1,4-Benzothiazine ATP-sensitive potassium channel openers: Modifications at the C-2 and C-6 positions

Article abstract of DOI:10.1021/jm400435a

ATP-sensitive potassium (K_ATP) channels play a prominent role in controlling cardiovascular function. In this paper, a novel series of 4-(1-oxo-2-cyclopentenyl)-1,4-benzothiazine derivatives modified at the C-2, and C-6 positions were synthesized as openers of vascular K_ATP channels. Most of the tested compounds evoked vasorelaxing effects on rat aortic rings and membrane hyperpolarization in human vascular smooth muscle cells, with potency similar or superior to that of the reference levcromakalim (LCRK). The selective K_ATP blocker glibenclamide antagonized the above vascular effects, confirming that K_ATP channels are closely involved in the mechanism of action. The experimental results confirmed the 1,4-benzothiazine nucleus as an optimal scaffold for activators of vascular K_ATP channels; moreover, the high level of potency exhibited by the 6-acetyl substituted benzothiazine 8, along with the lack of any significant interference with insulin secretion from pancreatic β-cells, paves the way to further develop a new series of potent activators of vascular K_ATP channels.

Full text of DOI:10.1021/jm400435a

Article
pubs.acs.org/jmc
1,4-Benzothiazine ATP-Sensitive Potassium Channel Openers:
Modifications at the C‑2 and C‑6 Positions
Alma Martelli,‡ Giuseppe Manfroni,*,† Paola Sabbatini,† Maria Letizia Barreca,† Lara Testai,‡
Michela Novelli,§ Stefano Sabatini,† Serena Massari,† Oriana Tabarrini,† Pellegrino Masiello,§
Vincenzo Calderone,*,‡ and Violetta Cecchetti†
†Dipartimento di Chimica e Tecnologia del Farmaco, Universita
̀
degli Studi di Perugia, Via del Liceo, 1, 06123 Perugia, Italy
‡Dipartimento di Farmacia, Universita
̀
degli Studi di Pisa, Via Bonanno 6, 56126 Pisa, Italy
§Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Universita
55, 56126 Pisa, Italy
̀
degli Studi di Pisa, Via Roma,
S
* Supporting Information
ABSTRACT: ATP-sensitive potassium (KATP) channels play a prominent role in controlling
cardiovascular function. In this paper, a novel series of 4-(1-oxo-2-cyclopentenyl)-1,4-benzothiazine
derivatives modified at the C-2, and C-6 positions were synthesized as openers of vascular KATP channels.
Most of the tested compounds evoked vasorelaxing effects on rat aortic rings and membrane
hyperpolarization in human vascular smooth muscle cells, with potency similar or superior to that of the
reference levcromakalim (LCRK). The selective KATP blocker glibenclamide antagonized the above
vascular effects, confirming that KATP channels are closely involved in the mechanism of action. The
experimental results confirmed the 1,4-benzothiazine nucleus as an optimal scaffold for activators of
vascular KATP channels; moreover, the high level of potency exhibited by the 6-acetyl substituted
benzothiazine 8, along with the lack of any significant interference with insulin secretion from pancreatic
β-cells, paves the way to further develop a new series of potent activators of vascular KATP channels.
capnic acidosis,8 many hormones and neurotransmitters,9
INTRODUCTION
■
caveolin-1,10 and even the gaseous modulator hydrogen
sulfide.11 KATP channels are inhibited by sulfonylurea agents
such as glibenclamide and tolbutamide, which are able to block
the channel at concentrations spanning from the low
nanomolar to the low micromolar range.7 In the past few
decades, the therapeutic potential of KATP channel opening in
cardiovascular diseases like hypertension led to the design of
several chemical entities able to activate these channels such as
benzopyrans like cromakalim,12 cyanoguanidines like pinaci-
dil,13 pyridyl nitrates like nicorandil,14 thioformamides like
aprikalim,15 benzothiadiazines like diazoxide,16 and recently
propylamines like iptakalim.17 These potassium channel
openers (KCOs) evoke membrane hyperpolarization with
consequent inactivation of voltage-operated calcium channels,
leading in turn to a reduction of intracellular calcium and
vasodilation. This hyperpolarization has been observed in
vascular smooth muscle cells18,19 and is antagonized by
glibenclamide but not by blockers of other potassium
channels.16 In coronary arteries of canine and rabbit hearts20
and in hamster microcirculation, KATP channels have been
found to play an important role in controlling the resting
vascular tone. In fact, in both these vascular beds, the
administration of glibenclamide evokes vasconstriction, indicat-
ing that KATP channels are activated even at resting
The ATP-sensitive potassium (KATP) channels represent an
efficient biological mechanism able to link the metabolic status
of cells with their excitability.1 In fact, they are inward rectifier
potassium channels showing a sensitivity to intracellular ATP
concentration.2 In particular, under conditions of satisfactory
phosphorylation potential, high intracellular levels of ATP are
the primary inhibitory factor on KATP channels. By contrast,
under conditions of reduced energetic metabolism, a decrease
in the ATP/ADP ratio and an increase in the ADP level
determine the activation of the KATP channel and thus an
outward flow of potassium ions with consequent membrane
hyperpolarization.1
From a structural point of view, KATP channels are
heteroctameric complexes formed by four pore-forming
subunits (Kir6) of the inward rectifier family of potassium
channels and by four larger regulatory proteins, the sulfonylurea
receptor subunits (SUR) that function as the sensor of the
ATP/ADP ratio.3 Different subtypes of Kir6 proteins (Kir6.1 or
Kir6.2) and SUR proteins (SUR1, SUR2A, or SUR2B) form
KATP channels in different tissues.4 In particular, vascular
smooth muscle expresses KATP channels mainly formed by
Kir6.1 and SUR2B.5 The KATP channels expressed in vascular
smooth muscle cells are of particular interest, as they contribute
to the control of membrane potential, arterial diameter, and
blood flow regulation.6 Vascular KATP channels are able to
control the vascular tone through the action of many
endogenous modulators such as levels of ATP/ADP,7 hyper-
Received: March 25, 2013
Published: May 10, 2013
© 2013 American Chemical Society
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conditions.21 Finally, a new KATP opener, iptakalim, has been
shown to induce arteriolar vasodilation and reduce the blood
pressure of hypertensive rodents and humans.17 Moreover,
iptakalim exerted protective effects against hypertensive damage
to target organs in rats and improved endothelial dysfunction
by selective activation of the Kir6.1/SUR2B subtype of KATP
channels expressed in the vascular endothelium.17 Presently, its
clinical trials for the treatment of mild−moderate hypertension
have been completed in China,22 providing an exciting base to
develop additional series of selective KATP channels openers
able to modulate the vascular tone and to be employed in
several cardiovascular diseases.
In our previous work on KCOs, we have found that the 1,4-
benzothiazine scaffold with a cyclopentenone moiety at the N-4
position, a gem-dimethyl group at the C-2 position, and an
electron-withdrawing substituent at the C-6 position led to
KCOs with the highest potencies.23 The lead compound of this
series is represented by derivative 1 bearing a CF3 group at the
C-6 position (Figure 1).
phenylsulfonyl27 or arylaminosulfonyl28 confered high potency.
Further variations of 6-substitution surprisingly showed that
alkyl groups, lacking any electron-withdrawing effects, could be
incorporated conferring approximately one-third of the activity
of the nitrile group.29
To date, the C-6 position has been marginally explored in the
benzothiazine KCOs.23 For this reason, we now report the
synthesis of new C-6 modified derivatives 5−11. In particular,
the CF3 group of the reference benzothiazine 1 was replaced by
groups having different electronic, steric, and hydrogen bond
acceptor/donor properties (Figure 1).
CHEMISTRY
■
The synthetic approach used to obtain the target compounds
2−9 involved the preparation of 1,4-benzothiazine intermedi-
ates 12−19, appropriately substituted at C-2 and C-6 position.
These were then functionalized at the N-4 position with a
cyclopentenone moiety by treatment with the crude product
obtained from reacting cyclopentanone and NBS in the
presence of a catalytic amount of dibenzoyl peroxide (Scheme
1).
a
Scheme 1
a
Figure 1. Chemical structure of 1,4-benzothiazine derivative 1 and
structural modifications at the C-2/(C-3) and C-6 positions.
Reagents and conditions: (i) [cyclopentanone, NBS, DBP, CCl4],
DIPEA, THF, reflux.
The structure−activity relationship (SAR) of this class of
KCOs has now been extended by exploring the C-2, C-3, and
C-6 positions, thus obtaining derivatives 2−11 (Figure 1). In
particular, taking the 1,4-benzothiazine derivative 123 as the
reference compound, the influence of the gem-dimethyl at the
C-2 position was examined by deleting one (compound 2) or
both methyl groups (compound 3). In addition, the gem-
dimethyl group was shifted from the C-2 to the C-3 position
(compound 4).
Regarding the C-6 position, it is known that the substituent
at this site has a great impact on modulating the biological
activity in the benzopyran KCOs.24 In particular, it has been
postulated that the C-6 substituent could contribute to receptor
affinity either by a direct interaction with the receptor site or
indirectly by withdrawing electrons from the benzene moiety of
the benzopyran nucleus, thus increasing charge transfer
interactions of the aromatic moiety with the receptor.25 In
early investigations, optimum substitution was attributed to
small, electron-withdrawing substituents such as NO2, CF3, and
CN.26 Later, it was found that even larger substituents such as
The target 6-amino-4-(1-oxo-2-cyclopentenyl)benzothiazine
derivative 10 was obtained from the corresponding 6-nitro
derivative 20,23 previously reported by us, through selective
reduction of nitro group with Zn/AcOH. Subsequent acylation
of 10 with AcCl in CH2Cl2 furnished the target 6-acetamido-4-
(1-oxo-2-cyclopentenyl)benzothiazine derivative 11 (Scheme
2).
a
Scheme 2
a
Reagents and conditions: (i) Zn, AcOH; (ii) AcCl, DIPEA, CH2Cl2.
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a
Scheme 3
a
Reagents and conditions: (i) HSC(Me)2CO2H, TBAB, Cs2CO3, DMSO; (ii) Fe, AcOH, H2O; (iii) LiAlH4, THF.
1,4-Benzothiazine intermediate 12 was prepared from
benzothiazinone 2130 by reduction with LiAlH4 (Scheme 3),
whereas benzothiazines 13,31 14,32 and 15,33 were prepared
following literature procedures.
6-Methylbenzothiazine 16 was obtained by reacting the 1-
chloro-4-methyl-2-nitrobenzene with 2-mercapto-2-methypro-
pionic acid in DMSO at 100 °C using Cs2CO3 as base and a
catalytic amount of tetra-n-butylammonium bromide (TBAB).
Reductive ring closure of the obtained nitroacid 22 gave
benzothiazinone 23, which was then reduced with LiAlH4
(Scheme 3).
Figure 2. ORTEP drawing of compound 25.
A different synthetic procedure was used to prepare 6-
diisobutylsulfonylbenzothiazine 17, which entails the introduc-
Following a similar procedure and employing AcCl,
tion of the C-6 substituent by electrophilic aromatic
substitution on the preformed 1,4-benzothiazine nucleus
(Scheme 4), where the C-6 position is the most favored.34
4-trifluoroacetyl intermediate 24 was converted into derivative
1
27 whose structure was confirmed by H NOESY NMR data
(Figure 3). Subsequent deprotection at C-4 position gave the
desired 6-acetylbenzothiazine 1835 in 71% overall yield (lit.
32%).
a
Scheme 4
Figure 3. NOESY experiment for 27 showed three main interactions:
CH3CO → H-5; CH3 → H-7; gem-CH3 → H-8.
Further modification of 6-acetylbenzothiazine 18 by
reduction with LiAlH4 in THF provided 6-ethylbenzothiazine
19 (Scheme 4).
a
Reagents and conditions: (i) (CF3CO)2O, Pyr, CH2Cl2; (ii) HSO3Cl;
(iii) Et3N, NH(i-Bu)2, THF; (iv) MeOH, K2CO3; (v) AcCl, AlCl3;
(vi) LiAlH4, THF.
RESULTS AND DISCUSSION
■
The KATP-opening activity of the synthesized compounds was
first tested by evaluating their vasorelaxant effect on
endothelium-denuded rat aortic rings precontracted with KCl
(25 mM) according to the protocol described in the
Experimental Section. The vasorelaxing activity data, expressed
as efficacy (%) and potency (pIC50), are reported in Table 1
along with those of reference compounds 1 and levcromakalim
(LCRK), a well-known KCO belonging to the benzopyran
class.
Thus, benzothiazine 1533 was initially protected at the N-4
position by using (CF3CO)2O and then reacted with
chlorosulfonic acid/AlCl3, affording the intermediate 25
regioselectively. The structure of 25 and therefore the reaction
regioselectivity have been confirmed by X-ray structural analysis
(Figure 2). Intermediate 25 was then elaborated to 6-
diisobutylsulfonylbenzothiazine 17 by reaction with N,N-
diisobutylamine in THF and in the presence of Et3N, followed
by the removal of the amine protecting group at the N-4
position by treatment with K2CO3 in MeOH.
All of the synthesized compounds exhibited full levels of
vasorelaxing efficacy, while a great variation in the potency
index could be observed among the compounds, thus
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a
Table 1. Vasorelaxant Activity of the 1,4-Benzothiazine Derivatives 2-11 Modified at the C-2, C-3, and C-6 Positions
bd
,
cde
, ,
compd
R2
H
R2′
R3, R3′
H
R6
Emax
,
%
pIC50
2
Me
CF3
CF3
CF3
H
94
2
8.08 0.07
(6.15 0.12)
6.27 0.09
(4.63 0.21)
7.18 0.03
(63 6)
94
(64 4)
3
H
H
H
Me
H
H
H
H
H
H
H
H
3
4
H
H
89
1
f
f
(NT)
90
(NT)
5
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
8
6.14 0.12
(4.68 0.22)
6.03 0.05
(4.71 0.09)
6.10 0.05
(61 7)
89
(78 3)
6
Me
2
7
SO2N(i-Bu)2
85
2
f
f
(NT)
88
(NT)
8
Ac
3
8.84 0.18
(7.25 0.26)
6.81 0.02
(5.19 0.09)
5.40 0.13
(77 2)
97
(69 8)
9
Et
2
10
NH2
NHAc
CF3
96
5
f
f
(NT)
(NT)
11
97 10
5.18 0.08
f
f
(NT)
(NT)
1
92
(96 4)
99
(93 3)
5
10.38 0.14
(8.08 0.07)
6.49 0.07
LCRK
5
(5.60 0.13)
a
b
Vasorelaxant potency was evaluated on endothelium denuded isolated aortic ring of male normotensive Wistar rats. Maximal vasorelaxing response
c
d
expressed as percentage of contractile tension. Values are the mean of 5−10 separate experiments
corresponding values obtained in the presence of glibenclamide are reported. Vasorelaxant potency expressed as negative log of the concentration
evoking a half-reduction of the contractile tone. NT = not tested in the presence of glibenclamide.
standard error. In parentheses, the
e
f
confirming the importance of the C-2 and C-6 substituents in
modulating the biological activity.
Among the substituents at the C-6 position, the acetyl-
substituted derivative 8 elicited the greatest potency, reaching a
vasorelaxant activity in the nanomolar range (Table 1). Since
the acetyl is a small and electron-withdrawing group, the result
is in agreement with the known SAR of the KCO benzopyran
class.24a Replacement of the acetyl with an acetamide
(derivative 11) resulted in a dramatic decrease in the activity.
This suggests (a) the importance of the electron-withdrawing
effect, which may occur only if the acetyl group is directly
linked to the aromatic ring and/or (b) the need for a precise
spatial arrangement to allow the substituent at C-6 to set up
hydrogen bond interactions with defined areas of the receptor.
Low activity was also observed with derivative 10 bearing a C-6
amino group, which is able to establish hydrogen bond
interactions but has electron donor properties. Despite the
presence of an electron-withdrawing group, the 6-diisobutyl-
sulfonamide derivative 7 did not show high vasorelaxant
potency. This result could be explained by considering the
steric hindrance of the ramified alkyl chains, supporting the
conclusion that the dimension of the C-6 group has some
influence on the potency. The replacement of the C-6
trifluomethyl group of compound 1 with a methyl (derivate
6), which is similar in shape but not in electronic effect,
reduced the activity by over 4 orders of magnitude. The same
behavior was also observed for the C-6 unsubstituted derivative
Although all the synthesized compounds showed a reduction
in potency when compared with the benzothiazine progenitor
1, it is noteworthy that the potency reached nanomolar levels
for the 2-monomethyl derivative 2 and the 6-acetyl derivative 8.
In particular, the potency of the latter was nearly 100-fold
higher than that exhibited by LCRK (Table 1). Furthermore,
derivatives 3−7 and 9 exhibited potency parameters com-
parable with that of LCRK. The 6-amino derivative 10 and the
6-acetamido derivative 11 had the lowest potency, more than
10-fold lower than that of LCRK.
In the modifications carried out at the C-2 position, the
removal of one or both methyl groups (compounds 2 and 3,
respectively) caused a progressive reduction of the vasorelaxant
potency. These findings are in agreement with those already
reported for the KCO benzopyran class,24c,36 confirming a
primary role played by the C-2 gem-dimethyl group in obtaining
highly active compounds. A reduction in potency was also
observed when the gem-dimethyl group was shifted from the C-
2 to the C-3 position (compound 4). However, it is worth
noting that the C-3 gem-dimethyl derivative 4 is as active as
LCRK while 2-monomethyl derivative 2 is about 10 times more
active than LCRK. The stereochemistry of the methyl group in
derivative 2 remains unassigned.
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5. Irrespective of the requirements for electron-withdrawing
capability and/or hydrogen bond interaction, the 6-ethyl
substituted analogue (compound 9) showed an unexpected
increase of potency, which was almost equivalent to that of
LCRK. However, the same behavior was also reported in the
early SAR studies on the KCO benzopyran class.29 In an
attempt to explain the singular activity of 6-ethyl derivative 9,
we have hypothesized that the 6-ethyl group could be at least
partially metabolized in tissue into active 6-acetyl derivative 8.
To support this hypothesis, the software MetaSite37 was used
to predict metabolic transformation of 9 related to cytochrome-
mediated reactions in phase I metabolism. The in silico results
indicated the 6-ethyl group as the primary site of metabolism
(computed score of 7) in all the considered tissues. In addition,
the metabolic pathway analysis underlined that the oxidation of
the ethyl group into the acetyl is predominant. On the contrary,
the MetaSite procedure applied to 6-methyl derivative 6
predicted the corresponding secondary alcohol, which does not
have electronic withdrawing properties, as the principal
metabolite.
In order to evaluate the involvement of the activation of KATP
channel in the mechanism of action, the vasorelaxing effects of
some selected benzothiazines were also tested in the presence
of glibenclamide, a well-established selective KATP-blocker able
to antagonize the effects of compound 1 and LCRK. This
sulfonylurea significantly antagonized the effects of all the
tested compounds, causing a marked and significant rightward
shift of the concentration−response curves of compounds 2, 3,
5, 6, 8, and 9 (Table 1, Figure 4). These pharmacological
results indicate that the activation of vascular KATP channels is
likely to account for the vasorelaxing properties of these
compounds.
benzothiazines caused a concentration-dependent decrease of
the fluorescence of HASMCs preincubated with DiBAC4(3),
indicating their hyperpolarizing effects on the membrane
potential (Figure 5).
Figure 5. Concentration dependent hyperpolarizing effects induced by
compounds 2, 8, and 9 in HASMCs. The effects represent the
decrease of relative fluorescence, expressed as a % of the maximal
effect induced by 100 μM LCRK. Each bar represents the mean value
of six independent measurements. Vertical bars represent the SEM.
Noteworthy, the effects of the three benzothiazines and of
the reference drug LCRK were strongly antagonized by
glibenclamide (Figure 6), indicating that such a membrane
Figure 6. Representative experiment of spectrofluorimetric recording
of change in membrane potential of HASMCs, induced by the tested
compounds. The addition of compound 8 (10 μM, arrow) caused a
significant decrease of relative fluorescence (black squares), indicating
a hyperpolarizing effect. This effect was completely prevented when
compound 8 was added in the presence of 1 μM glibenclamide (Gli,
white squares).
Figure 4. Concentration−vasorelaxing response curve for compound
8. Increasing concentrations of compound 8 were cumulatively added
to endothelium-denuded rat aortic rings KCl in 25 mM, in the absence
(black squares) or in the presence (white squares) of 1 μM
glibenclamide (Gli). The vertical bars represent the SEM.
hyperpolarization is almost completely due to the activation of
KATP channels expressed on the sarcolemmal membranes of
HASMCs. The maximal effect produced by all these
compounds was equivalent to that produced by LCRK
(Table 2). The submicromolar potency of compound 9 was
comparable with that of LCRK, while the potencies of
compounds 2 and 8 were improved by about 1.5 orders of
magnitude (Table 2). These results are consistent with the
experimental data obtained in the functional studies on rat
aortic rings (Table 1), where the potency of compound 9 was
equivalent to that of LCRK, while it was notably improved for
compounds 2 and 8. Nevertheless, the potency parameters
recorded on HASMCs were always lower than those observed
on rat aortic rings. This difference may reflect mechanistic
aspects (full vasorelaxing effect does not necessarily require full
An additional experimental approach was aimed to reach a
more complete pharmacodynamic characterization and to test
the pharmacological profile on human vascular KATP channels.
In particular, the effects of three benzothiazines and LCRK on
the membrane potential of cultured human aortic smooth
muscle cells (HASMCs) were tested by means of the
membrane-potential-sensitive dye bis(1,3-dibutylbarbituric
acid)trimethine oxonol (DiBAC4(3)), allowing an indirect
electrophysiological recording. Compounds 2 and 8 were
selected because of the high level of potency exhibited in the
functional tests on rat aortic rings, while compound 9 was
selected for its vasorelaxing potency almost comparable to that
of the reference drug LCRK (Table 1). The three selected
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a
In order to evaluate the effects of the activation of KATP
channels, diazoxide (80 μM) was selected as reference drug,
since it is known to produce inhibition of insulin release (and
even vasorelaxing effects).39 As expected, the HG-induced
secretion of insulin was almost half-reduced by diazoxide.
Glibenclamide antagonized the effects of diazoxide, indicating
that the diazoxide-mediated decrease of insulin release is
actually due to activation of KATP channels. Noteworthy,
neither compound 8 nor compound 9 (at those concentrations
evoking strong vasorelaxing effects) was able to significantly
influence the HG-induced release of insulin. Such experimental
evidence indicates that these new KATP activators are highly
vasoselective and that their vasorelaxing effects are unlikely to
be associated with significant metabolic adverse effects due to
impairment of insulin release and consequent hyperglycaemia.
In summary, SAR analysis of the 1,4-benzothiazine series
clearly shows a good overlapping with that of the benzopyran
KCOs. Most of the benzothiazine derivatives synthesized in this
study showed a potency similar or superior to that of LCRK.
These findings confirm the 1,4-benzothiazine nucleus as an
excellent scaffold to warrant good vasorelaxant activity
mediated by the activation of KATP channels. Moreover, the
high level of potency exhibited by the 6-acetyl substituted
benzothiazine 8, associated with the lack of any significant
interference with insulin secretion from pancreatic β-cells, paves
the way to the development of new series of powerful and safe
activators of vascular KATP channels.
Table 2. Hyperpolarizing Effects of Compounds 2, 8, and 9
bc
,
dc
,
e
compd
Emax
,
%
pEC50
Gli
2
106
103
98
5
5
6.87 0.06
6.93 0.02
5.13 0.03
5.27 0.08
+
+
+
+
8
9
3
LCRK
100
a
Hyperpolarizing effects were spectrofluorimetrically evaluated by
means of the membrane-potential-sensitive dye DiBAC4(3) on
HASMCs. Maximal response expressed as percentage of the maximal
hyperpolarization evoked by the reference drug LCRK (100 μM).
Values are the mean of six replicates standard error. Potency value
expressed as negative log of the concentration evoking a half-maximal
response. + indicates that the hyperpolarizing effects were
b
c
d
e
significantly antagonized by glibenclamide (Gli).
hyperpolarization) or may be due to difference of species and/
or methodological approaches.
Since the activation of pancreatic β-cell KATP channels, with
consequent inhibition of insulin release and possible hyper-
glycaemia, is one of the most usual and worrying adverse effects
of poorly selective KATP activators, a final experimental
approach was aimed at evaluating the influence of two selected
compounds, 8 and 9, on the release of insulin in isolated rat
pancreatic islets, according to standard procedures.38 The
results are shown in Figure 7. When exposed to low levels of
EXPERIMENTAL SECTION
■
Chemistry. All starting materials were commercially available,
unless otherwise indicated. Reagents and solvents were purchased
from common commercial suppliers and were used as such. Organic
solutions were dried over anhydrous Na2SO4 and concentrated with a
rotary evaporator at low pressure. All reactions were routinely checked
by thin-layer chromatography (TLC) on silica gel 60F254 (Merck) and
visualized by using UV or iodine. Column chromatography separations
were carried out on Merck silica gel 60 (mesh 70−230), and flash
chromatography separations were carried out on Merck silica gel 60
(mesh 230−400). Melting points were determined in capillary tubes
(Buchi Electrothermal model 9100) and are uncorrected. Yields were
of purified products and were not optimized. H NMR spectra were
̈
1
recorded at 200 or 400 MHz (BrukerAvance DRX-200 or 400,
respectively), and 2D 1H NMR NOESY was run in face sensitive
mode. Spectra were acquired at 298 K. Deuterochloroform was used as
solvent unless otherwise indicated. Chemical shifts (δ) are given in
ppm relative to TMS and calibrated using residual undeuterated
solvent as internal reference. Coupling constants (J) are reported in
Hz. Data processing was performed with standard Bruker software
XwinNMR, and the spectral data are consistent with the assigned
structures. GC−MS analyses were carried out with an HP 6890 gas
chromatograph (25 m dimethylsilicone capillary column) equipped
with an HP 5973 mass selective detector. The purity of the
compounds was determined by combustion analysis employing a
Fisons elemental analyzer, model EA1108CHN, and data for C, H, and
N are within 0.4% of the theoretical values (≥96%).
General Procedure for the Preparation of the Target
Derivatives 2−9. A mixture of freshly distilled cyclopentanone
(16.88 g, 17.75 mL, 30 mmol), NBS (35.78 g, 30 mmol), and a
catalytic amount of dibenzoyl peroxide in dry CCl4 (20 mL) was
refluxed for 3 h under nitrogen atmosphere, cooled, filtered, and
evaporated to dryness. The obtained dark oil was quickly poured into a
solution benzothiazine intermediate 12−19 (1 mmol) in dry THF (15
mL), and then freshly distilled DIPEA (3.86 g, 5.21 mL, 24 mmol) was
added dropwise. The resulting mixture was stirred at room
temperature for 10−15 h, and then Et2O was added. The dark
precipitate was filtered off, washing with Et2O. The filtrate was
sequentially washed with 1 N HCl, brine, dried, and evaporated to
Figure 7. Histograms reflect the insulin release detected after 1 h of
incubation in KRH buffer of batches of isolated rat pancreatic islets
exposed to low glucose (LG) (2.8 mM) or high glucose (HG) (16.7
mM) concentrations. Moreover, the effects of the KATP blocker
glibenclamide 1 μM (HG + Gli), the KATP activator diazoxide 80 μM
(HG + DZX), and their combination (HG + Gli + DZX) on the HG-
induced insulin release are reported. The histograms show the effects
of different concentrations of compounds 8 (HG + 8) and 9 (HG + 9)
on the HG-induced insulin release. The total islet insulin content, as
determined at the end of the incubation, was homogeneous among the
islet batches. Data are expressed as mean SEM (vertical bars) of at
least five replicas for each treatment. The asterisks indicate significant
differences, with respect to HG (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P ≤
0.001).
glucose (LG, 2.8 mM), the isolated islets exhibited a basal
insulin secretion (3.23 0.40 ng/mL). Stimulation with high
levels of glucose (HG, 16.7 mM) led to a significant 3-fold
increase in insulin release (9.36
0.68 ng/mL). Such a
secretagogue effect was further increased by the KATP-blocker
glibenclamide. Indeed, in the presence of HG and 1 μM
glibenclamide the insulin release achieved 12.89 0.98 ng/mL.
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dryness. The obtained residue was purified by flash column
chromatography, eluting with cyclohexane/EtOAc (95:5) to cyclo-
hexane/EtOAc (85:15). Additional purification method, yield, melting
point, and spectral data of each compound are given below.
6-Amino-2,2-dimethyl-4-(1-oxo-2-cyclopenten-2-yl)-3,4-di-
hydro-2H-1,4-benzothiazine (10). A solution of 6-nitro-4-cyclo-
pentenyl-2H-1,4-benzothiazine 2023 (0.1 g, 0.31 mmol) in AcOH (5
mL) and MeOH (5 mL) was added dropwise to a suspension of Zn
powder (0.11 g, 1.68 mmol) in water (1 mL) cooled to 0 °C. After the
addition was complete, the mixture was stirred at room temperature
for 30 min and then filtered over Celite. The filtrate was diluted with
water, basified with concentrated NH4OH, and extracted with EtOAc.
The organic layer was washed with brine, dried, and evaporated to
dryness. The crude dark residue was purified by flash column
chromatography, eluting with cyclohexane/EtOAc (8:2 to 1:1) to give
10 as a yellow oil (0.027 g, 30%). 1H NMR (200 MHz) δ 1.40 (6H, s,
CH3), 2.45−2.50 and 2.60−2.65 (each 2H, m, CH2CH2CO), 3.60
(2H, s, CH2), 6.20−6.30 (2H, m, H-5 and H-7), 6.85 (1H, d, J = 8.2
Hz, H-8), 7.15 (1H, t, J = 3.0 Hz, vinyl-H). GC−MS: m/z = 275 (19)
[M+ + 1], 274 (100) [M+], 232 (11), 231 (69), 203 (15), 177 (13).
Anal. (C15H18N2OS) C, H, N.
2-Methyl-4-(1-oxo-2-cyclopenten-2-yl)-6-trifluoromethyl-
3,4-dihydro-2H-1,4-benzothiazine (2). Recrystallized from petro-
1
leum ether/Et2O, 14% yield, white solid, mp 94−95 °C. H NMR
(400 MHz) δ 1.35 (3H, d, J = 6.6 Hz, CH3), 2.50−2.60 and 2.60−2.70
(each 2H, m, CH2CH2CO), 3.25 (1H, dd, J = 8.8, 13.2 Hz, CH2),
3.30−3.40 (1H, m, SCH), 4.20 (1H, dd, J = 2.7, 13.2 Hz, CH2), 6.95
(1H, t, J = 3.1 Hz, vinyl-H), 7.00−7.10 (2H, m, H-5 and H-7), 7.15
(1H, d, J = 8.1 Hz, H-8). GC−MS: m/z = 314 (15) [M+ + 1], 313
(68) [M+], 299 (10), 298 (53), 285 (23), 284 (100), 256 (12), 242
(20), 241 (10), 230 (11), 217 (10). Anal. (C15H14F3NOS) C, H, N.
4-(1-Oxo-2-cyclopenten-2-yl)-6-trifluoromethyl-3,4-dihy-
dro-2H-1,4-benzothiazine (3). Recrystallized from EtOH, 31%
1
yield, white solid, mp 120−121 °C. H NMR (200 Hz) δ 2.55−2.75
(4H, m, CH2CH2CO), 3.05−3.15 (2H, m, SCH2), 3.95−4.00 (2H, m,
CH2), 6.95 (1H, t, J = 2.9 Hz, vinyl-H), 7.05−7.15 (2H, m, H-5 and
H-7), 7.20−7.30 (1H, m, H-8). GC−MS: m/z = 299 (63) [M+], 285
(10), 284 (100), 243 (11), 242 (15). Anal. (C14H12F3NOS) C, H, N.
3,3-Dimethyl-4-(1-oxo-2-cyclopenten-2-yl)-6-trifluorometh-
yl-3,4-dihydro-2H-1,4-benzothiazine (4). Recrystallized from
6-Acetamido-2,2-dimethyl-4-(1-oxo-2-cyclopenten-2-yl)-
3,4-dihydro-2H-1,4-benzothiazine (11). A solution of acetyl
chloride (0.086 g, 0.078 mL, 0.99 mmol) in dry CH2Cl2 (3 mL)
was added dropwise and under nitrogen atmosphere to a solution of
cyclopentenyl derivative 10 (0.25 g, 0.87 mmol) and dry DIPEA (1.17
g, 1.58 mL, 9.05 mmol) in dry CH2Cl2 (10 mL) cooled to −10 °C.
After 10 min the mixture was poured into ice−water and acidified to
pH 5 with 0.2 N HCl. The organic layer was separated, washed with
water, dried, and evaporated to dryness. The crude brown residue was
crystallized from EtOH, giving the cyclopentenyl derivative 11 (0.19 g,
65%) as a yellow solid, mp 182−183 °C. 1H NMR (200 MHz) δ 1.35
(6H, s, CH3), 2.05 (3H, s, COCH3), 2.50−2.60 and 2.60−2.70 (each
2H, m, CH2CH2CO), 3.55 (2H, s, CH2), 6.65 (1H, dd, J = 2.2, 8.4 Hz,
H-7), 6.90 (1H, d, J = 8.4 Hz, H-8), 7.15 (1H, t, J = 3.0 Hz, vinyl-H),
7.25 (1H, d, J = 2.2 Hz, H-5), 7.35 (1H, bs, NH). GC−MS: m/z = 318
(19) [M+ + 2], 317 (55) [M+ + 1], 316 (95) [M+], 275 (19), 274
(53), 273 (100), 256 (19), 253 (10), 252 (15), 245 (18), 231 (11),
219 (18), 218 (11), 207 (20), 176 (11). Anal. (C17H20N2O2S) C, H,
N.
1
Et2O/petroleum ether, 20% yield, whitish solid, mp 87−88 °C. H
NMR (200 Hz) δ 1.30 (6H, s, CH3), 2.50−2.60 and 2.75−2.85 (each
2H, m, CH2CH2CO), 2.95 (2H, s, SCH2), 6.50 (1H, bs, H-5), 6.85−
6.95 (1H, m, H-7), 7.15 (1H, d, J = 8.3 Hz, H-8), 7.60 (1H, t, J = 2.8
Hz, vinyl-H). GC−MS: m/z = 327 (100) [M+], 312 (77), 284 (14),
271 (49), 230 (56), 217 (20). Anal. (C16H16F3NOS) C, H, N.
2,2-Dimethyl-4-(1-oxo-2-cyclopenten-2-yl)-3,4-dihydro-2H-
1
1,4-benzothiazine (5). 79% yield, yellow oil. H NMR (MeOH-d4)
(200 MHz) δ 1.30 (6H, s, CH3), 2.25−2.45 and 2.55−2.65 (each 2H,
m, CH2CH2CO), 3.45 (2H, s, CH2), 6.55 (1H, dt, J = 1.4, 8.1 Hz, H-
6), 6.70 (1H, dd, J = 1.5, 7.6 Hz, H-8), 6.75−6.85 (1H, m, H-7), 6.90
(1H, dd, J = 1.7, 7.6 Hz, H-5), 7.19 (1H, t, J = 3.1 Hz, vinyl-H). GC−
MS: m/z = 260 (14) [M+ + 1], 259 (79) [M+], 217 (15), 216 (100),
203 (12), 188 (18), 162 (14), 161 (13). Anal. (C15H17NOS) C, H, N
4-(1-Oxo-2-cyclopenten-2-yl)-2,2,6-trimethyl-3,4-dihydro-
2H-1,4-benzothiazine (6). Recrystallized from petroleum ether, 15%
yield, orange solid, mp 63−65 °C. 1H NMR (200 MHz) δ 1.40 (6H, s,
CH3), 2.20 (3H, s, ArCH3), 2.50−2.60 and 2.60−2.70 (each 2H, m,
CH2CH2CO), 3.60 (2H, s, CH2), 6.60−6.75 (2H, m, H-5 and H-7),
6.95 (1H, d, J = 8.0 Hz, H-8), 7.10 (1H, t, J = 3.0 Hz, vinyl-H). GC−
MS: m/z = 274 (26) [M+ + 1], 273 (100) [M+], 231 (26), 230 (100),
216 (20), 202 (30), 176 (23), 175 (20). Anal. (C16H19NOS) C, H, N.
N,N-Diisobutyl-2,2-dimethyl-4-(5-oxo-1-cyclopenten-1-yl)-
3,4-dihydro-2H-1,4-benzothiazine-6-sulfonamide (7). Recrystal-
lized from petroleum ether/Et2O, 15% yield, white solid, mp 115−116
°C. 1H NMR (200 MHz) δ 0.95 (12H, d, J = 6.6 Hz, CH2CH(CH3)2),
1.45 (6H, s, CH3), 1.85−2.00 (2H, m, CH2CH(CH3)2), 2.50−2.55
and 2.60−2.65 (each 2H, m, CH2CH2CO), 2.70 (4H, d, J = 7.5 Hz,
CH2CH(CH3)2), 3.60 (2H, s, CH2), 7.00−7.05 (1H, m, H-5), 7.10−
7.20 (3H, m, H-7, H-8 and vinyl-H). GC−MS: m/z = 450(29) [M+],
407 (35), 258 (100), 207 (10). Anal. (C23H34N2O3S2) C, H, N.
6-Acetyl-2,2-dimethyl-4-(1-oxo-2-cyclopenten-2-yl)-3,4-di-
hydro-2H-1,4-benzothiazine (8). Recrystallized from Et2O, 51%
yield, yellow solid, mp 131−132 °C. 1H NMR (200 MHz) δ 1.40 (6H,
s, CH3), 2.45 (3H, s, CH3CO), 2.50−2.55 and 2.55−2.60 (each 2H,
m, CH2CH2CO), 3.55 (2H, s, CH2), 7.05−7.15 (2H, m, H-8 and
vinyl-H), 7.30−7.40 (2H, m, H-5 and H-7). GC−MS: m/z = 301 (83)
[M+], 258 (100), 245 (16), 230 (19), 204 (14), 188 (12). Anal.
(C17H19NO2S) C, H, N.
2-Methyl-6-trifluoromethyl-3,4-dihydro-2H-1,4-benzothia-
zine (12). A solution of 2130 (0.80 g, 3.24 mmol) in dry THF (50
mL) was added dropwise and under nitrogen atmosphere to a
suspension of LiAlH4 (0.31 g, 8.17 mmol) in THF (20 mL) cooled to
0 °C. After the addition was complete, the mixture was refluxed for 3 h
and then cooled, and EtOAc was carefully added to destroy the excess
LiAlH4. The mixture was diluted with water and extracted with EtOAc.
The combined organic layers were washed with HCl, 2 N, brine, and
then dried and evaporated to dryness, yielding the title compound 12
as a whitish solid (0.49 g, 65%), which was used in the next step
without further purification, mp 106−107 °C. 1H NMR (400 MHz) δ
1.50 (3H, d, J = 6.7 Hz, CH3), 3.35 (1H, dd, J = 7.9, 11.8 Hz, CH2),
3.45−3.55 (1H, m, SCH), 3.75 (1H, dd, J = 2.8, 11.8 Hz, CH2), 4.30
(1H, bs, NH), 6.80 (1H, d, J = 1.7 Hz, H-5), 6.95 (1H, dd, J = 1.7, 8.1
Hz, H-7). 7.20 (1H, d, J = 8.1 Hz, H-8). GC−MS: m/z = 233 (100)
[M+], 232 (16), 218 (34), 217 (14), 214 (16), 205 (16), 204 (96), 200
(10), 198 (14), 185 (17), 184 (25), 172 (21), 157 (12), 145 (10).
2,2,6-Trimethyl-3,4-dihydro-2H-1,4-benzothiazine (16). The
title compound was prepared according to the procedure used for
compound 12 starting from 2,2,6-trimethylbenzothiazinone 23.
Compound 16 was obtained in 94% yield as a whitish solid, mp
1
67−68 °C. H NMR (200 MHz) δ 1.40 (6H, s, CH3), 2.20 (3H, s,
ArCH3), 3.25 (2H, s, CH2), 4.05 (1H, bs, NH), 6.35 (1H, s, H-5), 6.45
(1H, dd, J = 1.2, 7.8 Hz, H-7), 6.85 (1H, d, J = 7.8 Hz, H-8).
N,N-Diisobutyl-2,2-dimethyl-3,4-dihydro-2H-1,4-benzothia-
zine-6-sulfonamide (17). A mixture of 26 (2.07 g, 4.31 mmol) and
dry K2CO3 (1.24 g, 8.97 mmol) in dry MeOH (20 mL) was stirred for
20 min. The solvent was evaporated to dryness, and the obtained
residue was treated with water. The formed precipitate was filtered off
and dried to give 17 (1.66 g, 98%) as a whitish solid which was used in
2,2-Dimehyl-6-ethyl-4-(1-oxo-2-cyclopenten-2-yl)-3,4-dihy-
dro-2H-1,4-benzothiazine (9). Recrystallized from petroleum ether,
1
34% yield, white solid, mp 98−99 °C. H NMR (200 MHz) δ 1.15
(3H, t, J = 7.6 Hz, CH2CH3), 1.35 (6H, s, CH3), 2.30−2.65 (6H, m,
CH2CH2CO and CH2CH3), 3.60 (2H, s, CH2), 6.65−6.70 (2H, m, H-
5 and H-7), 6.95 (1H, d, J = 8.6 Hz, H-8), 7.05 (1H, t, J = 3.0 Hz,
vinyl-H). GC−MS: m/z = 288 (21) [M+ + 1], 287 (100) [M+], 245
(18), 244 (99), 231 (18), 216 (20), 190 (10). Anal. (C17H21NOS) C,
H, N.
1
the next step without further purification, mp 119−120 °C. H NMR
(400 MHz) δ 0.90 (12H, d, J = 6.6 Hz, CH2CH(CH3)2), 1.40 (6H, s,
CH3), 1.85−1.95 (2H, m, CH2CH(CH3)2), 2.85 (4H, d, J = 7.5
CH2CH(CH3)2), 3.25 (2H, s, CH2), 6.90 (1H, d, J = 1.7 Hz, H-5),
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6.95 (1H, dd, J = 1.7, 8.1 Hz, H-7), 7.05 (1H, d, J = 8.1 Hz, H-8).
GC−MS: m/z = 370 (50) [M+], 327 (63), 242 (41), 178 (100), 122
(13).
temperature for 2 h, then poured into ice−water and extracted with
EtOAc. The combined organic layers were washed with water, brine,
then dried and evaporated to dryness. The obtained solid residue was
purified by flash column chromatography, eluting with cyclohexane/
EtOAc, 9:1, to give 25 (4.48 g, 33%) as a yellowish solid, mp 88−89
6-Acetyl-2,2-dimethyl-3,4-dihydro-2H-1,4-benzothiazine
(18).32 The title compound was prepared according to the procedure
used for compound 17 starting from 4-trifluoroacetyl derivative 27.
The crude dark product was purified by flash column chromatography,
eluting with cyclohexane/EtOAc, 9:1, followed by crystallization from
petroleum ether/Et2O (9:1) to give 18 (75%) as colorless solid, mp
108−110 °C (lit., mp 109−110 °C).32 1H NMR (200 MHz) δ 1.45
(6H, s, CH3), 2.50 (3H, s, CH3CO), 3.25 (2H, d, J = 2.9 Hz, CH2),
4.25 (1H, bs, NH), 7.00 (1H, d, J = 8.1 Hz, H-8), 7.15 (1H, d, J = 1.8
Hz, H-5), 7.20 (1H, dd, J = 1.8, 8.1 Hz, H-7). GC−MS: m/z =
222(13) [M+ + 1], 221 (99) [M+], 179 (14), 178 (100), 166 (10), 136
(10), 135 (12).
1
°C. H NMR (400 MHz) δ 1.60 (6H, s, CH3), 4.00 (2H, bs, CH2),
7.45 (1H, d, J = 8.5 Hz, H-8), 7.85 (1H, d, J = 8.5 Hz, H-7), 8.30 (1H,
bs, H-5).
N,N-Diisobutyl-2,2-dimethyl-4-trifluoroacetyl-3,4-dihydro-
2H-1,4-benzothiazine-6-sulfonamide (26). A mixture of N,N-
diisobutylamine (0.69 g, 0.93 mL, 5.35 mmol) and Et3N (0.54 g, 0.74
mL, 5.35 mmol) in dry THF (10 mL) was added dropwise to a
solution of sulfonyl chloride derivative 25 (2.00 g, 5.35 mmol) in dry
THF (10 mL). The mixture was stirred at 0 °C for 1 h and at room
temperature for 3 h, then poured into ice−water and extracted with
EtOAc. The combined organic layers were washed with brine, dried,
and evaporated to dryness to give derivative 26 (2.49 g, 99%) as a light
yellow solid which was used in the next step without further
purification, mp 84−85 °C. 1H NMR (400 MHz) δ 0.90 (12H, d, J =
6.6 Hz, CH2CH(CH3)2), 1.50 (6H, s, CH3), 1.85−1.95 (2H, m,
CH2CH(CH3)2), 2.85 (4H, d, J = 7.5 Hz, CH2CH(CH3)2), 3.85 (2H,
bs, CH2), 7.20−7.35 (1H, m, H-8), 7.50−8.10 (2H, m, H-5 and H-7).
GC−MS: m/z = 466 (8) [M+], 425 (24), 424 (42), 423 (100), 340
(11), 339 (16), 338 (74), 290 (21), 275 (17), 274 (79), 218 (10), 190
(14).
6-Ethyl-2,2-dimethyl-3,4-dihydro-2H-1,4-benzothiazine (19).
The title compound was prepared according to the procedure used for
compound 12 starting from 6-acetylbenzothiazine 18. The crude
reaction product was purified by flash column chromatography, eluting
1
with cyclohexane/EtOAc, 9:1, to yield 19 as a brown oil (52%). H
NMR (200 MHz) δ 1.25 (3H, t, J = 7.6 Hz, CH2CH3), 1.45 (6H, s,
CH3), 2.55 (2H, q, J = 7.6 Hz, CH2CH3),), 3.25 (2H, s, CH2), 3.95
(1H, bs, NH), 6.40 (1H, d, J = 1.8 Hz, H-5), 6.55 (1H, dd, J = 1.8, 8.0
Hz, H-7), 6.90 (1H, d, J = 8.0 Hz, H-8). GC−MS: m/z = 208 (25)
[M+ + 1], 207 (100) [M+], 192 (40), 165 (18), 164 (95), 152 (21),
149 (21), 136 (15).
6-Acetyl-2,2-dimethyl-3,4-dihydro-4-trifluoroacetyl-2H-1,4-
benzothiazine (27). Under strictly dry conditions, freshly distilled
acetyl chloride (6.83 g, 6.19 mL, 87.01 mmol) was added dropwise to
a mechanically stirred mixture of 4-trifluoroacetylbenzothiazine 24
(8.00 g, 29.10 mmol) and AlCl3 (7.77 g, 58.27 mmol). After 1 h, an
additional amount of acetyl chloride (4.55 g, 4.12 mL, 57.96 mmol)
was added. The mixture was stirred for an additional 4 h and then
poured into ice−water. The obtained pink solid was filtered off,
washed with petroleum ether, and dried to afford 27 (5.72 g, 62%) as a
white solid which was used in the next step without further
purification, mp 66−68 °C. 1H NMR (200 MHz) δ 1.50 (6H, s,
CH3), 2.55 (3H, s, CH3CO), 3.85 (2H, bs, CH2), 7.15 (1H, d, J = 8.2
Hz, H-8), 7.75 (1H, dd, J = 2.1, 8.2 Hz, H-7), 7.95 (1H, d, J = 2.1 Hz,
2-Methyl-2-[(4-methyl-2-nitrophenyl)thio]propanoic Acid
(22). A catalytic amount of TBAB (5−10% w/w) was added to a
stirred solution of 4-chloro-3-nitrotoluene (3.00 g, 2.31 mL, 17.48
mmol) in dry DMSO (10 mL) heated at 100 °C. After 5 min, Cs2CO3
(17.10 g, 52.48 mmol) and 2-mercapto-2-methylpropanoic acid40
(4.19 g, 34.87 mmol) were added. After 2 h, the mixture was cooled,
poured into ice−water, washed with EtOAc, acidified to pH 2 with 12
N HCl, and extracted with EtOAc. The combined organic layers were
washed with brine, dried, and evaporated to dryness. The residue was
treated with water, filtered off, and dried, yielding 22 (4.03 g, 90%) as
a yellow ochre solid which was used in the next step without further
1
purification, mp 100−102 °C. H NMR (200 MHz) δ 1.50 (6H, s,
1
H-5). 2D H NMR NOESY showed three relevant NOE cross-peaks:
CH3), 2.45 (3H, s, ArCH3), 7.35 (1H, ddd, J = 0.7, 1.9, 8.0 Hz, H-5),
7.55 (1H, d, J = 8.0, H-6), 7.60 (1H, dd, J = 0.7, 1.9 Hz, H-3).
2,2,6-Trimethyl-2H-1,4-benzothiazin-3(4H)-one (23). Acti-
vated Fe powder (3.94 g, 70.56 mmol) was added portionwise to a
suspension of acid 22 (4.00 g, 15.67 mmol) in a mixture of AcOH (30
mL) and water (10 mL). After 1 h the reaction mixture was filtered
over Celite, washing with AcOH, and the filtrate was diluted with
water. The obtained precipitate was filtered off and washed with a
saturated solution of NaHCO3 and dried to give 23 (2.08 g, 64%) as
whitish solid which was used in the next step without further
CH3CO → H-5; CH3 → H-7; gem-CH3 → H-8. GC−MS: m/z = 318
(22) [M+ + 1], 317 (100) [M+], 302 (45), 274 (12), 248 (49), 220
(11), 206 (12), 178 (15).
Pharmacology. All the animal experimental procedures were
carried out following the guidelines of the European Community
Council Directive 86-609 and were approved by the Ethical
Committee of the University of Pisa, Italy, for animal experimentation.
Reagents and reference compounds are purchased from Sigma-Aldrich
unless otherwise indicated. ANOVA and Student t test were selected
as statistical analysis; P < 0.05 was considered a significant statistical
difference. Experimental data were analyzed by a computer fitting
procedure (software GraphPad Prism 4.0).
1
purification, mp 176−177 °C. H NMR (200 MHz) δ 1.50 (6H, s,
CH3), 2.35 (3H, s, ArCH3), 6.70 (1H, s, H-5), 6.85 (1H, d, J = 8.0 Hz,
H-7), 7.20 (1H, d, J = 8.0 Hz, H-8), 8.65 (1H, bs, NH).
Vasorelaxant Activity. To determine a possible vasodilator
mechanism of action, the compounds were tested on isolated thoracic
aortic rings of male normotensive Wistar rats (250−350 g). The rats
were sacrificed by cervical dislocation under light ether anesthesia and
bled. The aorta were immediately excised and freed of extraneous
tissues. The endothelial layer was removed by gently rubbing the
intimae surface of the vessels with a hypodermic needle. Then 5 mm
wide aortic rings were suspended under a preload of 2 g in 10 mL of
organ baths containing Tyrode solution (composition of saline in mM:
NaCl 136.8; KCl 2.95; CaCl2 1.80; MgSO4·7H2O 1.05; NaH2PO4
0.41; NaHCO3 11.9; glucose 5.5) thermostated at 37 °C and
continuously gassed with a mixture of O2 (95%) and CO2 (5%).
Changes in tension were recorded by means of an isometric transducer
(Basile model 7005) connected to a unirecord microdynamometer
(Basile model 7050). After an equilibration period of 60 min, the
endothelial removal was confirmed by administrating acetylcholine
(ACh) (10 μM) to KCl (25 mM) precontracted vascular rings. A
<10% relaxation of the KCl-induced contraction was indicative of an
acceptable lack of the endothelial layer, while the organs, showing a
2,2-Dimethyl-4-trifluoroacetyl-3,4-dihydro-2H-1,4-benzo-
thiazine (24). A solution of trifluoroacetic anhydride (18.75 g, 12.58
mL, 183.66 mmol) in dry CH2Cl2 (10 mL) was added dropwise to a
solution of 1,4-benzothiazine 1533 (10 g, 55.86 mmol) in dry CH2Cl2
(30 mL) and dry pyridine (8.81 g, 8.99 mL, 111.58 mmol) cooled to 0
°C. The mixture was stirred for 10 min and then poured into ice−
water. The organic layer was separated and evaporated to dryness. The
residue was dissolved in EtOAc and sequentially washed with 2 N
HCl, water, and brine, then dried and evaporated to dryness, giving 24
as a red oil (14.6 g, 97%) which was used in the next step without
1
further purification. H NMR (200 MHz) δ 1.50 (6H, s, CH3), 3.80
(2H, bs, CH2), 7.00−7.25 (4H, m, ArH). GC−MS: m/z = 276 (17)
[M+ + 1], 275 (100) [M+], 232 (11), 206 (50), 178 (11), 163 (23),
144 (13), 136 (24).
2,2-Dimethyl-4-trifluoroacetyl-3,4-dihydro-2H-1,4-benzo-
thiazine-6-sulfonyl Chloride (25). Chlorosulfonic acid (21.10 g,
12.04 mL, 181.1 mmol) was added dropwise under nitrogen
atmosphere to 4-trifluoroacetylbenzothiazine 24 (10.0 g, 36.30
mmol) cooled to 0 °C. The reaction mixture was stirred at room
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10% relaxation (i.e., significant presence of the endothelium), were
discarded. At 30−40 min after the confirmation of the endothelium
removal, the aortic preparations were contracted by treatment with a
single concentration of KCl (25 mM). When the contraction reached a
stable plateau, 3 times increasing concentrations of the tested
compounds or of the reference compound LCRK were added
cumulatively. Preliminary experiments showed that KCl-induced
contractions remained in a stable tonic state for at least 1 h. In
other sets of experiments, the potassium channel blocker glibencla-
mide (1 μM) was added before the KCl (25 mM) induced
contraction, and then selected compounds were administered. KCl
and ACh hychloride were dissolved in bidistilled water. Glibenclamide,
LCRK, and all the other synthesized derivatives were dissolved (10
mM) in DMSO and further diluted in bidistilled water. All the
solutions were freshly prepared immediately before the pharmaco-
logical experimental procedures. Previous experiments showed a
complete ineffectiveness of the administration of the vehicle. The
vasorelaxing efficacy was evaluated as the maximal vasorelaxing
response, expressed as the percentage (%) of the contractile tone
induced by KCl, 25 mM. The potency parameter was expressed as
pIC50, which was calculated as the negative logarithm of the molar
concentration of the test compounds, evoking a half reduction of the
contractile tone induced by 25 mM KCl. The efficacy and potency
parameters were expressed as the mean standard error, for 5−10
experiments.
Functional Studies. Cultured islets were picked up under a
dissecting microscope and preincubated for 30 min in a 24-well plate
(10 islets/well) in Krebs−Ringer−HEPES (KRH) buffer, pH 7.4,
containing 2.8 mM glucose and 1% bovine serum albumin. After
preincubation, the buffer was removed and islets were incubated for 1
h in the same buffer containing 2.8 or 16.7 mM glucose in the
presence or absence of various concentrations of either compound 8
(1, 10, and 100 nM) or compound 9 (10, 100, and 1000 nM),
dissolved in DMSO and properly diluted to obtain work
concentrations. The KATP blocker glibenclamide (1 μM) and the
KATP activator diazoxide (80 μM) were also used to modulate glucose-
stimulated insulin release and serve as suitable controls in this
experimental setting. At the end of the incubation period, the buffer
was taken for measurement of released insulin and 1 mL of cold
acidified EtOH (EtOH/H2O/concentrated HCl, 150:47:3, v/v) was
added into the wells for extraction and subsequent measurement of
islet insulin content. Determination of total insulin content was made
to verify homogeneity of islet batches in incubation experiments.
Insulin was measured by radioimmunoassay (rat insulin RIA kit,
Millipore, St. Charles, MO, U.S.).
ASSOCIATED CONTENT
■
S
* Supporting Information
Crystal structure analysis of compound 25, additional
references, and combustion analyss results for compounds 2−
11. This material is available free of charge via the Internet at
http://pubs.acs.org.
Vascular Smooth Muscle Hyperpolarizing Activity. Human
aortic smooth muscle cell (HASMC, Invitrogen) were cultured in
medium 231 (Invitrogen) supplemented with smooth muscle growth
supplement (SMGS, Invitrogen) and 1% of 100 units/mL penicillin
and 100 mg/mL streptomycin in tissue culture flasks at 37 °C in a
humidified atmosphere of 5% CO2.
AUTHOR INFORMATION
■
Corresponding Author
HASMCs were cultured up to about 90% confluence, and at 24 h
before the experiment cells were seeded onto a 96-well black plate,
clear bottom precoated with 1% gelatin (from porcine skin) at density
of 72 × 103 per well. After 24 h to allow cell attachment, the medium
was replaced and cells were incubated for 1 h with an appropriate
buffer (HEPES 20 mM, NaCl 120 mM, KCl 2 mM, CaCl2·2H2O 2
mM, MgCl2·6H2O, glucose 5 mM, pH 7.4, at room temperature)
containing the anionic DiBAC4(3) 2.5 μM. This probe spreads
between cellular and extracellular fluids in a membrane-potential-
dependent manner (following the Nernst rule), thus allowing
assessment of changes in membrane potential by means of
spectrofluorimetric recording at excitation and emission wavelengths
of 488 and 520 nm, respectively (multiwells reader, EnSpire, Perkin-
Elmer). In particular, an increase of fluorescence, corresponding to an
inward flow of the dye, reflects a membrane depolarization, while a
decrease in fluorescence, due to an outward flow of the dye, is linked
to membrane hyperpolarizing effects. After the assessment of baseline
fluorescence, the tested compounds or the reference drug LCRK was
added to evaluate the influence on membrane potential. The
involvement of specific ion channels is detected using a selective
channel blocker, glibenclamide, which was added 10 min before the
tested compounds and LCRK, when required.
Changes in fluorescence were calculated as relative fluorescence
decrease ((Ft − F0)/F0) where F0 is the basal fluorescence (before the
addition of the tested compounds or LCRK) and Ft is the fluorescence
at time t (after the addition of the tested compounds or LCRK). Then
changes in fluorescence were expressed as % of the maximal effect
produced by LCRK 100 μM. The potency parameter pEC50 is the
negative logarithm of the molar concentration of the test compounds,
evoking a half-maximal decrease of relative fluorescence. The
experiments were performed in six replicates.
*For G.M.: phone, +39-075-5855126; fax, +39-075-5855115; e-
mail, giuseppe.manfroni@unipg.it. For V.C.: phone, +39-050-
2219589; fax, +39-050-2219589; e-mail, calderone@farm.unipi.
it.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to Dr. Ferdinando Costantino for
kindly providing the X-ray data. The authors are grateful to
Roberto Bianconi for his excellent technical assistance.
ABBREVIATIONS USED
■
DiBAC4(3), bis(1,3-dibutylbarbituric acid)trimethine oxonol;
DIPEA, N,N-diisopropylethylamine; HASMC, human aortic
smooth muscle cell; KRH, Krebs−Ringer−HEPES; KATP,
ATP-sensitive potassium; Kir, inwardly rectifying potassium
channel; KCO, potassium channel opener; SAR, structure−
activity relationship; LCRK, levcromakalim; RPMI, Roswell
Park Memorial Institute; SUR, sulfonylurea receptor; TBAB,
tetra-n-butylammonium bromide
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