'), 132.0 (C-6), 133.1 (C-3'), 141.9
(C-1'), 148.6 (C-8a), 153.0 (NHCOOC(CH3)3), 154.9 (CHNHCONHAr). Anal.
(C23H28ClN3O4) theoretical: C, 61.95; H, 6.33; N, 9.42. Found: C, 61.60; H, 6.36; N, 9.46.
4.1.27.
R/S-N-4-Chlorophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-
1
dimethyl-2H-1-benzopyran-4-yl)urea (14n): m.p.: 113°C (dec); H NMR (DMSO-d6)
1.24 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.42 (s, 9H, NHCOOC(CH3)3), 1.70 (dd, J=13 Hz/11
Hz, 1H, 3-H), 2.08 (dd, J=13 Hz/6 Hz, 3-H), 4.91 (dd, J=16 Hz/9 Hz, 1H, 4-H), 6.50 (d, J=8.7
Hz, 1H, CHNHCONHAr), 6,63 (d, J=8.8 Hz, 1H, 8-H), 7.17 (d, J=6.7 Hz, 1H, 7-H), 7.29 (d,
J=8.7 Hz , 2H, 3'-H/5'-H), 7.40 (s, 1H, 5-H), 7.48 (d, J=8.6 Hz, 1H, 2'-H/6'-H), 8.64 (s, 1H,
CHNHCONHAr), 9.08 (s, 1H, NHCOOC(CH3)3). 13C NMR (DMSO-d6) δ 24.5 (CH3), 28.1
(C(CH3)3), 29.0 (CH3), 40.5 (C-3), 42.4 (C-4), 74.8 (C-2), 78.5 (C(CH3)3), 116.6 (C-8), 117.6
(C-5), 119.3 (C-2'/C-6'), 119.6 (C-7), 122.9 (C-4a), 124.6 (C-4'), 128.5 (C-3'/C-5'), 132.0 (C-
6), 139.4 (C-1'), 148.6 (C-8a), 153.0 (NHCOOC(CH3)3), 154.9 (CHNHCONHAr). Anal.
(C23H28ClN3O4) theoretical: C, 61.95; H, 6.33; N, 9.42. Found: C, 62.12; H, 6.39; N, 9.82.
4.1.28. R/S-N-3-Cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-
1
2H-1-benzopyran-4-yl)urea (14o): m.p.: 189-191°C; H NMR (DMSO-d6) δ 1.25 (s, 3H,
CH3), 1.36 (s, 3H, CH3), 1.42 (s, 9H, NHCOOC(CH3)3), 1.73 (dd, J=13 Hz/11 Hz, 1H, 3-H),
2.08 (dd, J=13 Hz/6 Hz, 3-H), 4.93 (dd, J=11 Hz/6 Hz, 1H, 4-H), 6.64 (d, J=8.8 Hz, 1H, 8-H),
6,70 (d, J=8.8 Hz, 1H, CHNHCONHAr), 7.17 (d, J=8.7 Hz, 1H, 7-H), 7.37 (d, J=8 Hz, 1H, 4'-
H), 7.41 (bs, 1H, 5-H), 7.46 (t, J=8 Hz, 1H, 5'-H), 7.64 (d, J=8 Hz, 1H, 6'-H), 8.01 (s, 1H, 2'-
H), 8.89 (s, 1H, CHNHCONHAr), 9.09 (s, 1H, NHCOOC(CH3)3); 13C NMR (DMSO-d6) δ
24.4 (CH3), 28.1 (C(CH3)3), 29.0 (CH3), 40.3 (C-3), 42.5 (C-4), 74.8 (C-2), 78.6 (C(CH3)3),
111.5 (C-3'), 116.6 (C-8), 117.6 (C-5), 119.0 (CN), 119.6 (C-7), 120.4 (C-2'), 122.5 (C-6'),
122.8 (C-4a), 124.7 (C-4'), 130.1 (C-5'), 132.0 (C-6), 141.3 (C-1'), 148.5 (C-8a), 152.9
20

ACCEPTED MANUSCRIPT
(NHCOOC(CH3)3), 154.9 (CHNHCONHAr). Anal. (C24H28N4O4) theoretical: C, 66.04; H,
6.47; N, 12.84. Found: C, 66.20; H, 6.53; N, 12.95.
4.1.29. R/S-N-4-Cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-
1
2H-1-benzopyran-4-yl)urea (14p): m.p.: 231-233°C; H NMR (DMSO-d6) δ 1.25 (s, 3H,
CH3), 1.36 (s, 3H, CH3), 1.42 (s, 9H, NHCOOC(CH3)3), 1.74 (t, J=12 Hz, 1H, 3-H), 2.08 (dd,
J=12 Hz/6 Hz, 3-H), 4.93 (dd, J=16 Hz/8 Hz, 1H, 4-H), 6.64 (d, J=7.7 Hz, 1H, 8-H), 6,72 (d,
J=8.3 Hz, 1H, CHNHCONHAr), 7.18 (d, J=6.5 Hz, 1H, 7-H), 7.39 (s, 1H, 5-H), 7.63 (d, J=7
Hz , 2H, 3'-H/5'-H), 7.70 (d, J=6.9 Hz, 1H, 2'-H/6'-H), 9.05 (s, 1H, CHNHCONHAr), 9.08 (s,
1H, NHCOOC(CH3)3); 13C NMR (DMSO-d6) δ 24.4 (CH3), 28.1 (C(CH3)3), 29.0 (CH3), 39.8
(C-3), 42.4 (C-4), 74.8 (C-2), 78.6 (C(CH3)3), 102.5 (C-4'), 116.7 (C-8), 117.6 (C-5/C-3'/C-
5'), 119.4 (CN), 119.6 (C-7), 122.6 (C-4a), 132.0 (C-6), 133.2 (C-2'/C-6'), 144.8 (C-1'), 148.5
(C-8a), 153.0 (NHCOOC(CH3)3), 154.6 (CHNHCONHAr). Anal. (C24H28N4O4) theoretical:
C, 66.04; H, 6.47; N, 12.84. Found: C, 65.94; H, 6.35; N, 12.45.
4.1.30. R/S-N-3-Chlorophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-
1
2H-1-benzopyran-4-yl)thiourea (15a): m.p. : 171-173°C; H NMR (DMSO-d6) δ 1.25 (s,
3H, CH3), 1.36 (s, 3H, CH3), 1.77 (t, J=12 Hz, 1H, 3-H), 2.16 (dd, J=13 Hz/6.3 Hz, 1H, 3-H),
3.63 (s, 3H, NHCOOCH3), 5.77 (dd, J=16.5 Hz/7.4 Hz, 1H, 4-H), 6,69 (d, J=8.8 Hz, 1H, 8-
H), 7.16 (d, J=7.2 Hz, 1H, 6'-H), 7.23 (d, J=8.2 Hz, 1H, 7-H), 7.32-7.37 (m, 2H, 4'-H/5'-H),
7.40 (s, 1H, 2'-H), 7.74 (s, 1H, 5-H), 8.25 (d, J=7.7 Hz, 1H, CHNHCSNHAr), 9.44 (s, 1H,
NHCOOCH3), 9.72 (s, 1H, CHNHCSNHAr); 13C NMR (DMSO-d6) δ 24.2 (CH3), 29.2
(CH3), 38.1 (C-3), 47.2 (C-4), 51.5 (OCH3), 75.0 (C-2), 116.8 (C-8), 117.4 (C-2'), 119.5 (C-
7), 121.6 (C-4'), 122.0 (C-4a), 122.6 (C-5), 123.8 (C-6'), 130.2 (C-5'), 131.7 (C-6), 132.7 (C-
3'), 140.7 (C-1'), 148.8 (C-8a), 154.1 (NHCOOCH3), 180.5 (CHNHCSNHAr). Anal.
(C20H22ClN3O3S) theoretical: C, 57.20; H, 5.28; N, 10.01; S, 7.64. Found: C, 57.39; H, 5.28;
N, 10.19; S, 7.63.
4.1.31. R/S-N-4-Chlorophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-
1
2H-1-benzopyran-4-yl)thiourea (15b): m.p. : 202-203°C; H NMR (DMSO-d6) δ 1.24 (s,
3H, CH3), 1.36 (s, 3H, CH3), 1.75 (t, J=12 Hz, 1H, 3-H), 2.15 (dd, J=13 Hz/6 Hz, 1H, 3-H),
3.63 (s, 3H, NHCOOCH3), 5.76 (bs, 1H, 4-H), 6,68 (d, J=8.8 Hz, 1H, 8-H), 7.23 (d, J=7.9 Hz,
1H, 7-H), 7.37 (d, J=8.8 Hz , 2H, 2'-H/6'-H), 7.39 (s, 1H, 5-H), 7.52 (d, J=8.8 Hz, 1H, 3'-H/5'-
21

ACCEPTED MANUSCRIPT
H), 8.18 (d, J=6.3 Hz, 1H, CHNHCSNHAr), 9.44 (s, 1H, NHCOOCH3) , 9.69 (s, 1H,
CHNHCSNHAr); 13C NMR (DMSO-d6) δ 24.2 (CH3), 29.2 (CH3), 38.1 (C-3), 47.2 (C-4),
51.5 (OCH3), 75.0 (C-2), 116.8 (C-8), 117.4 (C-5), 119.5 (C-7), 122.1 (C-4a), 125.0 (C-3'/C-
5'), 128.0 (C-4'), 128.4 (C-2'/C-6'), 131.6 (C-6), 138.1 (C-1'), 148.8 (C-8a), 154.2
(NHCOOCH3), 180.5 (CHNHCSNHAr). Anal. (C20H22ClN3O3S) theoretical: C, 57.20; H,
5.28; N, 10.01; S, 7.64. Found: C, 57.30; H, 5.23; N, 10.31; S, 7.77.
4.1.32. R/S-N-3-Cyanophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-
2H-1-benzopyran-4-yl)thiourea (15c) : m.p. 182-184°C; 1H NMR (DMSO-d6) δ 1.26 (s, 3H,
CH3), 1.37 (s, 3H, CH3), 1.77 (t, J=12 Hz, 1H, 3-H), 2.16 (dd, J=13 Hz/6 Hz, 3-H), 3.63 (s,
3H, NHCOOCH3), 5.77 (bs, 1H, 4-H), 6.69 (d, J=8.8 Hz, 1H, 8-H), 7.23 (d, J=8.4 Hz, 1H, 7-
H), 7.43 (s, 1H, 5-H), 7.51-7.56 (m, 2H, 4'-H/5'-H), 7.75 (d, J=7.7 Hz, 1H, 6'-H), 8.06 (s, 1H,
2'-H), 8,40 (d, J=5.4 Hz, 1H, CHNHCSNHAr), 9.42 (s, 1H, NHCOOCH3), 9.87 (s, 1H,
CHNHCSNHAr); 13C NMR (DMSO-d6) δ 24.2 (CH3), 29.2 (CH3), 38.1 (C-3), 47.3 (C-4),
51.4 (NHCOOCH3), 75.0 (C-2), 111.2 (C-3'), 116.9 (C-8), 117.4 (C-5), 118.6 (CN), 119.5 (C-
7), 121.9 (C-4a), 126.1 (C-2'), 127.6 (C-4'), 127.9 (C-6'), 129.9 (C-5'), 131.7 (C-6), 140.2 (C-
1'), 148.8 (C-8a), 154.1 (NHCOOCH3), 180.7 (CHNHCSNHAr). Anal. (C21H22N4O3S)
theoretical: C, 61.44; H, 5.40; N, 13.65; S, 7.81. Found: C, 61.33; H, 5.34; N, 13.69; S, 7.72.
4.1.33. R/S-N-4-Cyanophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-methoxycarbonylamino-
1
2H-1-benzopyran-4-yl)thiourea (15d) : m.p. : 216-217°C; H NMR (DMSO-d6) δ 1,26 (s,
3H, CH3), 1,37 (s, 3H, CH3), 1,76 (m, 1H, 3-H), 2,20 (dd, J=13.1 Hz/6.3 Hz, 1H, 3-H), 3,62
(s, 3H, NHCOOCH3), 5,75 (dd, 1H, J=17.3 Hz/7.9 Hz, 4-H), 6,70 (d, J=8.8 Hz, 1H, 8-H),
7,24 (d, J=8.2 Hz, 1H, 7-H), 7,39 (s, 1H, 5-H), 7,76 (d, J=8.9 Hz, 2H, 3’-H et 5’-H), 7,80 (d,
J=8.9 Hz, 2H, 2’-H et 6’-H), 8,50 (d, J=8.3 Hz, 1H, CHNHCSNHAr), 9,44 (s, 1H,
NHCOOCH3), 10,00 (s, 1H, CHNHCSNHAr); 13C NMR (DMSO-d6) δ 24.2 (CH3), 29.1
(CH3), 38.0 (C-3), 47.1 (C-4), 51.5 (OCH3), 75.0 (C-2), 104.9 (C-4'), 116.9 (C-8), 117.4 (C-
5), 119.1 (CN), 119.7 (C-7), 121.6 (C-4a), 121.7 (C-3'/C-5'), 131.7 (C-6), 132.8 (C-2'/C-6'),
143.9 (C-1'), 148.8 (C-8a), 154.2 (NHCOOCH3), 180.2 (CHNHCSNHAr). Anal.
(C21H22N4O3S) theoretical: C, 61.44; H, 5.40; N, 13.65; S, 7.81. Found: C, 61.37; H, 5.33; N,
13.63; S, 7.42.
4.1.34. R/S-N-3-Chlorophenyl-N'-(6-ethoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-
22

ACCEPTED MANUSCRIPT
1
1-benzopyran-4-yl)thiourea (15e): m.p. : 180-182°C; H NMR (DMSO-d6) δ 1.22 (t, J=7.1
Hz, 3H, CH2CH3), 1.25 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.76 (t, J=12.1 Hz, 1H, 3-H), 2.15
(dd, J=13 Hz/6.3 Hz, 1H, 3-H), 4.09 (q, J=7.1 Hz, 2H, CH2CH3), 5.76 (dd, J=16 Hz/7 Hz, 1H,
4-H), 6,68 (d, J=8.8 Hz, 1H, 8-H), 7.16 (d, J=7.3 Hz, 1H, 6'-H), 7.21 (d, J=8 Hz, 1H, 7-H),
7.32-7.35 (m, 2H, 4'-H/5'-H), 7.43 (s, 1H, 2'-H), 7.74 (s, 1H, 5-H), 8.25 (d, J=7.2 Hz, 1H,
CHNHCSNHAr), 9.41 (s, 1H, NHCOOCH2CH3), 9.73 (s, 1H, CHNHCSNHAr); 13C NMR
(DMSO-d6) δ 14.6 (CH2CH3), 24.2 (CH3), 29.2 (CH3), 38.1 (C-3), 47.2 (C-4), 59.9
(CH2CH3), 75.0 (C-2), 116.8 (C-8), 117.5 (C-2'), 119.6 (C-7), 121.6 (C-4'), 122.0 (C-4a),
122.6 (C-5), 123.8 (C-6'), 130.2 (C-5'), 131.8 (C-6), 132.7 (C-3'), 140.7 (C-1'), 148.7 (C-8a),
153.7 (NHCOOCH2CH3), 180.5 (CHNHCSNHAr). Anal. (C21H24ClN3O3S) theoretical: C,
58.12; H, 5.57; N, 9.68; S, 7.39. Found: C, 58.07; H, 5.55; N, 9.98; S, 7.22.
4.1.35. R/S-N-4-Chlorophenyl-N'-(6-ethoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-
1
1-benzopyran-4-yl)thiourea (15f): m.p. : 171-173°C; H NMR (DMSO-d6) δ 1.22 (t, J=7.1
Hz, 3H, CH2CH3), 1.24 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.75 (t, J=12.1 Hz, 1H, 3-H), 2.15
(dd, J=13 Hz/6.2 Hz, 1H, 3-H), 4.09 (q, J=7.1 Hz, 2H, CH2CH3), 5.76 (dd, J=16.4 Hz/7.5 Hz,
1H, 4-H), 6,67 (d, J=8.8 Hz, 1H, 8-H), 7.21 (d, J=7.4 Hz, 1H, 7-H), 7.36 (d, J=8.8 Hz, 2H, 2'-
H/6'-H), 7.40 (s, 1H, 5-H), 7.53 (d, J=8.8 Hz, 2H, 3'-H/5'-H), 8.21 (d, J=7.6 Hz, 1H,
CHNHCSNHAr), 9.41 (s, 1H, NHCOOCH2CH3), 9.74 (s, 1H, CHNHCSNHAr); 13C NMR
(DMSO-d6) δ 14.6 (CH2CH3), 24.2 (CH3), 29.2 (CH3), 38.1 (C-3), 47.2 (C-4), 59.9
(CH2CH3), 75.0 (C-2), 116.8 (C-8), 117.4 (C-5), 119.5 (C-7), 122.1 (C-4a), 125.0 (C-3'/C-5'),
128.0 (C-4'), 128.4 (C-2'/C-6'), 131.7 (C-6), 138.1 (C-1'), 148.7 (C-8a), 153.7
(NHCOOCH2CH3), 180.5 (CHNHCSNHAr). Anal. (C21H24ClN3O3S) theoretical: C, 58.12; H,
5.57; N, 9.68; S, 7.39. Found: C, 58.49; H, 5.62; N, 9.93; S, 7.40.
4.1.36. R/S-N-3-Cyanophenyl-N'-(6-ethoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-
1-benzopyran-4-yl)thiourea (15g): m.p. 192-194°C; 1H NMR (DMSO-d6) δ 1.22 (t, J=7 Hz,
3H, NHCOOCH2CH3), 1.25 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.77 (t, J=12 Hz, 1H, 3-H), 2.16
(dd, J=13 Hz/6 Hz, 3-H), 4.09 (q, J=7 Hz, 2H, NHCOOCH2CH3), 5.77 (bs, 1H, 4-H), 6.68 (d,
J=8.7 Hz, 1H, 8-H), 7.21 (d, J=8.2 Hz, 1H, 7-H), 7.45 (s, 1H, 5-H), 7.51-7.56 (m, 2H, 4'-H/5'-
H), 7.75 (d, J=7.6 Hz, 1H, 6'-H), 8.06 (s, 1H, 2'-H), 8,40 (d, J=7.2 Hz, 1H, CHNHCSNHAr),
9.39 (s, 1H, NHCOOCH2CH3), 9.86 (s, 1H, CHNHCSNHAr); 13C NMR (DMSO-d6) δ
NHCOOCH2CH3
24.7 (CH3), 29.7 (CH3), 38.6 (C-3), 47.8 (C-4), 60.4
23

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(NHCOOCH2CH3), 75.5 (C-2), 111.7 (C-3'), 117.3 (C-8), 117.9 (C-5), 119.1 (CN), 120.0 (C-
7), 122.4 (C-4a), 126.6 (C-2'), 128.0 (C-4'), 128.4 (C-6'), 130.4 (C-5'), 132.3 (C-6), 140.7 (C-
1'), 149.2 (C-8a), 154.2 (NHCOOCH2CH3), 181.2 (CHNHCSNHAr). Anal. (C22H24N4O3S)
theoretical: C, 62.24; H, 5.70; N, 13.20; S, 7.55. Found: C, 62.05; H, 5.66; N, 13.33; S, 7.42.
4.1.37. R/S-N-4-Cyanophenyl-N'-(6-ethoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-
1
1-benzopyran-4-yl)thiourea (15h): m.p. : 201-202°C; H NMR (DMSO-d6) δ 1.21 (t, J=7.1
Hz, 3H, CH2CH3), 1,26 (s, 3H, CH3), 1,37 (s, 3H, CH3), 1,76 (m, 1H, 3-H), 2,20 (dd, J=13.1
Hz/6.3 Hz, 1H, 3-H), 4.08 (q, J=7.1 Hz, 2H, CH2CH3), 5.74 (dd, J=16.4 Hz/7.5 Hz, 1H, 4-H),
6,69 (d, J=8.8 Hz, 1H, 8-H), 7,22 (d, J=8.1 Hz, 1H, 7-H), 7,41 (s, 1H, 5-H), 7,76 (d, J=8.9 Hz,
2H, 3’-H et 5’-H), 7,80 (d, J=8.9 Hz, 2H, 2’-H et 6’-H), 8,49 (d, J=8.3 Hz, 1H,
CHNHCSNHAr), 9,41 (s, 1H, NHCOOCH2CH3), 10,00 (s, 1H, CHNHCSNHAr); 13C NMR
(DMSO-d6) δ 14.5 (CH2CH3), 24.2 (CH3), 29.1 (CH3), 38.0 (C-3), 47.2 (C-4), 59.9
(CH2CH3), 75.0 (C-2), 104.9 (C-4'), 116.9 (C-8), 117.3 (C-5), 119.1 (CN), 119.7 (C-7), 121.6
(C-4a), 121.7 (C-3'/C-5'), 131.8 (C-6), 132.8 (C-2'/C-6'), 143.9 (C-1'), 148.8 (C-8a), 153.7
(NHCOOCH2CH3), 180.2 (CHNHCSNHAr). Anal. (C22H24N4O3S) theoretical: C, 62.24; H,
5.70; N, 13.20; S, 7.55. Found: C, 62.03; H, 5.64; N, 13.26; S, 7.36.
4.1.38.
R/S-N-3-Chlorophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-
1
isopropoxycarbonylamino-2H-1-benzopyran-4-yl)thiourea (15i): m.p. : 163-165°C; H
NMR (DMSO-d6) δ 1.23 (dd, J=6.2 Hz/2 Hz, 6H, CH(CH3)2), 1.25 (s, 3H, CH3), 1.36 (s, 3H,
CH3), 1.76 (t, J=12.1 Hz, 1H, 3-H), 2.15 (dd, J=13 Hz/6.3 Hz, 1H, 3-H), 4.85 (hept, J=6.2 Hz,
1H, CH(CH3)2), 5.76 (dd, J=15.7 Hz/8.5 Hz, 1H, 4-H), 6,67 (d, J=8.8 Hz, 1H, 8-H), 7.16 (d,
J=7.3 Hz, 1H, 6'-H), 7.19 (d, J=7 Hz, 1H, 7-H), 7.32-7.35 (m, 2H, 4'-H/5'-H), 7.45 (s, 1H, 2'-
H), 7.74 (s, 1H, 5-H), 8.24 (d, J=7.8 Hz, 1H, CHNHCSNHAr), 9.35 (s, 1H,
NHCOOCH(CH3)2), 9.73 (s, 1H, CHNHCSNHAr); 13C NMR (DMSO-d6) δ 22.0 (CH(CH3)2),
24.2 (CH3), 29.2 (CH3), 38.1 (C-3), 47.3 (C-4), 67.1 (CH(CH3)2), 75.0 (C-2), 116.8 (C-8),
117.3 (C-2'), 119.5 (C-7), 121.6 (C-4'), 121.9 (C-4a), 122.6 (C-5), 123.8 (C-6'), 130.2 (C-5'),
131.8 (C-6), 132.7 (C-3'), 140.7 (C-1'), 148.7 (C-8a), 153.3 (NHCOOCH(CH3)2), 180.5
(CHNHCSNHAr). Anal. (C22H26ClN3O3S) theoretical: C, 58.98; H, 5.85; N, 9.38; S, 7.16.
Found: C, 58.97; H, 5.78; N, 9.52; S, 6.81.
4.1.39.
R/S-N-4-Chlorophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-
24

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1
isopropoxycarbonylamino-2H-1-benzopyran-4-yl)thiourea (15j): m.p. : 184-186°C; H
NMR (DMSO-d6) δ 1.23 (dd, J=6.3 Hz/2.3 Hz, 6H, CH(CH3)2), 1.24 (s, 3H, CH3), 1.36 (s,
3H, CH3), 1.74 (t, J=12.1 Hz, 1H, 3-H), 2.15 (dd, J=13 Hz/6.2 Hz, 1H, 3-H), 4.85 (hept, J=6.2
Hz, 1H, CH(CH3)2), 5.75 (dd, J=15.9 Hz/8.8 Hz, 1H, 4-H), 6,67 (d, J=8.8 Hz, 1H, 8-H), 7.19
(d, J=7 Hz, 1H, 7-H), 7.36 (d, J=8.8 Hz , 2H, 2'-H/6'-H), 7.42 (s, 1H, 5-H), 7.52 (d, J=8.8 Hz,
2H, 3'-H/5'-H), 8.15 (d, J=8.2 Hz, 1H, CHNHCSNHAr), 9.35 (s, 1H, NHCOOCH(CH3)2) ,
9.68 (s, 1H, CHNHCSNHAr); 13C NMR (DMSO-d6) δ 22.0 (CH(CH3)2), 24.2 (CH3), 29.2
(CH3), 38.1 (C-3), 47.3 (C-4), 67.1 (CH(CH3)2), 75.0 (C-2), 116.8 (C-8), 117.4 (C-5), 119.5
(C-7), 122.0 (C-4a), 125.0 (C-3'/C-5'), 128.0 (C-4'), 128.4 (C-2'/C-6'), 131.8 (C-6), 138.1 (C-
1'), 148.7 (C-8a), 153.3 (NHCOOCH(CH3)2), 180.5 (CHNHCSNHAr). Anal.
(C22H26ClN3O3S) theoretical: C, 58.98; H, 5.85; N, 9.38; S, 7.16. Found: C, 58.83; H, 5.79; N,
9.58; S, 6.85.
4.1.40. R/S-N-3-Cyanophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-isopropoxycarbonylamino-
1
2H-1-benzopyran-4-yl)thiourea (15k): m.p.: 170-172°C; H NMR (DMSO-d6) δ 1.23 (d,
J=6.2 Hz, 6H, NHCOOCH(CH3)2), 1.25 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.77 (t, J=12 Hz,
1H, 3-H), 2.16 (dd, J=13 Hz/6 Hz, 3-H), 4.86 (dt, J=12.3 Hz/6 Hz, 1H, NHCOOCH(CH3)2),
5.77 (bs, 1H, 4-H), 6.67 (d, J=8.7 Hz, 1H, 8-H), 7.19 (d, J=8.2 Hz, 1H, 7-H), 7.47 (s, 1H, 5-
H), 7.50-7.56 (m, 2H, 4'-H/5'-H), 7.75 (d, J=7.7 Hz, 1H, 6'-H), 8.06 (s, 1H, 2'-H), 8,39 (d,
J=6.6 Hz, 1H, CHNHCSNHAr), 9.32 (s, 1H, NHCOOCH(CH3)2), 9.87 (s, 1H,
CHNHCSNHAr); 13C NMR (DMSO-d6) δ
NHCOOCH(CH3
, 24.7 (CH3),
29.7 (CH3), 38.6 (C-3), 47.8 (C-4), 67.6 (NHCOOCH(CH3)2), 75.5 (C-2), 111.7 (C-3'), 117.3
(C-8), 117.8 (C-5), 119.1 (CN), 120.1 (C-7), 122.3 (C-4a), 126.6 (C-2'), 128.0 (C-4'), 128.4
(C-6'), 130.4 (C-5'), 132.4 (C-6), 140.7 (C-1'), 149.1 (C-8a), 153.8 (NHCOOCH(CH3)2),
181.2 (CHNHCSNHAr). Anal. (C23H26N4O3S) theoretical: C, 62.99; H, 5.98; N, 12.78; S,
7.31. Found: C, 62.73; H, 5.98; N, 12.87; S, 7.38.
4.1.41. R/S-N-4-Cyanophenyl-N'-(3,4-dihydro-2,2-dimethyl-6-isopropoxycarbonylamino-
1
2H-1-benzopyran-4-yl)thiourea (15l): m.p. : 200-202°C; H NMR (DMSO-d6) δ 1.22 (dd,
J=6.2 Hz/2.5 Hz, 6H, CH(CH3)2), 1,25 (s, 3H, CH3), 1,37 (s, 3H, CH3), 1,75 (m, 1H, 3-H),
2,20 (dd, J=13.0 Hz/6.3 Hz, 1H, 3-H), 4.85 (hept, J=6.3 Hz, 1H, CH(CH3)2), 5.74 (dd, J=16.9
Hz/8.1 Hz, 1H, 4-H), 6,69 (d, J=8.8 Hz, 1H, 8-H), 7,20 (d, J=7.5 Hz, 1H, 7-H), 7,43 (s, 1H, 5-
H), 7,76 (d, J=8.8 Hz, 2H, 3’-H et 5’-H), 7,80 (d, J=8.8 Hz, 2H, 2’-H et 6’-H), 8,49 (d, J=8.2
25

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Hz, 1H, CHNHCSNHAr), 9,35 (s, 1H, NHCOOCH(CH3)2), 10,00 (s, 1H, CHNHCSNHAr);
13C NMR (DMSO-d6) δ 22.0 (CH(CH3)2), 24.2 (CH3), 29.1 (CH3), 38.0 (C-3), 47.2 (C-4),
67.1 (CH(CH3)2), 74.9 (C-2), 104.9 (C-4'), 116.9 (C-8), 117.5 (C-5), 119.1 (CN), 119.8 (C-7),
121.6 (C-4a), 121.7 (C-3'/C-5'), 131.8 (C-6), 132.8 (C-2'/C-6'), 144.0 (C-1'), 148.7 (C-8a),
153.3 (NHCOOCH(CH3)2), 180.2 (CHNHCSNHAr). Anal. (C23H26N4O3S) theoretical: C,
62.99; H, 5.98; N, 12.78; S, 7.31. Found: C, 63.07; H, 5.93; N, 12.84; S, 6.97.
4.2. Biological assays
(±)-Cromakalim (Tocris, UK), diazoxide (Sigma Chemical Co, USA) and verapamil (Sigma
Chemical Co, USA) were used as reference compounds. All experiments were performed
with aortae or pancreatic islets isolated from adult fed Wistar Rats (Charles River
Laboratories, Belgium). The laboratory animal care was approved by the local ethic's
committee of the Université Libre de Bruxelles.
4.2.1. Measurement of insulin secretion from incubated rat pancreatic islets
The method used to measure insulin release from incubated rat pancreatic islets was
previously described [27,34,40].
4.2.2. Measurement of myorelaxant activity on rat aorta rings
The method used to measure the myorelaxant effect of drugs on KCl-precontracted rat aortic
rings was previously described [34,38,40].
4.2.3. Measurements of 86Rb, 45Ca outflow and insulin release from perifused rat
pancreatic islets
The experimental conditions previously reported for measuring 86Rb, 45Ca outflow and insulin
release from perifused rat pancreatic islets were applied to compound 14o [27,40].
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4.2.4. Measurement of cytosolic Ca2+ concentration from isolated rat pancreatic islet
cells
The experimental conditions previously reported for measuring the cytosolic Ca2+
concentration (fura-2 fluorescence) from single rat pancreatic islet cells were applied to
compound 14o [27,40].
Supplementary data
1
Examples of H and 13C NMR spectra of target compounds from the two series (ureas -
thioureas) and bearing the same substituents (14g, 14h versus 15g, 15h; 14o, 14p versus 18,
9).
Author information
Corresponding Author
*Phone: + 32 4 366 43 65. Fax : + 32 4 366 43 62. E-mail : b.pirotte@ulg.ac.be
Acknowledgements
This study was supported in part by grants from the “National Fund for Scientific Research”
(F.N.R.S., Belgium) from which P. de Tullio is a Senior Research Associate and P. Lebrun is
a Research Director. The authors gratefully acknowledge the technical assistance of S.
Counerotte, F. Leleux, A.-M. Vanbellinghen and A. Van Praet.
Abbreviations used
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ADP: adenosine diphosphate; ATP: adenosine triphosphate; DCVC: dry column vacuum
chromatography; DMSO: dimethyl sulfoxide; EC50: half maximal effective concentration;
IC50: half maximal inhibitory concentration; KATP channel: ATP-sensitive potassium channel;
Kir: inwardly-rectifying potassium channel; NMR: nuclear magnetic resonance; PCO:
potassium channel opener; SUR: sulfonylurea receptor; TLC: thin layer chromatography;
TMS: tetramethylsilane.
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