2,2-Dimethyl-3,4-dihydro-2H-1,4-benzoxazines as isosteres of 2,2-dimethylchromans acting as inhibitors of insulin release and vascular smooth muscle relaxants

Article abstract of DOI:10.1039/c8md00593a

The present study describes the synthesis and biological evaluation of 4-phenylureido/thioureido-substituted 2,2-dimethyl-3,4-dihydro-2H-1,4-benzoxazines as isosteres of corresponding 2,2-dimethylchromans reported to be pancreatic β-cell K_ATP channel openers. The benzoxazines were found to be less active as inhibitors of the glucose-induced insulin release than their corresponding chromans, while the myorelaxant activity of some 4-arylureido-substituted benzoxazines was more pronounced than that exhibited by their chroman counterparts. The myorelaxant activity of the most potent benzoxazine 8e was further characterized on rat aortic rings precontracted by 30 mM KCl in the presence of glibenclamide (10 μM) or precontracted by 80 mM extracellular KCl. Our findings indicate that, on vascular smooth muscle cells, the benzoxazine 8e mainly behaved as a calcium entry blocker.

Full text of DOI:10.1039/c8md00593a

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RESEARCH ARTICLE
2
,2-Dimethyl-3,4-dihydro-2H-1,4-benzoxazines as isosteres of
,2-dimethylchromans acting as inhibitors of insulin release and
vascular smooth muscle relaxants
2
Received 00th January 20xx,
Accepted 00th January 20xx
a
a,b
a
b
DOI: 10.1039/x0xx00000x
Bernard Pirotte,* Xavier Florence, Eric Goffin, and Philippe Lebrun
www.rsc.org/
The present study describes the synthesis and biological evaluation of 4-phenylureido/thioureido-substituted 2,2-
dimethyl-3,4-dihydro-2H-1,4-benzoxazines as isosteres of corresponding 2,2-dimethylchromans reported to be pancreatic

-cell KATP channel openers. The benzoxazines were found to be less active as inhibitors of the glucose-induced insulin
release than their corresponding chromans, while the myorelaxant activity of some 4-arylureido-substituted benzoxazines
was more pronounced than that exhibited by their chroman counterparts. The myorelaxant activity of the most potent
benzoxazine 8e was further characterized on rat aortic rings precontracted by 30 mM KCl in the presence of glibenclamide
(10 µM) or precontracted by 80 mM extracellular KCl. Our findings indicate that, on vascular smooth muscle cells, the
benzoxazine 8e mainly behaved as a calcium entry blocker.
mainly examined as antihypertensive agents or potential
1
5
1
. Introduction
bronchodilators.
A typical example of ATP-sensitive potassium channel opener (PCO)
is the 2,2-dimethylchroman (±)-cromakalim [reported in Figure 1 as
the levorotatory isomer levcromakalim (1)], which is known to be
much more potent on the smooth muscle SUR2B-type than on the
pancreatic endocrine SUR1-type KATP channel.
Although the configuration of the 3- and 4-positions of the chroman
ring of cromakalim were found to be critical for myorelaxant activity
ATP-sensitive potassium channels (KATP channels) located at the
plasma membrane of excitable cells are ion channels linking cell
metabolism to membrane excitability.
1
-3
16
KATP channels are involved in multiple physiological processes,
among which the control of insulin release from pancreatic -cells
4-6
and the modulation of vascular smooth muscle tone.
The
(
levcromakalim was the most active among four possible isomers)
structure of those channels at the molecular level has been
reported to be octameric complexes of four pore-forming inwardly
1
7
,
many other examples of PCOs structurally related to cromakalim
+
have been developed; among which compounds devoid of chiral
centers at the 3- and 4-positions. The cromakalim analogue Ro31-
rectifying K (K 6.x) channels (K 6.1 or K 6.2) and four regulatory
ir
ir
ir
7-9
sulfonylurea receptor (SURx) subunits (SUR1, SUR2A or SUR2B).
6
930 (2, Figure 1) is a PCO devoid of chiral carbon atoms, indicating
Several modulators of the KATP channels are currently used in
clinical practice (i.e. hypoglycemic sulfonylureas as oral antidiabetic
that a planar sp2 configuration at the 4-position can be tolerated
18
10
and doesn’t negatively impact its pharmacological profile.
drugs) and original KATP channel openers are expected to become
promising therapeutic agents provided that they are able to exert
Moreover, this planar carbon atom at the 4-position can be
replaced by a nitrogen atom giving rise to potent PCOs belonging to
11
high potency and selectivity for a single KATP channel subtype.
Selective openers of the pancreatic -cell KATP channel subtype
SUR1/Kir6.2) have been proposed for the prevention and/or
management of type 1 - type 2 diabetes, congenital hyperinsulinism
2
,2-dimethyl-3,4-dihydro-2H-1,4-benzoxazines, such as YM934 (3,
19
20
Figure 1) tightly related to 2, and such as compound 4 (Figure 1)
(
bearing an alkylamide residue at the 4-position. This series of
heterocyclic compounds may be viewed as isosteres of 2,2-
dimethylchromans for which the chiral center at the 4-position has
been suppressed. Incidentally, 2,2-dimethyl-3,4-dihydro-2H-1,4-
benzothiazine isosteres (replacement of the oxygen atom at the 1-
position with a sulfur atom) such as compound 5 (Figure 1) were
1
2-14
and treatment of insulinoma.
Likewise, selective openers of the
smooth muscle cell KATP channel subtype (SUR2B/Kir6.1) have been
reported to be among the most myorelaxant K channel openers
ATP
2
1
ever discovered.
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Fig. 1 Chemical structure of KATP channel openers belonging to 2,2-dimethylchromans (1, 6, 7), 2,2-dimethylchromens (2), 2,2-dimethyl-
,4-dihydro-2H-1,4-benzoxazines (3, 4) and 2,2-dimethyl-3,4-dihydro-2H-1,4-benzothiazines (5).
In previous works, we described novel series of cromakalim ring and to the six protons of the two methyl groups linked at the 2-
analogues as potential K channel openers belonging to 2,2- position for the arylurea compounds 8a-d logically appeared as
3
ATP
2
2-30
dimethylchromans.
The introduction of a phenylurea or a singlets (around 3.27 ppm for –CH - and around 1.37 ppm for the
2
phenylthiourea residue at the 4-position of the chroman ring led to two -CH ), the picture was clearly different for their arylthiourea
3
compounds such as 6 and 7 (Figure 1), which, in contrast to counterparts 8e-h. In the latter case, the two methylenic protons at
cromakalim, were found to exert a strong opening activity on the the 3-position were differencied and a coupling was observed
22,26
pancreatic SUR1-type KATP channels.
(doublets at around 3.15 ppm and 3.38 ppm). Moreover, the two
The present study was an attempt to examine the impact of the methyl groups at the 2-position appeared at two different chemical
introduction of such phenylurea/thiourea moieties, at the 4- shifts (singlets around 1.34 and 1.48). It is tempting to speculate
position of 2,2-dimethyl-3,4-dihydro-2H-1,4-benzoxazines (Figure that, for compounds 8e-h, the thiourea group adopted a permanent
2
), on insulin secreting cells activity and vascular smooth muscle position in the space that differently influenced the electronic
tone. Both biological responses could reflect a potential interaction environment of the two methylenic protons and the two methyl
with KATP channels or voltage-sensitive calcium channels.
groups. The higher length of the C=S bound, compared to the C=O,
bound should be responsible for a lack of free rotation around the
N-N single bound.
2
. Results and discussion
2
.2. Biological results
2
.1. Chemistry
The biological effects of the newly synthesized 2,2-dimethyl-3,4-
dihydro-2H-1,4-benzoxazines 8a-h, of the corresponding 2,2-
dimethylchromans 6, 7, 14a-d and the reference compounds (±)-
cromakalim and diazoxide, on the glucose-induced insulin release
from rat pancreatic islets and on the contractile activity of K -
depolarized rat aorta rings are reported in Table 1.
The synthesis of the target compounds (8a-h) is described in
scheme 1. Starting from 2-amino-4-chlorophenol (9), ring closure
reaction with 2-bromo-2-methylpropionyl bromide led to the 2,2-
dimethylbenzoxazine-3-one (10). The amide function of 10 was
reduced by means of borane-tetrahydrofuran complex to provide
the corresponding secondary amine (11), which reacted with
nitrous acid to be converted into the 4-nitroso-substituted 2,2-
dimethyl-3,4-dihydro-2H-1,4-benzoxazine (12). Reduction of the
nitroso group with zinc metal in acetic acid gave access to the 4-
amino-substituted benzoxazine (13). The latter reacted with the
appropriate phenyl isocyanate or phenyl isothiocyanate to provide
the corresponding 4-phenyl(thio)ureido-substituted 2,2-dimethyl-
+
The benzoxazines were systematically found to be less active than
their corresponding chromans on the glucose-induced insulin
release, while the myorelaxant activity on rat aorta was found to be
more pronounced with the 4-arylurea-substituted benzoxazines
than with the corresponding 4-arylurea-substituted chromans (see
8g and 8h versus 6 and 14d). None of them, however, expressed
the myorelaxant properties of the 2,2-dimethylchroman reference
compound (±)-cromakalim (Table 1).
3
,4-dihydro-2H-1,4-benzoxazines (8a-h).
1
An interesting observation was conducted with the H-NMR data
obtained with the 4-arylurea-substituted compounds 8a-d
compared to their 4-arylthiourea-substituted counterparts 8e-h
Looking at the activity on insulin secretion (10 µM), it was observed
that, for thioureas 8a and 8b, the para position for the CN
substituent was preferred to the meta position, while the situation
was found to be different in the urea series (see 8e and 8f).
(
see experimental section 3.1). Although the signals attributed to
the two methylenic protons at the 2-position of the benzoxazine
2
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Fig. 2 General formula of the newly synthesized 4-arylureido/thioureido-substituted 6-chloro-2,2-dimethyl-3,4-dihydro-2H-1,4-
benzoxazines (8).
By comparing the myorelaxant activity of 4-arylurea-substituted presence of glibenclamide (10 µM) or precontracted by 80 mM
benzoxazines 8e-h with
their
4-arylthiourea-substituted extracellular KCl.
counterparts 8a-d, it was clearly observed that the arylureas were
more potent than the corresponding arylthioureas. Such a
comparison was less obvious on the pancreatic endocrine tissue.
The concomitant presence of the KATP channel blocker
glibenclamide (10 µM) in the bathing solution failed to affect the
myorelaxant properties of 8e (P > 0.05, Table 2); as it can be
2
9,37
The most active 2,2-dimethylbenzoxazine on rat pancreatic islets observed with calcium entry blockers such as verapamil.
By
was the 4-cyanophenylthiourea-substituted compound 8b, which contrast, the KATP channel blocker glibenclamide induced a marked
was the isosteric analogue of the most active 4- reduction in the vasorelaxant response to the potassium channel
cyanophenylthiourea-substituted 2,2-dimethylchroman 7 (Table 1). opener (±)-cromakalim (Table 2).
Both compounds were clearly more potent on the insulin releasing
When the aorta rings were precontracted by 80 mM KCl, the
vasorelaxant potency of compound 8e was not significantly affected
process than the reference compound diazoxide (Table 1).
Although most 2,2-dimethylchromans reported in table 1 expressed (P > 0.05); as previously reported for the calcium entry blocker
2
9,37
some tissue selectivity, being more potent on the endocrine tissue verapamil.
By contrast, and under the same experimental
than on the smooth muscle tissue, this feature did not apply for the conditions, the myorelaxant effect of the potassium channel opener
majority of corresponding 2,2-dimethyl-3,4-dihydro-2H-1,4- (±)-cromakalim was drastically reduced (Table 2). On the whole,
benzoxazines
(Table
1).
The
4-arylthiourea-substituted these findings indicate that, on vascular smooth muscle cells, 8e
benzoxazines 8a and 8b expressed some pancreatic endocrine mainly behaved as a calcium entry blocker.
tissue selectivity, while the corresponding 4-arylurea-substituted
analogues 8e and 8f were found to be active on both tissues in the
same range of concentrations (Table 1). Compounds 8g and 8h
were also equipotent on pancreatic -cells and vascular cells.
3
. Experimental section
3.1. Chemistry
A critical information can be deduced from the virtual logP value
(
‘average’ logP value) calculated for each compound (Table 1). As
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
expected, the isosteric replacement of a –CH- moiety by a –N- atom
was responsible for an increase in the hydrophilicity. Likewise, the
thiourea derivatives were found to be more lipophilic than the
corresponding urea derivatives in both series of compounds.
1
13
a Bruker Avance 500 spectrometer ( H: 500 MHz; C: 125 MHz)
Finally, regarding the perspective of development of new
therapeutic drugs, the newly synthesized benzoxazines appeared to
exhibit a more favorable hydrophilic/lipophilic balance compared to
using DMSO-d as solvent and tetramethylsilane (TMS) as internal
6
standard; chemical shifts are reported in δ values (ppm) relative to
internal TMS. The abbreviation s = singlet, d = doublet, t = triplet, q
the previously described chromans.
Indeed, the 2,2-
=
quadruplet, m = multiplet and bs = broad signal are used
dimethylchromans previously synthesized exhibited an estimated
average logP higher than 4, and sometimes close to 5, being at the
limit of the criterion defined by the Lipinski’s “rule of five” for
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
31
acceptable oral bioavailability.
each final 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).
In order to decipher the mechanism of action of the most potent
myorelaxant benzoxazine 8e, its vasorelaxant activity was further
characterized on rat aortic rings precontracted by 30 mM KCl in the
3.1.1. 6-Chloro-2,2-dimethyl-2H-benzoxazin-3(4H)-one (10)
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Scheme 1: Synthetic pathway to 4-phenyl(thio)ureido-substituted 6-chloro-2,2-dimethyl-3,4-dihydro-2H-1,4-benzoxazines 8.
Reagents: i: 1. Br-CO-C(CH ) -Br, Na CO , CHCl , H O; 2. K CO , DMF, 80°C; ii : BH , THF; iii NaNO , AcOH; iv : Zn, AcOH; v : 3-/4-chloro-
3
2
2
3
3
2
2
3
3
2
/
3-/4-cyanophenyl iso(thio)cyanate, CH Cl .
2 2
The title compound was obtained in two steps. Firstly, 2-amino-4- 3.1.3. 6-Chloro-3,4-dihydro-2,2-dimethyl-4-nitroso-2H-
chlorophenol (9) (14.36 g, 0.1 mol) was suspended in a mixture of benzoxazine (12)
CHCl (600 mL) in a saturated solution of sodium carbonate (350 6-Chloro-2,2-dimethyl-3,4-dihydro-2H-benzoxazine (11) (5.79 g,
3
mL). The medium was then cooled to 0 °C and 2-bromo-2- 9,29 mmol) was dissolved in methanol and acetic acid (3.62 mL,
methylpropionyl bromide (18.54 mL, 0.15 mol) was added 63.22 mmol) was added. The solution was cooled to 0 °C and an
dropwise. The mixture was stirred 4 hours at room temperature. aqueous solution of NaNO (3.51 g, 50.82 mmol) was cautiously
2
The organic layer was decanted, washed with water, dried over added. The mixture was stirred overnight at room temperature.
MgSO and evaporated under reduced pressure.
After completion of the reaction, the mixture was adjusted to
4
Secondly, the crude product was solubilized in DMF (250 mL) and pH=10 with sodium hydroxide 10%. The title compound was
K CO (13.82 g, 0.1 mol) was added. The mixture was then stirred at precipitated by water, collected by filtration, washed by water and
2
3
-1 1
8
0 °C during 3 hours. At the end of the reaction, water (250 mL) was dried (5.98 g, 78 %): mp: 65-67 °C; IR (KBr) υ: 1494 (N=O) cm ; H
added. The resulting precipitate was collected by filtration and the NMR (DMSO-d , 500 MHz): δ: 1.27 (s, 6H, CH ), 3.95 (s, 2H, CH ),
6
3
2
filtrate was extracted by CHCl . The organic layer was dried over 7.08 (d, 1H, 8-H), 7.32 (dd, 1H, 7-H), 7.97 (d, 1H, 5-H). Anal.
3
MgSO and evaporated under reduced pressure. The precipitate (C H ClN O ) theoretical: C, 52.99; H, 4.89; N, 12.36. Found: C,
4
10 11
2 2
and the crude material were crystallized in boiling ether. The title 52.82; H, 4.89; N, 12.29.
compound was collected by filtration, washed by ether and dried
(
16.93g, 80 %): mp: 164-166 °C; IR (KBr) υ: 1687 (C=O), 3132 (N-H) 3.1.4. 4-Amino-6-chloro-3,4-dihydro-2,2-dimethyl-2H-benzoxazine
-1 1
cm ; H NMR (DMSO-d , 500 MHz): δ: 1.40 (s, 6H, CH ), 6.89 (s, 1H, (13)
6
3
7
-H), 6.96 (s, 2H, 5-H et 8-H), 10.73 (s, 1H, -NH ). Anal. (C H ClNO ) 6-Chloro-2,2-dimethyl-4-nitroso-3,4-dihydro-2H-benzoxazine (12) (5
2 10 10 2
theoretical: C, 56.75; H, 4.76; N, 6.62. Found: C, 56.94; H, 4.68; N, g, 22,06 mmol) was dissolved in ice-cooled methanol (140 mL).
6
.58.
Acetic acid (24 mL, 419 mmol) and zinc powder (7.07 g, 108 mmol)
were added. The solution was stirred 15 minutes at room
temperature. When reaction ended, the mixture was adjusted to
3.1.2. 6-Chloro-3,4-dihydro-2,2-dimethyl-2H-benzoxazine (11)
A borane tetrahydrofuran complex solution 1M (106 mL, 106 mmol) pH=10 with concentrated ammonia. The title compound was
was added to a ice-cooled solution of 6-chloro-2,2-dimethyl-2H- extracted by ethyl acetate. The organic layers were pooled, dried
benzoxazin-3(4H)-one (10) (15 g, 47.24 mmol) in anhydrous over MgSO4 and evaporated under reduced pressure. The crude
tetrahydrofuran. The mixture was stirred overnight at room product was purified by chromatography on a silica gel 60 column
temperature. Water was then added to quench the reaction. The eluted by methylene chloride. Pure fractions were pooled and the
title compound was extracted by ethyl acetate. The organic layer solvent was evaporated under reduced pressure (3.57 g, 76 %): mp:
-1 1
was dried over MgSO and was evaporated under reduced pressure. 79-81 °C; IR (KBr) υ: 3347 (N-H ) cm ; H NMR (DMSO-d , 500 MHz):
4
2
6
The crude product was used in the next step without further δ: 1.27 (s, 6H, CH ), 3.08 (s, 2H, CH ), 4.46 (s, 2H, NH ), 6.55-6.61 (m,
3
2
2
-1 1
purification (10.93 g, 78 %): IR (KBr) υ: 3385 (N-H) cm ; H NMR 2H, 7-H et 8-H), 7.97 (d, 1H, 5-H). Anal. (C H ClN O) theoretical: C,
10
13
2
(
DMSO-d , 500 MHz): δ: 1.22 (s, 6H, CH ), 3.00 (s, 2H, CH ), 6.47 (dd, 56.48; H, 6.16; N, 13.17. Found: C, 56.55; H, 6.02; N, 12.92.
6 3 2
1
H, 7-H), 6.59 – 6.61 (m, 2H, 5-H et 8-H), 7.06 (bs, 1H, -NH).
3.1.5. 6-Chloro-4-(3-cyanophenylaminothiocarbonylamino)-3,4-
dihydro-2,2-dimethyl-2H-benzoxazine (8a)
4
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Table 1: Estimated logP (Average logP) values and effects of original 4-phenyl(thio)ureido-substituted 6-chloro-2,2-dimethyl-3,4-
DOI: 10.1039/C8MD00593A
dihydro-2H-1,4-benzoxazines, 4-phenyl(thio)ureido-substituted 6-chloro-2,2-dimethyl-3,4-dihydro-2H-chromans, (±)-cromakalim and
+
diazoxide on the glucose-induced insulin release from rat pancreatic islets and on the contractile activity of K depolarized rat aorta
rings.
b
Residual insulin secretion (%)
Myorelaxant activity
1
2
a
Compounds
X
R
R
Average log P
c
1
0 µM
1 µM
EC (µM)
5
0
8
8
8
a
b
c
S
S
S
S
CN
Cl
3.83
3.84
4.71
4.71
27.0 ± 1.3 (31)
10.8 ± 0.6 (23)
71.8 ± 3.8 (31)
81.4 ± 4.7 (30)
83.1 ± 4.8 (23)
73.3 ± 3.0 (22)
>30 (4)
>30 (4)
>30 (6)
>30 (6)
CN
Cl
-
-
8d
8
8
8
e
f
g
O
O
O
O
CN
Cl
3.42
3.43
4.31
4.32
35.7 ± 2.5 (14)
43.1 ± 2.8 (16)
46.0 ± 3.0 (15)
56.2 ± 3.5 (16)
79.5 ± 4.6 (15)
79.0 ± 5.4 (32)
92.5 ± 3.7 (23)
89.5 ± 5.1 (21)
1.9 ± 0.6 (5)
3.7 ± 1.0 (4)
9.9 ± 2.7 (7)
13.3 ± 2.6 (4)
CN
Cl
8h
d
d
d
1
4a
7
S
S
S
S
O
O
CN
Cl
4.04
4.04
4.90
4.90
4.55
4.55
15.8 ± 0.9 (18)
10.6 ± 0.7 (24)
16.2 ± 1.6 (22)
66.2 ± 2.5 (31)
47.8 ± 2.8 (29)
89.5 ± 3.7 (23)
87.9 ± 3.0 (16)
0.60 ± 0.06 (4)
d
e
d
e
e
f
d
CN
Cl
>10.0 (4)
>10.0 (4)
e
14b
e
e
1
6
4c
8.9 ± 0.7 (15)
>10.0 (5)
f
f
Cl
25.0ꢀ±ꢀ1.3ꢀ(24)
34.6ꢀ±ꢀ1.9ꢀ(21)
81.0ꢀ±ꢀ4.0ꢀ(23)
85.4ꢀ±ꢀ5.4ꢀ(22)
>30ꢀ(4)
f
f
f
14d
Cl
>300ꢀ(4)
g
g
(
±)-cromakalim
diazoxide
n.d.
n.d.
96.4 ± 6.5 (16)
80.8 ± 3.7 (32)
-
-
0.13 ± 0.01 (7)
26.1 ± 2.9 (4)
g
g
a
b
Average logP values calculated according to the ALOGPS 2.1 program (ref. 25). Percentage of residual insulin release from rat
c
pancreatic islets incubated in the presence of 16.7 mM glucose (mean ± SEM (n)). EC : drug concentration giving 50% relaxation of the
3
5
0
d
e
0 mM KCl-induced contraction of rat aorta rings (mean ± SEM (n)). Published compounds and results (ref. 22). Published compounds
f
g
and results (ref. 27). Published compounds and results (ref. 26). Published results (ref. 37).
13
3
-Cyanophenyl isothiocyanate (415 mg, 2.59 mmol) was added to a 1H, NH), 10.14 (s, 1H, NH). C NMR (DMSO-d , 125 MHz): δ: 24.3
6
solution
of
4-amino-6-chloro-3,4-dihydro-2,2-dimethyl-2H- (CH ), 26.0 (CH ), 57.8 (CH ), 75.2 (C-2), 110.7 (C-3’), 114.3 (C-5),
3 3 2
benzoxazine (13) (500 mg, 2.35 mmol) in methylene chloride (5 mL). 118.0 (C-8), 118.5 (CN), 121.3 (C-7), 124.0 (C-6), 128.2 (C-2’), 128.6
The solution was stirred 24 hours at room temperature. The (C-6’), 129.4 (C-5’), 130.0 (C-4’), 134.8 (C-4a), 139.9 (C-1’), 142.6 (C-
product was collected by filtration, washed with n-hexane an dried 8a), 180.3 (CS). Anal. (C H ClN OS) theoretical: C, 57.98; H, 4.60;
18
17
4
(
3
1
560 mg, 64 %): mp: 202-204 °C; IR (KBr) υ: 1543 (C=S), 2228 (C≡N), N, 15.03; S, 8.60. Found: C, 57.86; H, 4.53; N, 15.15; S, 8.56.
300 (N-H) cm ; H NMR (DMSO-d , 500 MHz): δ: 1.34 (s, 3H, CH ),
6 3
-1 1
.48 (s, CH ), 3.16 (d, J = 11.0 Hz, 1H, CH ), 3.39 (d, J = 10.9 Hz, 1H,
3
2
CH ), 6.77 (d, J = 1.3 Hz, 1H, 5-H), 6.79 (d, J = 8.6 Hz, 1H, 8-H), 6.86 3.1.6. 6-Chloro-4-(4-cyanophenylaminothiocarbonylamino)-3,4-
2
(
d, J = 7.6 Hz, 1H, 7-H), 7.55 (t, J = 7.9 Hz, 1H, 5’-H), 7.63 (d, J = 7.6 dihydro-2,2-dimethyl-2H-benzoxazine (8b)
Hz, 1H, 6’-H), 7.94 (d, J = 7.8 Hz, 1H, 4’-H), 8.13 (s, 1H, 2’-H), 9.84 (s,
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Table 2. Myorelaxant effects of 8e and (±)-cromakalim on 30 mM or 80 mM KCl-precontracted rat aorta rings incubated in the absence
DOI: 10.1039/C8MD00593A
or presence of glibenclamide.
Myorelaxant activity
a
EC (µM)
5
0
Compounds
KCl 30 mM
KCl 30 mM
+
KCl 80 mM
b
1
0 µM Gliben.
3
3
.0 ± 0.7 (4)
.1 ± 0.5 (6)
3.9 ± 0.5 (8)
8e
5.0 ± 1.2 (6)
c
c
0
0
.22 ± 0.07 (6)
.17 ± 0.01 (4)
28.8 ± 6.0 (5)
(±)-Cromakalim
c
c
1
37.7 ± 10.4 (10)
a
b
c
EC : drug concentration giving 50% relaxation (mean ± SEM (n)). Gliben.: glibenclamide. Published results (ref. 37).
50
4
-Cyanophenyl isothiocyanate (415 mg, 2.59 mmol) was added to a (C-2), 114.2 (C-5), 118.0 (C-8), 121.2 (C-7), 123.5 (C-6’), 124.0 (C-6),
solution of 4-amino-6-chloro-3,4-dihydro-2,2-dimethyl-2H- 124.5 (C-2’), 124.8 (C-4’), 129.6 (C-5’), 132.1 (C-3’), 134.9 (C-4a),
benzoxazine (13) (500 mg, 2.35 mmol) in methylene chloride (5 mL). 140.4 (C-1’), 142.6 (C-8a), 180.1 (CS). Anal. (C H Cl N OS)
17
17
2 3
The solution was refluxed for 1 hour. Thereafter, the mixture was theoretical: C, 67.63; H, 5.68; N, 12.45; S, 9.50. Found: C, 67.41; H,
cooled to room temperature. The resulting precipitate was 5.69; N, 12.36; S, 9.69.
collected by filtration, washed with n-hexane and dried (641 mg,
7
3 %): mp: 212-214 °C; IR (KBr) υ: 1541 (C=S), 2228 (C≡N), 3296 (N-
-1 1
3
.1.8. 6-Chloro-4-(4-chlorophenylaminothiocarbonylamino)-3,4-
H) cm ; H NMR (DMSO-d , 500 MHz): δ: 1.33 (s, 3H, CH ), 1.48 (s,
CH ), 3.15 (d, J=11.0 Hz, 1H, CH ), 3.38 (d, J = 10.7 Hz, 1H, CH ), 6.75
6
3
dihydro-2,2-dimethyl-2H-benzoxazine (8d)
The title compound was obtained as described for 14, starting from
1
3
2
2
(
s, 1H, 5-H), 6.79 (d, J = 8.4 Hz, 1H, 8-H), 6.87 (d, J = 7.7 Hz, 1H, 7-H),
3 (500 mg, 2.35 mmol) and 4-chlorophenyl isothiocyanate (439
7
9
2
5
6
.80 (d, J = 8.7 Hz, 2H, 3’-H/5’-H), 7.93 (d, J = 7.9 Hz, 2H, 2’-H/6’-H),
13
mg, 2.59 mmol) (530 mg, 59 %): mp: 205-207 °C; IR (KBr) υ: 1533
.91 (s, 1H, NH), 10.20 (s, 1H, NH). C NMR (DMSO-d , 125 MHz): δ:
6
-1 1
(
C=S), 3268 (N-H) cm ; H NMR (DMSO-d , 500 MHz): δ: 1.33 (s, 3H,
6
4.2 (CH ), 26.0 (CH ), 57.7 (CH ), 75.2 (C-2), 106.6 (C-4’), 114.3 (C-
3
3
2
CH ), 1.47 (s, CH ), 3.16 (d, J = 11.0 Hz, 1H, CH ), 3.38 (d, J = 10.3 Hz,
1
6
), 118.0 (C-8), 118.9 (CN), 121.4 (C-7), 124.0 (C-6), 124.2 (C-2’/C-
’), 132.3 (C-3’/C-5’), 134.8 (C-4a), 142.7 (C-8a), 143.3 (C-1’), 179.8
3
3
2
H, CH ), 6.75 (s, 1H, 5-H), 6.78 (d, J = 8.5 Hz, 1H, 8-H), 6.85 (d, J =
2
.6 Hz, 1H, 7-H), 7.40 (d, J = 8.8 Hz, 2H, 3’-H/5’-H), 7.62 (d, J = 8.7
(
CS). Anal. (C H ClN OS) theoretical: C, 57.98; H, 4.60; N, 15.03; S,
18 17 4
13
Hz, 2H, 2’-H/6’-H), 9.71 (s, 1H, NH), 9.99 (s, 1H, NH). C NMR
8
.60. Found: C, 57.87; H, 4.53; N, 15.16; S, 8.56.
(
DMSO-d , 125 MHz): δ: 24.3 (CH ), 26.0 (CH ), 57.7 (CH ), 75.1 (C-
6 3 3 2
2
6
), 114.1 (C-5), 118.0 (C-8), 121.2 (C-7), 124.0 (C-6), 126.8 (C-2’/C-
’), 127.9 (C-3’/C-5’), 129.1 (C-4’), 134.9 (C-4a), 137.9 (C-1’), 142.6
3
.1.7. 6-Chloro-4-(3-chlorophenylaminothiocarbonylamino)-3,4-
dihydro-2,2-dimethyl-2H-benzoxazine (8c)
The title compound was obtained as described for 8b, starting from
(
C-8a), 180.2 (CS). Anal. (C H Cl N OS) theoretical: C, 67.63; H,
17 17 2 3
5
.68; N, 12.45; S, 9.50. Found: C, 67.40; H, 5.67; N, 12.37; S, 9.65.
1
2
3
1
3
3 (500 mg, 2.35 mmol) and 3-chlorophenyl isothiocyanate (340 µL,
.59 mmol) (467 mg, 52 %): mp: 207-209 °C; IR (KBr) υ: 1540 (C=S),
-1 1
3.1.9. 6-Chloro-4-(3-cyanophenylaminocarbonylamino)-3,4-
297 (N-H) cm ; H NMR (DMSO-d , 500 MHz): δ: 1.33 (s, 3H, CH ),
6
3
dihydro-2,2-dimethyl-2H-benzoxazine (8e)
-Cyanophenyl isocyanate (373 mg, 2.35 mmol) was added to a
solution of 4-amino-6-chloro-3,4-dihydro-2,2-dimethyl-2H-
.47 (s, 3H, CH ), 3.15 (d, 1H, CH ), 3.38 (d, 1H, CH ), 6.75-6.85 (m,
3
2
2
3
H, 5-H, 7-H et 8-H), 7.23 (d, 1H, 4’-H), 7.36 (t, 1H, 5’-H), 7.56 (d, 1H,
’-H), 7.81 (s, 1H, 2’-H), 9.73 (s, 1H, NH), 10.04 (s, 1H, NH).
6
1
benzoxazine (13) (500 mg, 2.35 mmol) in methylene chloride (5 mL).
The solution was stirred 30 minutes at room temperature. The
resulting precipitated product was collected by filtration, washed
with n-hexane an dried (822 mg, 98 %): mp: 194-196 °C; IR (KBr) υ:
H NMR (DMSO-d , 500 MHz): δ: 1.33 (s, 3H, CH ), 1.47 (s, CH ), 3.15
6
3
3
(
d, J = 11.0 Hz, 1H, CH ), 3.38 (d, J = 10.7 Hz, 1H, CH ), 6.75 (s, 1H, 5-
2 2
H), 6.78 (d, J = 8.5 Hz, 1H, 8-H), 6.86 (d, J = 6.9 Hz, 1H, 7-H), 7.23 (d,
J = 7.0 Hz, 1H, 6’-H), 7.36 (t, J = 8.1 Hz, 1H, 5’-H), 7.57 (d, J = 7.6 Hz,
1
-1 1
13
1
698 (C=O), 2227 (C≡N), 3363 (N-H) cm ; H NMR (DMSO-d , 500
6
H, 4’-H), 7.81 (s, 1H, 2’-H), 9.73 (s, 1H, NH), 10.04 (s, 1H, NH).
C
MHz): δ: 1.37 (s, 6H, CH ), 3.28 (s, 2H, CH ), 6.75 (s, 2H, 7-H/8-H),
NMR (DMSO-d , 125 MHz): δ: 24.3 (CH ), 26.0 (CH ), 57.7 (CH ), 75.2
3
2
6
3
3
2
6
| J. Name., 2012, 00, 1-3
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6
.85 (s, 1H, 5-H), 7.42 (d, J = 7.7 Hz, 1H, 4’-H), 7.48 (t, J = 7.9 Hz, 1H, 3.2. Biological assays
View Article Online
5
’-H), 7.81 (d, J = 7.9 Hz, 1H, 6’-H), 8.03 (s, 1H, 2’-H), 8.72 (s, 1H,
DOI: 10.1039/C8MD00593A
13
NH), 9.09 (s, 1H, NH). C NMR (DMSO-d ) δ 25.1 (CH ), 58.0 (CH ), (±)-Cromakalim (Tocris, United Kingdom) and diazoxide ((Sigma
6
3
2
7
(
5
(
4.7 (C-2), 111.3 (C-3’), 113.0 (C-5), 117.7 (C-8), 118.8 (CN), 120.3 Chemical Co, USA) were used as reference compounds. All
C-7), 121.4 (C-2’), 123.5 (C-6’), 124.2 (C-6), 125.5 (C-4’), 130.0 (C- experiments were performed with aortae or pancreatic islets
’), 135.8 (C-4a), 139.7 (C-1’), 140.9 (C-8a), 155.1 (CO). Anal. isolated from adult fed Wistar Rats (Charles River Laboratories,
C H ClN O ) theoretical: C, 60.59; H, 4.80; N, 15.70. Found: C, Belgium). The experimental procedure using animals was approved
18
17
4 2
6
0.67; H, 4.65; N, 15.74.
by the ethical and animal welfare committee of the Université libre
de Bruxelles (CEBEA n° 493N) in accordance with the Belgian
legislation for the animals protection and welfare (Royal Order of
3.1.10. 6-Chloro-4-(4-cyanophenylaminocarbonylamino)-3,4-
th
th
dihydro-2,2-dimethyl-2H-benzoxazine (8f)
the 15 May 2001 modified by the Royal Order of the 29 May
The title compound was obtained as described for 8e, starting from 2013).
1
2
2
3 (500 mg, 2.35 mmol) and 4-cyanophenyl isocyanate (373 mg,
.59 mmol) (721 mg, 86 %): mp: 195-197 °C; IR (KBr) υ: 1693 (C=O), 3.2.1. Measurement of insulin secretion from incubated rat
-1 1
219 (C≡N), 3370 (N-H) cm ; H NMR (DMSO-d , 500 MHz): δ: 1.37 pancreatic islets.
6
(
s, 6H, CH ), 3.28 (s, 2H, CH ), 6.74 (s, 2H, 7-H/8-H), 6.84 (s, 1H, 5-H), Pancreatic islets were isolated by the collagenase method and
3 2
7
.72 (s, 4H, 2’-H/3’-H/5’-H/6’-H), 8.76 (s, 1H, NH), 9.25 (s, 1H, NH). freshly isolated islets were used for measurements of insulin
C NMR (DMSO-d , 125 MHz): δ: 25.1 (CH ), 57.9 (CH ), 74.7 (C-2), secretion. Groups of 10 islets, each derived from the same batch of
1
3
6
3
2
1
1
1
03.5 (C-4’), 113.0 (C-5), 117.7 (C-8), 118.6 (C-2’/C-6’), 119.3 (CN), islets, were pre-incubated for 30 min at 37 °C in 1 ml of a
20.0 (C-7), 124.2 (C-6), 133.1 (C-3’/C-5’), 135.8 (C-4a), 142.1 (C-8a), physiological salt medium (in mM: NaCl 115, KCl 5, CaCl 2.5, MgCl
2
2
44.0 (C-1’), 154.7 (CO). Anal. (C H ClN O ) theoretical: C, 60.59; 1, NaHCO 24) supplemented with 2.8 mM glucose, 0.5% (w/v)
18
17
4
2
3
H, 4.80; N, 15.70. Found: C, 60.35; H, 4.73; N, 15.34.
albumin (RIA grade) and equilibrated against a mixture of O (95 %)
2
and CO (5%). The islets were then incubated at 37 °C for a further
2
3
.1.11. 6-Chloro-4-(3-chlorophenylaminocarbonylamino)-3,4-
90 min in 1 ml of the same medium containing 16.7 mM glucose
and, in addition, either the reference compounds or the required
dihydro-2,2-dimethyl-2H-benzoxazine (8g)
The title compound was obtained as described for 8e, starting from chroman derivative. The release of insulin was measured
1
2
3
3
6
3 (500 mg, 2.35 mmol) and 3-chlorophenyl isocyanate (316 µL, radioimmunologically using rat insulin as a standard. Residual
.59 mmol) (757 mg, 88 %): mp: 181-183 °C; IR (KBr) υ: 1692 (C=O), insulin secretion was expressed as a percentage of the value
-1 1
371 (N-H) cm ; H NMR (DMSO-d , 500 MHz): δ: 1.37 (s, 6H, CH ), recorded in control experiments (100%); that is in the absence of
6
3
32-34
.27 (s, 2H, CH ), 6.74 (s, 2H, 7-H/8-H), 6.83 (s, 1H, 5-H), 6.86 (d, J = drug and presence of 16.7 mM glucose.
2
.9 Hz, 1H, 7-H), 7.02 (dd, J = 7.9 Hz/1.5 Hz, 1H, 4’-H), 7.28 (t, J = 8.1
Hz, 1H, 5’-H), 7.43 (d, J = 8.0 Hz, 1H, 6’-H), 7.74 (d, J = 1.8 Hz, 1H, 2’- 3.2.2. Measurement of myorelaxant activity on rat aorta rings.
13
H), 8.62 (s, 1H, NH), 8.94 (s, 1H, NH). C NMR (DMSO-d ) δ 25.0 The rat thoracic aorta was removed, cut into transverse rings (3-4
6
(
CH ), 57.9 (CH ), 74.7 (C-2), 113.0 (C-5), 117.2 (C-6’), 117.6 (C-2’), mm), and adhering fat and connective tissue was detached. After
3 2
1
18.2 (C-8), 119.6 (C-7), 121.7 (C-4’), 124.2 (C-6), 130.2 (C-5’), 133.0 removal of the endothelium, the segments were suspended under
(
C-3’), 135.9 (C-4a), 141.1 (C-1’), 141.7 (C-8a), 155.0 (CO). Anal. 1.5 g tension in an organ bath containing 20 ml of a buffered
(
C H Cl N O ) theoretical: C, 55.75; H, 4.68; N, 11.47. Found: C, physiological solution (in mM: NaCl 118, KCl 4.7, CaCl 2.5, NaHCO
3
17
17
2
3
2
2
5
5.84; H, 4.56; N, 11.45.
25, KH PO 1.2, MgSO 1.2, glucose 5). The solution was maintained
2 4 4
at 37 °C and continuously oxygenated with a mixture of 95% O and
2
3.1.12. 6-chloro-4-(4-chlorophenylaminocarbonylamino)-3,4-
5% CO . After equilibration for 60 min, isometric contractions were
2
dihydro-2,2-dimethyl-2H-benzoxazine (8h)
The title compound was obtained as described for 8e, starting from was induced by increasing the extracellular concentration of K (30
measured with a force-displacement transducer. Contractile activity
+
1
2
3
3
8
3 (500 mg, 2.35 mmol) and 4-chlorophenyl isocyanate (331 µL, mM or 80 mM KCl). When the tension was stabilized, drugs were
.59 mmol) (663 mg, 77 %): mp: 180-182 °C; IR (KBr) υ: 1690 (C=O), added cumulatively until maximal relaxation or until a maximum
-1 1
362 (N-H) cm ; H NMR (DMSO-d , 500 MHz): δ: 1.37 (s, 6H, CH ), concentration of 200 µM. Some experiments were repeated in the
6
3
.27 (s, 2H, CH ), 6.73 (s, 2H, 7-H/8-H), 6.83 (s, 1H, 5-H), 7.30 (d, J = continuous presence of 10 µM glibenclamide (Sigma Chemical Co,
2
.9 Hz, 2H, 3’-H/5’-H), 7.56 (d, J = 8.9 Hz, 2H, 2’-H/6’-H), 8.56 (s, 1H, USA) in the physiological medium. The contractile responses were
13
NH), 8.87 (s, 1H, NH). C NMR (DMSO-d , 125 MHz): δ: 25.5 (CH ), expressed as the percentage of the contractile response to KCl (100
6
3
5
2
1
8.4 (CH ), 75.1 (C-2), 113.3 (C-5), 118.1 (C-8), 120.0 (C-7), 120.8 (C- %). The EC50 values (concentration evoking 50% inhibition of the
2
’/C-6’), 124.6 (C-6), 126.1 (C-4’), 128.9 (C-3’/C-5’), 136.4 (C-4a), plateau phase induced by KCl) were assessed from concentration–
39.0 (C-1’), 142.2 (C-8a), 155.5 (CO). Anal. (C H Cl N O ) response curves using Datanalyst software (EMKA Technologies,
17
17
2
3
2
33-35
theoretical: C, 55.75; H, 4.68; N, 11.47. Found: C, 56.06; H, 4.62; N, France).
1.11.
1
3.3. Statistical evaluation
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Journal Name
F.M. Ashcroft, Annu. Rev. Neurosci., 1988, 11, 97-118.
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M.A. Burke, R.K. Mutharasan, and H. Ardehali, Circ. Res., 2008,
The statistical significance of differences between mean data was
assessed by using the non-paired Student’s t-test. The biological
results were considered as statistically different when p value was
DOI: 10.1039/C8MD00593A
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02, 164-176.
C.G. Nichols, Nature, 2006, 440, 470-476.
D.L. Cook and C.N. Hales, Nature, 1984, 311, 271-273.
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<0.05.
3.4. Predicted partition coefficients
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,
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S. Seino, H. Takahashi, T. Takahashi and T. Shibasaki, Diabetes
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36-441.
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corresponding 4-phenylureido/thioureido-substituted
dimethylchromans was described and the impact of such structural
modification on the pharmacological profile of the new drugs
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releasing process and the vasorelaxant properties of the original
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C. Moreau, H. Jacquet, A.L. Prost, N. D'hahan, M. Vivaudou,
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P.M. Paciorek, D.T. Burden, Y.M. Burke, I.S. Cowlrick, R.S.
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The benzoxazines were found to be less active than their
corresponding chromans on the insulin secreting cells, but some 4-
arylureido-substituted benzoxazines appeared to be more potent as
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S. Khelili, N. Kihal, M. Yekhlef, P. de Tullio, P. Lebrun and B.
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The most active myorelaxant compound, i.e. the 4-arylureido-
subsituted benzoxazine 8e, was selected for further
characterization of its mechanism of action. Altogether, our
pharmacological observations suggest that 8e behaved as a calcium
entry blocker rather than as a KATP channel opener.
Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
This study was supported in part by grants from the “National Fund
for Scientific Research” (F.N.R.S., Belgium) from which P. Lebrun is a P. Lebrun, P. Arkhammar, M.-H. Antoine, Q.-A. Nguyen, J.B.
Research Director. The authors gratefully acknowledge the
technical assistance of S. Counerotte, F. Leleux, and A.-M.
Vanbellinghen.
Hansen and B. Pirotte, Diabetologia, 2000, 43, 723-732.
P. Lebrun, B. Becker, N. Morel, P. Ghisdal, M.-H. Antoine, P. de
Tullio and B. Pirotte, Biochem. Pharmacol., 2008, 75, 468-
475.
B. Pirotte, P. de Tullio, S. Boverie, C. Michaux and P. Lebrun, J.
Med. Chem., 2011, 54, 3188-3199.
B. Becker, M.-H. Antoine, Q.-A. Nguyen, B. Rigo, K.E. Cosgrove,
P.D. Barnes, M.J. Dunne, B. Pirotte and P. Lebrun, Br. J.
Pharmacol., 2001, 134, 375-385.
Notes and references
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I. Tetko, J. Gasteiger, R. Todeschini, A. Mauri, D. Livingstone, P.
Ertl, V. Palyulin, E. Radchenko, N. Zefirov, A. Makarenko, V.
Tanchuk and V. Prokopenko, J. Comput.-Aided Mol. Des.
View Article Online
DOI: 10.1039/C8MD00593A
2
005, 19, 453-463.
B. Pirotte, X. Florence, E. Goffin and P. Lebrun, ChemMedChem.,
017, 12, 1810-1817.
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DOI: 10.1039/C8MD00593A
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R1
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H
N
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N
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N
S
N
H
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H
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O
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X
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