Novel 1,4-benzothiazine derivatives as large conductance Ca 2+-activated potassium channel openers

Article abstract of DOI:10.1021/jm701605f

The design and synthesis of a novel class of 1,4-benzothiazines targeted for the large-conductance calcium-activated potassium channels (BK) are presented. In vitro functional characterization of BK channel opening activity was assessed by measuring the r

Full text of DOI:10.1021/jm701605f

J. Med. Chem. 2008, 51, 5085–5092
5085
Novel 1,4-Benzothiazine Derivatives as Large Conductance Ca2+-Activated Potassium Channel
Openers
Vincenzo Calderone,‡ Roberto Spogli,† Alma Martelli,‡ Giuseppe Manfroni,† Lara Testai,‡ Stefano Sabatini,† Oriana Tabarrini,†
and Violetta Cecchetti*,†
Dipartimento di Chimica e Tecnologia del Farmaco, UniVersita` degli Studi di Perugia, Via del Liceo, 1, 06123 Perugia, Italy and
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, UniVersita` degli Studi di Pisa, Via Bonanno 6, 56126 Pisa, Italy
ReceiVed December 20, 2007
The design and synthesis of a novel class of 1,4-benzothiazines targeted for the large-conductance calcium-
activated potassium channels (BK) are presented. In vitro functional characterization of BK channel opening
activity was assessed by measuring the relaxation of isolated rat aortic rings precontracted with KCl 20
mM. The results of this study show that the 1,4-benzothiazine heterocyclic nucleus is a suitable backbone
for designing novel BK-openers; indeed, some of these new 1,4-benzothiazine derivatives had a vasorelaxant
potency comparable or superior to that of reference BK-activator NS-1619 (1).
Introduction
The large-conductance calcium-activated potassium (BKa or
BKCa or maxi-K) channels belong to the wide family of voltage-
activated potassium channels and exhibit a very high single-
channel conductance.1,2 They are characteristically activated by
the concerted influences of membrane depolarization and
increases in cytosolic Ca2+ levels.3 These features are deter-
minant for their role as feedback regulators of the activity of
voltage-dependent calcium channels. BK channels are present
in many cell types, including neurons and smooth muscle cells,
where they are involved in a variety of physiological processes.4
Hence, in the nervous system, BK channels regulate neuronal
excitability and neurotransmitter release,5 while in smooth
muscle, they play a key role in setting the contractile tone of
vascular,6,7 bronchotracheal,7,8 urethral,9 uterine,10 or gas-
trointestinal11 smooth musculature. These transmembrane pro-
teins exist as a complex of four R-subunits which form the
channel and may be coassembled with four ꢀ regulatory
Figure 1. Chemical structures of reference compounds and design
concept.
subunits.4ThepresenceofmultiplesplicevariantsofR-subunits12,13
and multiple subtypes of ꢀ-subunits (ꢀ1-ꢀ4)14–17 generates
A number of naturally derived compounds29 or small synthetic
considerable diversity within the BK family that may be tissue-
and organ-specific.4,18 In view of these properties and as a
consequence of their central role in regulating cell activity, BK
channels are particularly appealing as a therapeutic drug
target.19–23
molecules20,21,24,25 have been identified as BK-openers. The
benzimidazolone derivatives, typified by NS-1619 (1) and NS-
004 (2),30 (Figure 1) were the pioneer BK-activators and have
been the reference models that have led to the design of several
novel synthetic BK-openers. As a consequence, the majority
of small synthetic BK-activators have several structural features
in common.20,21,24,25 These include two aromatic rings linked
via a spacer unit that may be either a heterocyclic or acyclic
moiety, usually a urea function; the heterocyclic spacer is
sometimes fused to one of the aromatic rings. In addition, a
key feature of many BK-openers is the 5-halo-2-hydroxy or
5-halo-2-methoxy substitution pattern present on one of the
aromatic rings; the other aromatic ring often displays electron-
withdrawing groups such as a trifluoromethyl group or a chlorine
atom.
In particular, agents that activate BK channels (BK-openers
or -activators) stabilize the cell by increasing the efflux of
potassium ions, leading to hyperpolarization and thus decrease
the cell excitability and/or cause smooth muscle relaxation. Thus,
BK channel openers could offer a novel therapeutic approach
to several diseases associated with both the central nervous
system and smooth muscle such as stroke, epilepsy, bladder
overactivity, asthma, and hypertension to mention a few.24–28
* To whom correspondence should be addressed. Phone: +39 075 585
5153. Fax: +39 075 585 5115. E-mail: violetta.cecchetti@unipg.it.
†
Dipartimento di Chimica e Tecnologia del Farmaco, Universita` degli
Recently, we found that the 1,4-benzothiazine nucleus is a
versatile skeleton to obtain new potassium channel activators.
In fact, 2,2-dimethyl-3,4-dihydro-2H-1,4-benzothiazine deriva-
tives, suitably functionalized at the N-4 and the C-6 positions
as in compounds of type A (Figure 1), were identified by us as
highly potent ATP-sensitive potassium (KATP) channel activators
Studi di Perugia.
‡
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
nologie, Universita` degli Studi di Pisa.
a Abbreviations: BK, large-conductance calcium-activated potassium
channel; KATP, ATP-sensitive potassium channel; LDA, lithium diisopro-
pylamide; TEA, tetraethylammonium chloride; IbTX, iberiotoxin; SAR,
structure-activity relationship; ACh, acetylcholine.
10.1021/jm701605f CCC: $40.75
2008 American Chemical Society
Published on Web 08/05/2008

5086 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Calderone et al.
Table 1. Structure, Vasorelaxing Potency and Efficacy of the Compounds Reported in This Studya
b
d
compd
n
R6
R4
X
R2′
R3′
R5′
Emax ( SEM,c %
pIC50 ( SEMc
3e
4e
5
6
7
0
0
0
0
0
0
0
0
1
0
0
0
0
0
H
CF3
H
H
H
H
H
H
H
H
H
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CS
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
42 ( 3
39 ( 9
100
77 ( 5
44 ( 4
NCf
NCf
OMe
OMe
OH
OMe
OH
OMe
OMe
OMe
OH
OMe
OMe
OMe
Br
Br
Br
Cl
Cl
Br
Br
Br
Br
Br
Br
H
4.80 ( 0.14
7.40 ( 0.12
NCf
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
8
9
NEg
NEg
34 ( 4
75 ( 7
34 ( 10
67 ( 15
NCf
10
11
12
13
14
15
16
17a
17b
18
1
4.30 ( 0.07
H
NCf
Me
Me
H
H
H
6.21 ( 0.18
26 ( 3
<25
100
NCf
CH2
CH2
NCf
4.65 ( 0.04
NEg
NEg
70
5.20 ( 0.20
100
5.30
a The synthesized compounds were administered to isolated endothelium-denuded rat aortic rings, precontracted by KCl 20 mM. b Maximal vasorelaxing
effect expressed as the % of contractile tension. c Standard error of a mean 5-10 separate experiments. d Vasorelaxant potency expressed as the negative log
of the concentration evoking a half-reduction of the contractile tone. e The preparation is reported in ref 33. f The parameter could not be calculated because
of the low efficacy (∼ or <50%). g The compound was ineffective.
Scheme 1a
exhibiting vasodilator potency on rat aortic rings in the
subnanomolar range.31,32
On the basis of this finding and considering the structural
features of the known BK-activators, we explored the potential
of 1,4-benzothiazine nucleus for designing and synthesizing a
new series of type B derivatives targeted for BK channels
(Figure 1 and Table 1). The rational pathway used to adapt the
general pharmacophore model of benzimidazolone series to the
1,4-benzothiazine nucleus began with compound 3,33 which has
the minimum structural requirements of the BK pharmacophore
model, that is, two aromatic areas linked by a 2H-1,4-thiazin-
3(4H)-one spacer. The insertion of the electron-withdrawing
trifluoromethyl group at the C-6 position led to derivative 4.33
Both of these 2-aryl-1,4-benzothiazine derivatives, as well as
their analogues synthesized in this study, generally have at least
one stereogenic center at the C-2 position of the benzothiazine
nucleus, which was unresolved in the first approach of this
pharmacological survey.
a Reagents and conditions: (i) DMF; (ii) K2CO3, DMF, 70-80 °C.
and erythro (17b) diastereoisomers were obtained, which were
separated and then evaluated as enantiomeric mixtures.
Compounds 5-9 were then synthesized to evaluate the
influence of 5-halo-2-methoxyl or 5-halo-2-hydroxyl substitution
pattern at the C-2 phenyl moiety; compound 10, having an
additional bromine atom, was also synthesized.
Chemistry
The 2-aryl-2H-1,4-benzothiazine-3(4H)-one derivatives 5, 6,
8, and 10 were synthesized, analogously to compounds 333 and
Recent studies by Li et al.34,35 demonstrated that the acidic
properties of the amide function are an important parameter for
the pharmacological activity of BK-openers. Thus, to evaluate
if the acidity of the amide function of these 1,4-benzothiazine
derivatives is related to their biological activity, it was enhanced
by oxidation of the thiazine sulfur atom to sulfoxide as in
compound 11, or suppressed by N-methylation as in derivatives
12 and 13. The importance of the carbonyl moiety as a hydrogen
bond site was also investigated through the 3-thioxo derivative
14 and 3-deoxo derivatives 15 and 16. In addition, the distance
between the 1,4-benzothiazine nucleus and the C-2 phenyl group
was changed by inserting a hydroxymethylene or a vinyl bridge
as in compounds 17 and 18, respectively. In the case of
compound 17, which has two chiral centers, both threo (17a)
4
33 described in the literature, by reacting 2-aminobenzenethiol
or 2-amino-4-(trifluoromethyl)benzenethiol hydrochloride and
an appropriate R-bromoarylacetic ester (Scheme 1).
The R-bromoarylacetic esters 19 and 20 were obtained by
radical bromination of ethyl (2-methoxyphenyl)acetate36 together
with undesired R,R-dibromo derivative 21 (Scheme 2). The
R-bromoarylacetic ester 24 was instead prepared from (2-
methoxyphenyl)acetic acid through chlorination, esterification
and radical bromination (Scheme 2).
(5-Bromo-2-methoxyphenyl)benzothiazinone derivative 6 was
used as starting material to afford different target derivatives 7,
11-16 (Scheme 3). In particular, the de-O-methylation of 6
using BBr3 in CH2Cl2 at room temperature afforded phenol
derivative 7 almost quantitatively. The same reaction carried

1,4-Benzothiazines as Ca2+-ActiVated K Channel Openers
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5087
Scheme 2a
using lithium diisopropylamide (LDA) prepared in situ; the
diastereoisomeric separation was carried out by flash chroma-
tography. The threo/erythro configuration was assigned on the
basis of 1H NMR coupling constant values for the C-2
benzothiazine proton and the benzylic one in agreement with
that reported in the literature.38,39
Benzylidene derivative 18 was then obtained as Z-isomer in
a one-pot procedure from the diastereoisomeric mixture 17
(Scheme 4), which was first mesylated at room temperature and
then warmed to reflux. No trace of the E isomer was found.
The Z configuration was attributed on the basis of the vinyl
proton chemical shift. The Z-stereoselectivity is probably due
to steric factors as reported in cases of other benzylidene
derivatives.38
a Reagents and conditions: (i) Br2, hυ, CCl4, reflux; (ii) SO2Cl2, THF,
-10 °C; (iii) EtI, K2CO3, DMF, 50 °C.
Results and Discussion
Scheme 3a
For all of the synthesized compounds 3-18, the BK-opening
activity was evaluated in vitro as the vasorelaxing effect on
endothelium-denuded rat aortic rings precontracted with KCl
(20 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 benzimidazolone derivative 1 chosen as reference
compound.
Although this functional test cannot be considered as an
exhaustive pharmacological characterization, it should be viewed
as a satisfactory pharmacological tool for a preliminary iden-
tification of a BK-mediated mechanism, being that the vasore-
laxing activity of BK-openers is one of the best known and
characteristic pharmacodynamic features of this class of com-
pounds. In addition, previous studies have demonstrated a good
correlation between iberiotoxin (IbTX)-sensitive vasorelaxing
effects (recorded by functional tests) and direct activation of
vascular BK channels (recorded by patch clamp experiments)
and functional studies on vascular smooth muscle preparations
continue to be largely used in the pharmacological characteriza-
tion of BK-activating molecules.40–43
a Reagents and conditions: (i) BBr3, CH2Cl2; (ii) Al2O3 (TsOH, H2O),
Oxone 1 mol equiv, CHCl3; (iii) MeI, t-BuOK, DMF; (iV) Lawesson’s
reagent, MW; (V) THF-BH3 complex, THF, reflux; (Vi) LiAlH4, THF.
The biological data indicate that some of the synthesized
compounds had a nearly complete vasorelaxing effect; some
had a low efficacy, and only a few showed complete inef-
fectiveness. With respect to potency, compounds such as 5, 10,
16, and 17b had pIC50 values close to that of reference
benzimidazolone 1, while derivatives 6 and 12 were significantly
more potent.
Scheme 4a
Obviously, this evidence does not exclude a possible contri-
bution of mechanisms of action other than the activation of BK
channels. Moreover, the relative similarity of the pore region,
observed in many potassium channel subtypes, could be another
cause of “nonselectivity” for several potassium channel openers
(e.g., pinacidil, cromakalim, etc.). This problem could be
satisfactorily resolved by using selective blockers.
On the basis of these considerations, the most potent
compounds 6 and 12 were submitted to additional experimental
protocols to further investigate their mechanism of action. In
particular, the vasorelaxing activities of compounds 6 and 12
were significantly antagonized by tetraethylammonium chloride
(TEA) (10 mM; a nonselective blocker of potassium channels).
It should be noted that in these experimental procedures,
vascular smooth muscle is depolarized by KCl 20 mM causing
inward calcium currents, needed to obtain the contractile effect
and, consequently, to study a vasorelaxing activity. The presence
of TEA can cause a further depolarization which could be a
“nonspecific” factor that reduces the activity of the vasodilator
agents. However, the presence of such a simple “functional
a Reagents and conditions: (i) LDA, -55 °C/25 °C, 5-bromo-2-
methoxybenzaldehyde, THF; (ii) (a) MsCl, Et3N, CH2Cl2, (b) 40 °C.
out on compound 8 gave the para-chlorophenol derivative 9
(not shown in the Schemes). Sulfoxide 11 was obtained as a
diastereoisomeric mixture by oxidizing 6 with oxone in CH2Cl2
and in the presence of Al2O3. N-alkylation of 6 with MeI and
t-BuOK in DMF yielded derivative 12, which was further de-
O-methylated to phenol derivative 13 using BBr3 in CH2Cl2 at
room temperature. The reaction of 6 using Lawesson’s reagent
and catalyzed by domestic microwaves in neat condition
afforded the thioxo derivative 14. The reduction of the lactam
carbonyl group of 6 by using BH3-THF complex in THF at
reflux afforded the deoxo derivative 15, while derivative 16 was
directly obtained by using LiAlH4 in THF at room temperature.
The threo and erythro diastereoisomer derivatives, 17a and
17b respectively, were prepared (Scheme 4) via an aldol-like
condensation of 6-(trifluoromethyl)-2H-1,4-benzothiazin-3(4H)-
one37 and commercial 5-bromo-2-methoxybenzaldehyde by

5088 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Calderone et al.
CF3 moiety at the C-6 position, in agreement with the current
benzimidazolone SAR.
The role of the amidic function was evaluated by restricting
the substitution pattern to a CF3 at the C-6 position and a
5-bromo-2-methoxphenyl group at the C-2 position, which were
found to be optimal in the preliminary set. N-methylation of
the amide gave derivative 12, which showed a decreased potency
of one logarithmic unit and a slightly decreased efficacy when
compared to 6. The oxidation of the sulfur atom to sulfoxide,
in order to increase the acidity of the amidic proton, was
detrimental because derivative 11 showed no activity at all. The
bioisosteric replacement of the carbonyl function by thiol-
carbonyl one gave derivative 14, which had no activity, and
carbonyl reduction to methylene unit gave derivative 15, which
likewise was inactive. These results suggest that, in this case,
the amidic function is of primary importance for obtaining active
compounds. As reported above, the acidic properties of the
amide function are considered as a possible factor that affect
the pharmacological activity of some BK-openers. Moreover,
it must be pointed out that many BK-activators exhibit a
carbonyl moiety in the spacer unit, suggesting that this structural
requirement may play a positive role in the interaction with the
biological target. In this study, the suppression by methylation
of the hydrogen bond donor capacity of the lactam group in
the 2H-1,4-thiazin-3(4H)-one spacer gave the active compound
12. In contrast, the removal of the carbonyl function seemed to
produce a negative impact on the vasorelaxant activity, as shown
by the pharmacological profiles exhibited by the couple of
analogues 6 and 15. These results indicate that, at least in the
1,4-benzothiazine BK-openers, the carbonyl function is more
important for the activity than the amidic hydrogen.
The introduction of a hydroxymethylene spacer unit at the
C-2 position gave rise to the diastereoisomeric derivatives threo
17a and erythro 17b. The erythro derivative has a potency
intermediate between that of 6 and reference benzimidazolone
derivative 1; the threo derivative was instead ineffective. This
behavior could be explained by considering that the threo
derivative can give an intramolecular hydrogen bond39 between
the C-3 carbonyl function and the hydroxyl group that could
be detrimental to activity. The introduction of a double bond in
C-2 afforded Z-benzylidene 18, which was found to be inef-
fective, indicating that the presence of a rigid spacer between
the two aromatic areas annihilates the activity.
Figure 2. Concentration-vasorelaxing effect curves for compound 6
in control conditions (squares) and in the presence of TEA 10 mM
(triangles) or IbTX 100 nM (circles). Vertical bars indicate the standard
error.
antagonism” or “nonspecific” interferences can be excluded
because in previous studies it was demonstrated that TEA was
able to antagonize the effects of some well-known BK-activators
while not inhibiting the vasorelaxing effects of vasodilators
acting through a different mechanism of action. Moreover, as
above indicated, a good correlation was observed between TEA-
and IbTX-sensitive vasorelaxing effects (recorded by functional
tests) and direct activation of vascular BK potassium channels
(recorded by patch clamp experiments).43 Finally, the specific
involvement of BK channels in this pharmacological effect was
also investigated by using IbTX, a well-established selective
BK blocker. In the presence of this toxin, the vasorelaxing effect
of compound 6 was significantly antagonized, suggesting that
the activation of BK channel could account for the vasorelaxing
properties of these compounds (Figure 2).
These preliminary biological data indicate that the 1,4-
benzothiazine nucleus can be considered as a suitable replace-
ment of the benzimidazolone nucleus because some of the 1,4-
benzothiazine derivatives reported in this study showed BK-
mediated vasorelaxant activities that, in some cases, were higher
than that of derivative 1.
Different activity levels were observed in compounds 6-9
having a 5-halo-2-hydroxy- or 5-halo-2-methoxy-phenyl ring
at the C-2 position. Derivative 6, in which the second aromatic
portion is a 5-bromo-2-methoxyphenyl group, has a high level
of vasorelaxing efficacy (77%) and was 100-times more potent
than the reference compound 1. Its 5-bromo-2-hydroxy coun-
terpart 7 was instead inactive, as were the 5-chloro-2-methoxy
and 5-chloro-2-hydroxy derivatives 8 and 9, respectively. These
results are quite surprising and difficult to interpret considering
that these moieties have generally been associated with a high
activity of BK-activators. Compounds 3 and 4, having a naked
phenyl ring at the C-2 position, were also inactive. The
introduction of a second bromine atom at the C-3′ position of
6 led to derivative 10, which was 1000 times less potent than
the parent compound. This result is in sharp contrast with the
structure-activity relationship (SAR) that was obtained for the
bisphenol-based structure BK-openers in which multihalogen
substitution such as 2,4-dichloro- or 2,4-dibromo-phenols was
pivotal for obtaining highly active compounds.35 The above
observed divergences with respect to the SAR based on
benzimidazolones and related compounds led to speculate either
the existence of a peculiar binding mode at the same hypothetical
receptor site or a different binding site for these benzothiazine
derivatives.
Conclusion
In summary, the main observation that has emerged from this
study is that the 1,4-benzothiazine heterocyclic nucleus is a
suitable backbone for designing novel BK-activators; indeed,
some of these new 1,4-benzothiazine derivatives showed a
vasorelaxant potency comparable or superior to that of reference
BK-activator 1. The observed vasorelaxant activity was due to
the activation of BK channels because it was antagonized by
IbTX, a selective blocker; the highest potency was displayed
by the 6-trifluoromethylbenzothiazinone derivative 6 having the
5-bromo-2-methoxyphenyl group linked at the C-2 position of
the 1,4-benzothiazine nucleus.
Although many structural characteristics of these benzothi-
azine derivatives seem to line up with the general pharmacoph-
oric elements typical of most synthetic BK-openers, the
preliminary SAR defined for this novel series showed some
peculiar divergences with respect to that defined for the
benzimidazolone series and related compounds, leading to
speculation of a different mode of channel protein recognition.
This fact provides an interesting rationale for the further
On the other hand, the lower activity of C-6 unsubstituted
derivative 5 when compared to 6 shows the importance of a

1,4-Benzothiazines as Ca2+-ActiVated K Channel Openers
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5089
crystallized from EtOH to yield derivative 8 (45%) as white needles;
mp 169-170 °C. 1H NMR (200 MHz, CDCl3) 3.86 (s, 3H, OCH3),
5.18 (s, 1H, SCH), 6.86 (d, 1H, J ) 8.8 Hz, H-3′), 7.15 (d, 1H, J
) 2.5 Hz, H-6′), 7.17-7.22 (m, 1H, H-5), 7.23-7.29 (m, 2H,
H-Ar), 7.38 (d, 1H, J ) 8.1 Hz, H-8), 9.72 (bs, 1H, NH). GC-MS
m/z (%): 375 (40) [M+ + 1], 373 (100) [M+ - 1], 354 (6), 340
(11), 330 (19), 312 (10), 204 (31), 167 (9), 155 (11), 142 (15), 125
(9), 111 (4), 91 (5).
development of a 1,4-benzothiazine-based series of derivatives,
in order to obtain a more detailed understanding of the SAR of
this novel class of BK-openers.
Experimental Section
Reagents and solvents were purchased from common commercial
suppliers and were used as received. Organic solutions were dried
over anhydrous Na2SO4 and concentrated with a Bu¨chi rotary
evaporator at low pressure. Yields were of purified product. All
starting materials were commercially available unless otherwise
indicated. All reactions were routinely checked by thin-layer
chromatography (TLC) on silica gel 60 F254 (Merck) and visualized
by using UV. Column chromatography separations were carried
out on Merck silica gel 60 (mesh 70-230) and flash chromatog-
raphy on Merck silica gel 60 (mesh 230-400). Melting points were
determined in capillary tubes (Bu¨chi Electrotermal model 9100)
and are uncorrected. 1H NMR spectra were recorded on Bruker
AC-200 (200 MHz) or Avance DRX 400 (400 MHz) spectrometers
in the indicated solvent. Chemical shifts are given in ppm (δ)
relative to tetramethylsilane as internal standard. The spectral data
are consistent with the assigned structures. GC-MS analyses were
carried out with an HP 6890 gas chromatograph (25 m dimethyl
silicone capillary column) equipped with an HP 5973 mass selective
detector. Elemental analyses were performed on a Carlo Erba
elemental analyzer, model 1106, and data for C, H, and N are within
( 0.4% of the theoretical values.
2-(5-Bromo-2-hydroxyphenyl)-6-trifluoromethyl-2H-1,4-ben-
zothiazin-3(4H)-one (7). Compound 6 (0.500 g, 1.2 mmol) was
added in small portions under nitrogen atmosphere to a 1 M solution
of BBr3 in CH2Cl2 (15 mL, 15 mmol). The reaction was stirred for
4 h, quenched with water and then CHCl3 (20 mL) was added.
The organic layer was washed with water and brine, dried, and
evaporated to dryness. The residue was crystallized from cyclo-
hexane/EtOAc (8:2) to yield derivative 7 (0.422 g, 87%) as a whitish
1
solid; mp 242-243 °C. H NMR (200 MHz, acetone-d6) 5.16 (s,
1H, SCH), 6.92 (d, 1H, J ) 8.5 Hz, H-3′), 7.20-7.40 (m, 3H,
H-Ar), 7.50-7.60 (m, 2H, H-Ar), 9.30 (bs, 1H, OH), 10.17 (bs,
1H, NH). GC-MS m/z (%): 404 (8) [M+], 403 (46) [M+ - 1], 401
(45) [M+ - 2], 375 (16), 373 (15), 267 (17), 266 (100), 201 (28),
199 (30), 173 (10), 175 (8), 145 (8), 143 (10), 63 (10).
2-(5-Chloro-2-hydroxyphenyl)-6-trifluoromethyl-2H-1,4-ben-
zothiazin-3(4H)-one (9). It was prepared following the same
procedure used to prepare 7 starting from derivative 8. The crude
product was crystallized from cyclohexane/EtOAc (8:2) to yield
1
derivative 9 (75%) as a whitish solid; mp 237-239 °C. H NMR
2-(5-Bromo-2-methoxyphenyl)-2H-1,4-benzothiazin-3(4H)-one
(5). A solution of R-bromoarylacetic ester 19 (0.704 g, 2.0 mmol)
and 2-aminobenzenethiol (0.250 g, 2.0 mmol) in DMF (3 mL) was
stirred at room temperature for 30 h. The reaction mixture was
then poured into ice-water. The precipitated solid was filtered off
and recrystallized from EtOH to afford 5 (0.420 g, 60%) as a white
(200 MHz, acetone-d6) 5.18 (s, 1H, SCH), 6.98 (d, 1H, J ) 8.6
Hz, H-3′), 7.14 (d, 1H, J ) 2.5 Hz, H-6′), 7.19 (dd, 1H, J ) 2.5
and 8.6 Hz, H-4′), 7.32-7.37 (m, 1H, H-7), 7.51-7.56 (m, 2H,
H-Ar), 9.26 (s, 1H, OH), 10.13 (bs, 1H, NH). GC-MS m/z (%):
361(20) [M+], 359 (48), 330 (9), 326 (10), 204 (24), 193 (13), 192
(100), 167 (19), 161 (40), 141 (13), 112 (11), 77 (13), 75 (11), 51
(7).
1
solid; mp 226-228 °C. H NMR (200 MHz, DMSO-d6) 3.78 (s,
3H, OCH3), 4.94 (s, 1H, SCH), 6.94-7.10 (m, 4H, H-Ar),
7.19-7.27 (m, 2H, H-Ar), 7.46 (dd, 1H, J ) 2.5 and 8.8 Hz,
H-Ar), 11.0 (bs, 1H, NH). GC-MS m/z (%): 351 (100) [M+ + 1],
349 (100), 318 (8), 316 (6), 308 (8), 290 (8), 288 (6), 242 (5), 227
(9), 207 (38), 188 (6), 186 (7), 171 (7), 136 (44), 120 (10), 91 (9).
2-(3,5-Dibromo-2-methoxyphenyl)-6-trifluoromethyl-2H-1,4-
benzothiazin-3(4H)-one (10). The title compound was prepared from
2-amino-4-(trifluoromethyl)benzenethiol hydrochloride and R-bro-
moarylacetic ester 20 by a procedure similar to that described for
the preparation of derivative 5. The crude product was crystallized
from EtOH to yield derivative 10 (46%) as a crystalline whitish
2-(5-Bromo-2-hydroxyphenyl)-4-methyl-6-trifluoromethyl-2H-
1,4-benzothiazin-3(4H)-one (13). It was prepared following the same
procedure employed for the preparation of 7 starting from derivative
12. The crude product was crystallized from cyclohexane/EtOAc
(8:2) to yield the derivative 13 (56%) as a cream-colored solid;
mp 167-168 °C. 1H NMR (200 MHz, CDCl3) 3.55 (s, 3H, NCH3),
4.98 (s, 1H, SCH), 6.75 (d, 1H, J ) 8.5 Hz, H-3′), 7.20-7.30 (m,
3H, H-Ar), 7.40-7.47 (m, 1H, H-Ar), 7.65 (d, 1H, J ) 8.1 Hz,
H-8), 7.95 (bs, 1H, OH). GC-MS m/z (%): 419 (48) [M+ + 1],
417 (47) [M+ - 1], 386 (22), 384 (22), 218 (43), 215 (11), 214
(20), 208 (10), 207 (17), 206 (100), 204 (17), 186 (14), 184 (12),
175 (22), 162 (26), 157 (11), 77 (16).
1
solid; mp 193-194 °C. H NMR (200 MHz, DMSO-d6) 3.70 (s,
3H, OCH3), 5.24 (s, 1H, SCH), 7.25-7.32 (m, 3H, H-Ar), 7.51
(d, 1H, J ) 8.4 Hz, H-8), 7.85 (d, 1H, J ) 2.4 Hz, H-4′), 11.15
(bs, 1H, NH). GC-MS m/z (%): 499 (55) [M+ + 1], 497 (100)
[M+], 495 (52) [M+ - 1], 455 (10), 454 (19), 453 (10), 375 (9),
373 (8), 293 (13), 291 (19), 289 (9), 266 (17), 249 (6), 204 (50),
207 (33), 184 (6), 75 (10).
2-(5-Bromo-2-methoxyphenyl)-6-trifluoromethyl-2H-1,4-ben-
zothiazin-3(4H)-one 1-oxide (11). Six g of Al2O3 (type 507 C
neutral, Fluka) were equilibrated with the atmosphere into a 150
mL round-bottomed flask at 120 °C for at least 48 h; it was then
cooled to 25 °C and a solution of p-toluensulfonic acid (0.08 mmol/
g) in CH2Cl2 (20 mL) was added. After evaporation of the solvent
at reduced pressure, water (0.5 mL) was added and the adsorbent
was tumbled on a rotary evaporator at atmospheric pressure until
uniformly free-flowing. A solution of 6 (1.00 g, 2.4 mmol) in CHCl3
(100 mL) was added under stirring followed by Oxone (2.95 g,
4.8 mmol). The slurry was stirred at 25 °C for 48 h. The adsorbent
was then filtered off by washing with EtOAc. The organic layer
was washed with saturated aqueous solution of FeSO4, dried, and
evaporated to dryness. The residue was purified by flash chroma-
tography using a step gradient of cyclohexane/EtOAc (8:2 to 4:6)
as eluant to afford 11 (0.33 g, 32%) as a diastereoisomeric mixture
in about 60/40 ratio. 1H NMR (400 MHz, acetone-d6) 3.86 (s, 1.2H,
OCH3), 3.96 (s, 1.8H, OCH3), 5.52 (s, 0.6H, SCH), 5.77 (s, 0.4H,
SCH), 7.13 (d, 0.4H, J ) 8.8 Hz, H-3′), 7.15 (d, 0.6H, J ) 8.8 Hz,
H-3′), 7.31 (d, 0.6H, J ) 2.4 Hz, H-Ar), 7.55-7.68 (m, 3H,
H-Ar), 7.80 (d, 0.4H, J ) 2.5 Hz, H-Ar), 7.91 (d, 0.6H, J ) 7.7
Hz, H-Ar), 8.11 (d, 0.4H, J ) 6.9 Hz, H-Ar), 10.50 (bs, 1H,
NH). Anal. (C16H11BrF3NO3S) C, H, N.
2-(5-Bromo-2-methoxyphenyl)-6-trifluoromethyl-2H-1,4-ben-
zothiazin-3(4H)-one (6). A solution of 2-amino-4-(trifluorometh-
yl)benzenethiol hydrochloride (2.30 g, 10 mmol) in DMF (5 mL)
was added dropwise to a suspension of R-bromoarylacetic ester 19
(3.52 g, 10 mmol) and K2CO3 (2.76 g, 20 mmol) in DMF (5 mL),
and the resulting mixture was stirred at 70-80 °C for 4.5 h. After
cooling, the mixture was poured into ice-water and the precipitate
solid was filtered off, dried, and recrystallized from MeOH to afford
6 (3.43 g, 82%) as a white solid; mp 184-187 °C. 1H NMR (200
MHz, acetone-d6) 3.90 (s, 3H, OCH3), 5.15 (s, 1H, SCH), 7.05 (d,
1H, J ) 8.8 Hz, H-3′), 7.32 (d, 1H, J ) 2.5 Hz, H-6′), 7.34-7.41
(m, 1H, H-7), 7.47-7.56 (m, 3H, H-Ar), 10.25 (bs, 1H, NH). GC-
MS m/z (%): 419 (100) [M+], 417 (99), 404 (6), 402 (7), 400 (8),
376 (15), 374 (15), 295 (10), 204 (25), 188 (8), 186 (6), 157 (6),
118 (6).
2-(5-Chloro-2-methoxyphenyl)-6-trifluoromethyl-2H-1,4-ben-
zothiazin-3(4H)-one (8). The title compound was prepared following
the same procedure used to preparation of 6 except R-bromoary-
lacetic ester 24 was used instead of 19. The crude product was

5090 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Calderone et al.
2-(5-Bromo-2-methoxyphenyl)-4-methyl-6-trifluoromethyl-2H-
1,4-benzothiazin-3(4H)-one (12). A mixture of derivative 6 (0.100
g, 0.24 mmol) and t-BuOK (0.030 g, 0.27 mmol) in dry DMF (5
mL) was stirred for 15 min. A solution of MeI (0.02 mL, 0.26
mmol) in dry DMF (5 mL) was then added dropwise. After 8 h,
the mixture was poured into ice-water, acidified to pH ≈ 1 with
1 N HCl, and then extracted with EtOAc. The combined organic
layers were washed with brine, dried, and evaporated to dryness.
Crystallization from cyclohexane/EtOAc (8:2) of the crude product
afforded 12 as a white solid (0.093 g, 90%); mp 145-146 °C. 1H
NMR (200 MHz, CDCl3) 3.52 (s, 3H, NCH3), 3.77 (s, 3H, OCH3),
4.97 (s, 1H, SCH), 6.72 (d, 1H, J ) 8.8 Hz, H-3′), 7.10 (d, 1H, J
) 2.3 Hz, H-6′), 7.20-7.44 (m, 4H, H-Ar). GC-MS m/z (%): 434
(30) [M+ + 2], 433 (100) [M+ + 1], 432 (28) [M+], 430 (91)
[M+ - 1], 400 (25), 398 (21), 387 (10), 291 (6), 218 (56), 215
(13), 214 (29), 212 (12), 210 (12), 207 (53), 204 (23), 202 (12),
188 (33), 186 (35), 157 (8), 91 (11).
9.4 mmol) in dry THF (20 mL) was cooled to -78 °C and a 1.6 M
n-BuLi solution in hexane (5.9 mL, 9.4 mmol) was added dropwise.
The mixture was stirred for 30 min and then a solution 6-(triflu-
oromethyl)-2H-1,4-benzothiazin-3(4H)-one37 (1.00 g, 4.3 mmol)
in dry THF (20 mL) was added. The resulting dark-red mixture
was warmed to -55 °C, and a solution of 5-bromo-2-methoxy-
benzaldehyde (0.925 g, 4.3 mmol) in dry THF (20 mL) was added
dropwise. During the addition, the solution color changed from dark-
red to orange to yellow. The mixture was stirred at -55 °C for 30
min, and then at room temperature for an additional 3 h. Quenching
with aqueous saturated solution of NH4Cl (60 mL), extraction with
Et2O, drying, and solvent evaporation to dryness gave a mixture
of the diastereomeric aldols, which were separated by flash
chromatography eluting with benzene/EtOAc (9:1).
Fractions containing the faster moving compound, upon evapora-
tion, gave threo-isomer derivative 17a (0.344 g, 18%) as white
1
needles; mp 224-225 °C. H NMR (400 MHz, CDCl3) 3.38 (d,
1H, J ) 5.0 Hz, CHOH), 3.79 (s, 3H, OCH3), 4.03 (d, 1H, J ) 4.5
Hz, SCH), 5.56 (dd, 1H, J ) 4.5 and 5.0 Hz, CHOH), 6.71 (d, 1H,
J ) 8.8 Hz, H-3′), 7.08 (bs, 1H, H-Ar), 7.30-7.50 (m, 2H, H-Ar),
7.60-7.61 (m, 1H, H-Ar), 9.01 (bs, 1H, NH). Anal.
(C17H13BrF3NO3S) C, H, N. The evaporation of fractions containing
the later isomer gave erythro-isomer 17b (0.792 g, 36%) as a white
2-(5-Bromo-2-methoxyphenyl)-6-trifluoromethyl-2H-1,4-ben-
zothiazine-3(4H)-thione (14). Compound 6 (0.301 g, 0.72 mmol)
and Lawesson’s reagent (0.291 g, 0.72 mmol) were mixed in a
glass tube. The glass tube was then placed in an alumina bath inside
a domestic microwave oven (350 W) and irradiated for 1 min. The
mixture was chromatographed on silica flash eluting with cyclo-
hexane/EtOAc (8:2) to yield the thioxo derivative 14 as a yellowish
solid (0.162 g, 52%); mp 212 dec °C. 1H NMR (400 MHz, acetone-
d6) 3.95 (s, 3H, OCH3), 5.58 (s, 1H, SCH), 7.05 (d, 1H, J ) 8.8
Hz, H-3′), 7.10 (d, 1H, J ) 2.4 Hz, H-6′), 7.41-7.44 (m, 2H,
H-Ar), 7.50 (d, 1H, J ) 8.2 Hz, H-8), 7.78-7.83 (m, 1H, H-5),
12.01 (bs, 1H, NH). Anal. (C16H11BrF3NOS2) C, H, N.
1
solid; mp 199-200 °C. H NMR (400 MHz, CDCl3) 3.84 (s, 3H,
OCH3), 3.86 (d, 1H, J ) 7.5 Hz, CHOH), 3.92 (d, 1H, J ) 8.5 Hz,
SCH), 5.02 (dd, 1H, J ) 7.5 and 8.5 Hz, CHOH), 6.77 (d, 1H, J
) 8.7 Hz, H-3′), 7.10 (bs, 1H, H-Ar), 7.26-7.28 (m, 1H, H-Ar),
7.34-7.39 (m, 3H, H-Ar), 9.10 (bs, 1H, NH). Anal.
(C17H13BrF3NO3S) C, H, N.
(2Z)-2-(5-Bromo-2-methoxybenzylidene)-6-trifluoromethyl-2H-
1,4-benzothiazin-3(4H)-one (18). A solution of MsCl (0.046 g, 0.40
mmol) in CH2Cl2 (15 mL) and then Et3N (0.044 g, 0.44 mmol) in
CH2Cl2 (15 mL) were added at room temperature to a solution of
diastereoisomeric mixture 17 (0.180 g, 0.40 mmol) in CH2Cl2 (50
mL). The reaction mixture was stirred for 15 min, then warmed to
40 °C for 1 h. After cooling, the mixture was washed with water,
dried, and evaporated to dryness. The residue was purified by
crystallization from CH2Cl2 to afford the derivative 18 as a pale-
yellow solid (0,115 g; 67%); mp 255-256 °C. 1H NMR (200 MHz,
DMSO-d6) 3.83 (s, 3H, OCH3), 7.09 (d, 1H, J ) 8.8 Hz, H-3′),
7.32 (d, 1H, J ) 8.5 Hz, H-5′), 7.56-7.67 (m, 3H, H-6′, H-7, H-8),
7.85 (s, 1H, H-vinyl), 11.3 (bs, 1H, NH). Anal. (C17H11BrF3NO2S)
C, H, N.
2-(5-Bromo-2-methoxyphenyl)-6-trifluoromethyl-3,4-dihydro-
2H-1,4-benzothiazine (15). A 1 M solution of THF-BH3 complex
(0.90 mL, 0.90 mmol) was added dropwise to a solution of
derivative 6 (0.205 g, 0.49 mmol) in dry THF (10 mL) at 0 °C.
The solution was refluxed for 2 h, cooled, carefully diluted with
MeOH (15 mL), and again refluxed for an additional 30 min. After
cooling, the reaction was acidified with 2 N HCl refluxed for 1 h,
cooled, basified with 10% NaOH, and finally extracted with EtOAc.
The combined organic layers were washed with brine, dried, and
evaporated to dryness to give 15 (0.186 g, 94%) as a semisolid.
1H NMR (200 MHz, CDCl3) 3.50-3.60 and 3.65-3.70 (each m,
1H, CH2), 3.85 (s, 3H, OCH3), 4.31 (bs, 1H, NH), 4.82 (dd, 1H, J
) 3.0 and 7.8 Hz, SCH), 6.74-6.81 (m, 2H, H-5 and H-3′),
6.85-6.92 (m, 1H, H-7), 7.14 (d, 1H, J ) 8.1 Hz, H-8), 7.40 (dd,
1H, J ) 2.3 and 8.6 Hz, H-4′), 7.45 (d, 1H, J ) 2.3 Hz, H-6′).
GC-MS m/z (%): 406 (16) [M+ + 2], 405 (83) [M+ + 1], 404
(17) [M+], 403 (82) [M+ - 1], 385 (5), 291 (4), 25 (11), 204 (100),
201 (56), 184 (9), 171 (14), 118 (8), 90 (6).
Ethyl Bromo-(5-bromo-2-methoxyphenyl)acetate (19), Ethyl
Bromo-(3,5-dibromo-2-methoxyphenyl)acetate (20) and Ethyl Di-
bromo-(5-bromo-2-methoxyphenyl)acetate (21). A solution of Br2
(0.32 mL, 6.3 mmol) in CCl4 (20 mL) was added dropwise at room
temperature to a solution of ethyl (2-methoxyphenyl)acetate36 (0.408
g, 2.1 mmol) in CCl4 (20 mL). The reaction mixture was irradiated
with a 300 W lamp and refluxed for 2 h. After cooling, the mixture
was quenched with an aqueous solution of Na2S2O3, then the organic
layer was separated and evaporated to dryness. The obtained oil
residue was dissolved in EtOAc and sequentially washed with
aqueous saturated solution of NaHCO3 and brine. The organic layer
was then dried and evaporated to dryness, and the residue was
subjected to flash chromatography eluting with cyclohexane/EtOAc
(95:5) to afford the fastest moving tribromo derivative 20, followed
by the intermediate moving dibromo derivative 19 and the slowest
moving one 21.
2-(2-Methoxyphenyl)-6-trifluoromethyl-3,4-dihydro-2H-1,4-ben-
zothiazine (16). A solution of derivative 6 (0.400 g, 0.96 mmol) in
dry THF (10 mL) was added dropwise to a suspension of LiAlH4
(0.181 g, 4.78 mmol) in dry THF (15 mL). The mixture was stirred
for 4 h, and then EtOAc (15 mL) was carefully added. After 10
min, the mixture was filtered through a pad of celite and the filter
washed with EtOAc (50 mL). The filtrate was evaporated under
reduced pressure, and the resulting solid was stirred with aqueous
HCl 0.2 N (40 mL) and then extracted with EtOAc (60 mL). The
organic layer was separated and washed consecutively with water
and brine, dried, and the solvent removed under reduced pressure.
The crude product was purified by flash chromatography eluting
with cyclohexane/EtOAc (9:1) to yield the desired compound 16
1
Compound 19, white solid (0.318 g, 43%); mp 52-53 °C. H
1
as a yellow solid (0.065 g, 21%); mp 88-89 °C. H NMR (200
NMR (200 MHz, CDCl3) 1.30 (t, 3H, J ) 7.1 Hz, CH2CH3), 3.89
(s, 3H, OCH3), 4.29 (q, 2H, J ) 7.1 Hz, CH2CH3), 5.80 (s, 1H,
CHCO), 6.80 (d, 1H, J ) 8.8 Hz, H-3), 7.45 (dd, 1H, J ) 2.4 and
8.8 Hz, H-4), 7.79 (d, 1H, J ) 2.4 Hz, H-6).
MHz, methanol-d4) 3.45 (dd, 1H, J ) 8.2 and 12.2 Hz, CHRHꢀ),
3.63 (dd, 1H, J ) 12.2 and 3.1 Hz, CHRHꢀ), 3.78 (s, 3H, OCH3),
4.66 (s, 1H, NH), 4.73 (dd, 1H, J ) 3.1 and 8.2 Hz, SCH),
6.69-6.79 (m, 4H, H-Ar), 7.03 (d, 1H, J ) 8.0 Hz, H-8),
7.18-7.26 (m, 2H, H-Ar). GC-MS m/z (%): 325 (94) [M+], 306
(5), 292 (6), 216 (7), 204 (100), 184 (7), 121 (53), 107 (5), 91
(24), 77 (5).
1
Compound 20, semisolid (0.150 g, 17%). H NMR (200 MHz,
CDCl3) 1.28 (t, 3H, J ) 7.1 Hz, CH2CH3), 3.91 (s, 3H, OCH3),
4.23 (q, 2H, J ) 7.1 Hz, CH2CH3), 5.73 (s, 1H, CHCO), 7.67 and
7.77 (each d, 1H, J ) 2.4 Hz, H-Ar).
1
2-[(5-Bromo-2-methoxyphenyl)hydroxymethyl]-6-trifluorometh-
yl-2H-1,4-benzothiazin-3(4H)-one (17a, 17b). A nitrogen-flushed
solution of freshly distilled diisopropylamine (0.951 g, 1.32 mL,
Compound 21, whitish solid; (0.190, 21%); mp 92-94 °C. H
NMR (200 MHz, CDCl3) 1.31 (t, 3H, J ) 7.1 Hz, CH2CH3), 3.88
(s, 3H, OCH3), 4.34 (q, 2H, J ) 7.1 Hz, CH2CH3), 6.79 (d, 1H, J

1,4-Benzothiazines as Ca2+-ActiVated K Channel Openers
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5091
) 8.7 Hz, H-3), 7.52 (dd, 1H, J ) 2.4 and 8.7 Hz, H-4), 8.14 (d,
1H, J ) 2.4 Hz, H-6).
was dissolved (100 mM) in EtOH 95% and further diluted in
bidistilled water, KCl was dissolved (2 M) in Tyrode solution, TEA
(Sigma) was dissolved (10 mM) in Tyrode solution, IbTX (Sigma)
was dissolved (100 nM) in bidistilled water. All the synthesized
derivatives 3-18 were dissolved (10 mM) in DMSO and further
diluted in bidistilled water. All the solutions were freshly prepared
immediately before the pharmacological 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 20 mM. When the limit
concentration of 10 µM (the highest concentration that could
be administered) of the tested compounds did not reach the
maximal effect, the efficacy parameter represented the vasore-
laxing response, expressed as the percentage of the contractile
tone induced by KCl 20 mM, evoked by this limit concentration.
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 20 mM KCl. The pIC50 could not be calculated
for those compounds that had an efficacy parameter close to or
less than 50%. The efficacy and potency parameters were
expressed as the mean ( standard error for 5-10 experiments.
The student t-test was selected for the statistical analysis; P <
0.05 was considered a significant statistical difference. Experi-
mental data were analyzed by a computer fitting procedure
(software Graph Pad Prism 3.0).
Ethyl Bromo-(5-chloro-2-methoxyphenyl)acetate (24). Sulfuryl
chloride (5.7 mL, 0.70 mmol) was added dropwise over 1 h to a
stirred solution of (2-methoxyphenyl)acetic acid (8.30 g, 50 mmol)
in dry THF (50 mL) cooled to -10 °C. After the complete addition,
the reaction mixture was poured into ice-water and the obtained
slurry was stirred for 3 h. The obtained white precipitate was
collected by filtration, washed with water and dried to afford (5-
chloro-2-methoxyphenyl)acetic acid 2244 (9.33 g, 93%) as an off-
white solid; mp 125-127 °C.
A solution of 22 (8.22 g, 41 mmol) in dry DMF (20 mL) was
added dropwise to a stirred suspension of anhydrous K2CO3 (11.3
g, 82 mmol) and dry DMF (30 mL). EtI (35 mL, 44 mmol) was
then added dropwise. The reaction mixture was warmed to 50 °C
for 3 h and then poured into water and extracted with CH2Cl2. The
combined organic layers were sequentially washed with 0.1 N HCl
and brine, then dried and evaporated to dryness. The residue was
purified by vacuum distillation (90 °C, 25 mmHg) to yield ethyl
(5-chloro-2-methoxyphenyl)acetate 23 as dark-brown viscous oil
(8.34 g, 89%) suitable for the next step without any further
purification.1H NMR (200 MHz, CDCl3) 1.25 (t, 3H, J ) 7.1 Hz,
CH2CH3), 3.57 (s, 2H, CH2CO), 3.79 (s, 3H, OCH3), 4.15 (q, 2H,
J ) 7.1 Hz, CH2CH3), 6.81 (d, 1H, J ) 8.5 Hz, H-3), 7.13-7.23
(m, 2H, H-Ar).
Compound 23 (7.32 g, 32 mmol) was converted to the title
compound by using the same procedure as for 19, except that a
longer reaction time (10 h) was required. The obtained crude residue
was purified by flash chromatography eluting with cyclohexane/
CH2Cl2 (1:1) to afford the bromoderivative 24 (4.13 g, 42%) as a
Acknowledgment. We thank Professor Arnaldo Fravolini
for critical reading of the manuscript and support. The technical
assistance of R. Bianconi is gratefully acknowledged.
1
white solid; mp 48-49 °C. H NMR (200 MHz, CDCl3) 1.23 (t,
3H, J ) 7.1 Hz, CH2CH3), 3.85 (s, 3H, OCH3), 4.20 (q, 2H, J )
7.1 Hz, CH2CH3), 5.71 (s, 1H, CHCO), 6.74 (d, 1H, J ) 8.8 Hz,
H-3), 7.21 (dd, 1H, J ) 2.6 and 8.8 Hz, H-4), 7.56 (d, 1H, J ) 2.6
Hz, H-6).
Supporting Information Available: A Table of combustion
analysis data for target compounds 11, 14, 17a-b, and 18. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Vasorelaxant Activity. All the experimental procedures were
carried out following the guidelines of the European Community
Council Directive 86-609. 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 aortae 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.
Five mm wide aortic rings were suspended, under a preload of 2 g,
in 20 mL 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), thermostatted 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
administering acetylcholine (ACh) (10 µM) to KCl (20 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 relaxation g10% (i.e., significant
presence of the endothelium), were discarded. Thirty to forty min
after the confirmation of the endothelium removal, the aortic
preparations were contracted by treatment with a single concentra-
tion of KCl (20 mM). When the contraction reached a stable plateau,
3-fold increased concentrations of the tested compounds or of the
reference compound 1 were added cumulatively. Preliminary
experiments showed that both the KCl (20 and 60 mM)-induced
contractions remained in a stable tonic state for at least 40 min. In
other sets of experiments, the nonselective potassium channel
blocker TEA (10 mM) or the BK-selective blockers IbTX (100
nM) were added, after the KCl (20 mM)-induced contraction,
followed by the administration of selected compounds.
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