Highly potent 1,4-benzothiazine derivatives as KATP-channel openers

Article abstract of DOI:10.1021/jm030791q

A series of 1,4-benzothiazines, suitably functionalized at the N-4 and C-6 positions, arising from the replacement of a benzopyran-based structure of cromakalim with a 1,4-benzothiazine nucleus, has been synthesized as potassium channel openers (KCOs). Mo

Full text of DOI:10.1021/jm030791q

3
670
J . Med. Chem. 2003, 46, 3670-3679
High ly P oten t 1,4-Ben zoth ia zin e Der iva tives a s KATP -Ch a n n el Op en er s1
,
§
‡
§
§
§
‡
Violetta Cecchetti,* Vincenzo Calderone, Oriana Tabarrini, Stefano Sabatini, Enrica Filipponi, Lara Testai,
§
‡
§
Roberto Spogli, Enrica Martinotti, and Arnaldo Fravolini
Dipartimento di Chimica e Tecnologia del Farmaco, Universit a` degli Studi di Perugia, Via del Liceo, 1, 06123 Perugia (Italy)
and Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Sezione di Farmacologia, Universit a` degli Studi
di Pisa, Via Bonanno 6, 56126 Pisa (Italy)
Received J anuary 28, 2003
A series of 1,4-benzothiazines, suitably functionalized at the N-4 and C-6 positions, arising
from the replacement of a benzopyran-based structure of cromakalim with a 1,4-benzothiazine
nucleus, has been synthesized as potassium channel openers (KCOs). Most of the tested
compounds show high vasorelaxant potency that is considerably higher than that of the
reference levcromakalim (LCRK). In the presence of the well-established selective KATP blocker,
glibenclamide, the vasorelaxing effects were antagonized in a competitive fashion, indicating
the involvement of the KATP channel in their pharmacological effect. Some aspects of the
structure-activity relationship associated with the N-4 and C-6 substituents are discussed.
The highest level of activity was achieved with a cyclopentenone ring at the N-4 position coupled
with an electron-withdrawing group such as nitro, trifluoromethyl, or cyano at the C-6 position.
Compounds 4c, 5c, and 6c displayed a vasorelaxant potency at least 10 000 times greater than
that of LCRK, thus becoming the most potent KCOs identified to date.
In tr od u ction
SUR2A, and SUR2B) gives rise to the diversity and
organ-specific distribution of KATP channels which may
provide a rational basis for identifying tissue-selective
KCOs.
Interest in the KCOs was triggered in the early 1980s
6
Potassium channel openers (KCOs), or activators, are
a group of compounds with a broad spectrum of poten-
tial therapeutic applications2 due to the critical role
played by potassium channels in a wide variety of
physiological processes including the regulation of action
potential, neuronal excitability, stimulus-secretion cou-
pling, cell volume, and epithelial electrolyte transport.
Most of the KCOs identified to date are known to
interact with the KATP channels that are widely distrib-
uted in various tissues and whose function is mainly
regulated by changes in the intracellular levels of
nucleotides, particularly ATP and MgADP which inhibit
and stimulate channel activity, respectively. Thus, the
KATP channels provide a vital link between cellular
7
8
when it was discovered that cromakalim, pinacidil,
9
and nicorandil relaxed smooth vascular muscle via a
mechanism involving the opening of KATP channels.
Thus, these agents were first indicated for the treatment
of hypertension and angina pectoris, but this has since
been extended to include other diseases involving
smooth muscle contraction such as asthma, urinary
incontinence, and baldness. These agents are also
thought to afford cellular protection against cardiac
1
0
1
1
12
1
3
ischemia, independent of their vasodilating actions,
1
4
and to have antilipemic effects.
3
metabolism and electrical activity. The opening of KATP
From a chemical point of view, KATP channel openers
belong to several different structural classes, the main
ones being benzopyrans, cyanoguanidines, and thiofor-
mamides. Among these, the most investigated class is
that of the benzopyrans whose prototype is cromakalim
channels results in an efflux of potassium ions from the
cell with a simultaneous transmembrane hyperpolar-
ization that restricts calcium entry through voltage-
dependent (L-type) calcium channels and inhibits in-
tracellular calcium release, thereby dampening cellular
excitability.
The molecular framework of a KATP channel is a
heterooctomeric complex composed of four pore-forming
subunits of the inward rectifier family of potassium
channels (Kir 6.0) and four larger regulatory proteins,
the sulfonylurea receptor subunits (SUR). Recent evi-
dence has shown that the KCOs stimulate KATP channel
activity by binding to the SUR subunit. The existence
of isoforms of both pore-forming Kir subunits (Kir 6.1
and Kir 6.2) and the regulatory protein SUR (SUR1,
(
CRK) or rather, its biologically active (-)3S,4R enan-
tiomer, levcromakalim (LCRK) (Figure 1).
In many structure-activity studies the different
positions of the benzopyran ring have been variously
substituted permitting the optimal activity to be cor-
relate with a specific set of structural characteristics
and stereochemical features in the molecule.15 Thus,
many potent benzopyran derivatives have been identi-
fied.
4
5
The benzopyran nucleus itself has also been modified
in both the aromatic ring and in the pyran moiety. When
1
6
the CRK aromatic ring was replaced by pyridine and
*
To whom correspondence should be addressed. Phone: + 39 075
1
7
16b
thiophene systems, potent pyran[3,2-c]pyridines and
5
855153. Fax: + 39 075 5855115. E-mail: viola@unipg.it.
§
17a
Dipartimento di Chimica e Tecnologia del Farmaco, Universit a` di
thieno[3,2-b]pyrans
were identified.
Perugia.
‡
The replacement of the pyran nucleus with different
heterocyclic and nonheterocyclic systems often produced
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Bio-
tecnologie, Universit a` di Pisa.
1
0.1021/jm030791q CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/12/2003

1
,4-Benzothiazines as KATP-Channel Openers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3671
Sch em e 1a
a
Reagents: (a) HSC(Me)2CO2H, K2CO3, DMF, 100 °C; (b)
FeSO4/NH4OH; (c) BrC(Me)2CO2Et, K2CO3, DMF, 100 °C; (d)
LiAlH4, THF; (e) BH3‚HF; (f) CuCN, DMF, 150 °C.
based SAR, they have been suitably functionalized with
a gem-dimethyl group at the C-2 position and an
electron-withdrawing group, such as Br, NO2, CN, or
CF3, at the C-6 position. Furthermore, lactam rings (a ,
b), acyclic amides (d and f), cyclopentenone (c), and acyl
chains (e, g, and h ), all bearing the usual oxo function
at different distances from the benzothiazine nucleus,
were selected as N-4 substituents (Table 1). Chloroacetyl
(h ) and allyl (i) substituents were also inserted in
analogy to Prasad’s compounds.
F igu r e 1.
moderately active compounds when compared with the
benzopyran series.
These structure-activity studies indicated that, in
general, the replacement of the pyran oxygen with NH
1
8
or CH2 to produce tetrahydroquinolines and tetrahy-
1
8
dronaphthalenes, respectively, as well as the ring
expansion to benzoxepines19 has a detrimental effect on
the potency. A slight reduction in potency was observed
with the elimination of the pyran oxygen as in the
indane20 derivatives or by replacing it with a carbonyl
function as in tetrahydronaphthalen-1-ones. The sub-
stitution of the pyran oxygen with a sulfur atom yielded
the benzothiopyran 1 (Figure 1) which retained nearly
all of the potency when compared to its benzopyran
analogue even if the oxidation of sulfur atom to sulfoxide
or sulfone is detrimental.22
Moreover, in backward analysis and for comparative
purposes, some examples of 1,4-benzothiazines (2A-F ,
2
9
Table 2), previously described by Prasad, were also
resynthesized and assayed to check whether the re-
ported antihypertensive activity could be due to the
activation of KATP channels.
2
1
Ch em istr y
The general synthetic approach used to prepare the
target compounds 3-7, involved the construction of the
1,4-benzothiazine ring having a gem-dimethyl group at
C-2 and an electron-withdrawing substituent at C-6,
followed by a suitable functionalization at the N-4
position.
Advantageous replacements of the pyran ring were
2
3
obtained with oxazine systems since some of 1,3- and
,4-benzoxazine derivatives24 are potent KCOs. The
more extensively studied 1,4-benzoxazines produced
1
The synthetic procedures for building the 1,4-ben-
zothiazine nucleus to obtain the intermediates 12-15
are illustrated in Scheme 1. Thus, 2,5-dibromonitroben-
zene was treated with 2-mercapto-2-methylpropanoic
2
5
YM-934 (Figure 1) which is a potent vasodilator.
A systematic study focused on pyran heterocyclic
replacement was recently described by Matsumoto et
2
6
30
al. Among the modification made, the 1,4-benzothiaz-
ine nucleus was found a promising new system for
KCOs.27 This finding had already been reported by us
and has now been amply demonstrated and discussed
in depth in this paper.
acid followed by reductive cyclization with FeSO4/
NH OH afforded the 6-bromobenzothiazinone 9 which
4
1
was then reduced with LiAlH to give 6-bromobenzothi-
4
azine intermediate 12. Similarly, the reaction of 2-mer-
capto-2-methylpropanoic acid with 2-fluoro-5-nitroa-
niline directly gave the 6-nitrobenzothiazinone 10 from
which 6-nitrobenzothiazine intermediate 13 was ob-
tained by selective reduction of the 2-oxo function with
borane-tetrahydrofuran complex. The preparation of
6-trifluoromethylbenzothiazine 14 was achieved by
reacting of 2-amino-4-(trifluoromethyl)benzenethiol with
ethyl 2-bromo-2-methylpropanoate followed by a reduc-
tion with LiAlH4. The 6-cyanobenzothiazine 15 was
obtained by treating 6-bromobenzothiazine 12 with
CuCN in DMF.
In an effort to find new KCO chemotypes, we exam-
ined the effect of replacing the benzopyran ring with a
1
,4-benzothiazine nucleus. We have already successfully
exploited the 1,4-benzothiazine nucleus as a scaffold for
building compounds that are active on the cardiovas-
cular system. In addition, we have taken into account
2
8
that the simple 1,4-benzothiazine derivatives 2 (Figure
1
), reported many years ago by Prasad,29 showed anti-
hypertensive properties in experimental animals.
Thus, in this study we report the synthesis and the
biological evaluation of a new series of 1,4-benzothiazine
derivatives 3-7 (Figure 1). As suggested by benzopyran-
Functionalization at the N-4 position was achieved
as depicted in Schemes 2, 3, and 5. Treatment of

3
672 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Cecchetti et al.
Ta ble 1. Vasorelaxant Activity of 1,4-Benzothiazine Derivatives 3-7 Synthesized in This Studya
a
Vasorelaxant activity was evaluated in aortic ring precontracted with 20 mM KCl (see Experimental Section). b Maximal vasorelaxing
response expressed as percentage of contractile tension. c Standard error of the mean of 5-10 separate experiments. Vasorelaxant potency
expressed as negative log of the concentration evoking a half-reduction of the contractile tone. Parameter recorded in the presence of 1
µM glibenclamide. Parameter recorded in the presence of 60 mM KCl. The parameter could not be calculated because of the low efficacy
d
e
f
g
h
i
(
≈ or <50%). Significantly different from the respective control. Vasorelaxant efficacy produced by the limit concentration 0.1 mM of
j
k
the test compound. The compound was ineffective. 4-Allyl-2,2-dimethyl-6-trifluomethyl-2H-1,4-benzothiazine-3(4H)-one.
Ta ble 2. Vasorelaxant Activity of 1,4-Benzothiazine
Derivatives 2A-F
and 6c (Scheme 2). Intermediates 12 and 13 were also
converted into target compounds 3e, 3g, 3h and 4e, 4g,
a
4
h , by acylation in CH2Cl2 and in the presence of Et3N
with acetyl chloride, 3-fluorobenzoyl chloride, and chlo-
roacethyl chloride, respectively (Scheme 2).
The synthesis of compounds 3a , 3b, 3d , 3f, 4a , 4d ,
and 4f was achieved (Scheme 3) through 4-aminoben-
zothiazine intermediates 18 and 19 obtained from
benzothiazines 12 and 13, respectively, by nitrosation
and successive reduction of the resulting 4-nitroso
compounds 16 and 17. The reduction step was carried
out with Zn and AcOH/MeOH to convert the 6-bromo-
Emaxb
SEM, %
(
pIC50d
(
c
SEMc
compd
X
R4
R6
Ae
B
27 ( 5f
NCg
2
2
CO
CO
H
H
H
CF3
e
93 ( 3f
4.54 ( 0.042
f,h
h
8
1 ( 21
4.55 ( 0.19
2
2
2
2
Ce
CO
CO
CH2CHdCH2
H
31 ( 7f
f
NCg
e
D
CH2CHdCH2 CF3 90 ( 3
4.83 ( 0.021
4
-nitroso derivative 16 to its 4-amino counterpart 18,
i
j
j
E
CH2 COCH2Cl
CH2 CSNHCH2Ph
H
H
NE
NE
NE
NE
i
j
j
while a selective reduction with formamidinesulfinic
acid was necessary to reduce the 6-nitro-4-nitroso
derivative 17 to 4-amino-6-nitro derivative 19. The
F
a-d,g,j
See corresponding footnotes in Table 1. See ref 29a. f See
footnote i in Table 1. See footnote e in Table 1. See ref 29b.
e
h
i
4
-amino intermediates 18 and 19 were then acylated
with 4-chlorobutyrryl chloride or 5-chlorovaleryl chloride
followed by ring closure with t-BuOK to give the
pyrrolidone derivatives 3a and 4a and pyperidone
derivative 3b. Acylation with acetyl chloride or 3-fluo-
robenzoyl chloride gave instead the acetamido deriva-
intermediates 12-15, in THF and in the presence of
Et3N, with the crude product, obtained by reacting
cyclopentanone and N-bromosuccinimide in the pres-
ence of a catalytic amount of dibenzoyl peroxide, gave
the target 3-oxo-1-cyclopentenyl derivatives 3c, 4c, 5c,

1
,4-Benzothiazines as KATP-Channel Openers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3673
Sch em e 2a
Sch em e 4a
a
Reagents: (a) MCPBA (1 equiv), CH2Cl2; (b) MCPBA (2 equiv),
CH2Cl2; (c) CuCN, NMP, reflux.
Sch em e 5a
a
Reagents: (a) [cyclopentenone, NBS, DBP, CCl4] Et3N, THF,
reflux; (b) acyl chloride, Et3N, CH2Cl2.
Sch em e 3a
a
Reagents: (a) ICH2CHdCH2, NaH, DMF dry; (b) LiAlH4, THF.
with KCl (20 mM) according to a 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 LCRK.
The results of the pharmacological tests indicate that
many of the synthesized compounds had a nearly
complete vasorelaxing efficacy, with only a few mol-
ecules showing a low efficacy or complete ineffective-
ness. With respect to potency, compounds such as 3c,
a
Reagents: (a) NaNO2, AcOH/MeOH; (b) Zn, AcOH/MeOH, 0
°
C; (c) HNdC(NH2)SO2H, MeOH/NaOH; (d) acyl chloride, Et3N,
CH2Cl2, 0 °C; (e) t-BuOK, DMF, 0 °C.
3
d , and 3f had appreciable pIC50 values. The potency
reached very interesting levels for other molecules, such
as 4d , 4f, and 6a , while compounds 4c, 5c, and 6c had
surprisingly high pIC50 values.
The mechanism of action was investigated in some
selected compounds. In high depolarization conditions,
tives 3d and 4d and benzoylamido derivatives 3f and
f, respectively.
Oxidation of pyrrolidinone derivative 3a with 1 or 2
4
equiv of MCPBA gave sulfoxide analogue 3a a and
sulfone analogue 3a b, respectively (Scheme 4). 6-Cyano
derivative 6a , which was required for a direct compari-
son of our novel series to LCRK, was also obtained by
reacting compound 3a with CuCN in DMF (Scheme 4).
Finally, for a direct comparison between our new
series of 1,4-benzothiazines and the old ones described
by Prasad, the 3-oxo-4-allyl derivative 7i and its 3-deoxo
counterpart, 5i, were also prepared (Scheme 5) by
alkylating 3-oxo-1,4-benzothiazine 11 with allyl iodide,
followed by reduction with LiAlH4 in THF.
(
aortic rings precontracted with KCl 60 mM) the
vasorelaxing efficacy of 3a , 3c, 4c, 4d , 4f, 5c, and 6c
was dramatically decreased (Table 1), in perfect agree-
ment with the pharmacodynamic profile expected for
3
1
KCO. The possible involvement of the KATP channel
in this pharmacological effect was also investigated by
using glibenclamide, a well-established selective KATP
blocker. In the presence of this sulfonylurea, the va-
sorelaxing effects of 3a , 3c, 3f, 4c, 4d , 4f, 5c, and 6c
were antagonized in a clearly competitive fashion, with
an almost parallel rightward displacement of the con-
centration-response curves and an almost full recovery
of the maximal effect (Table 1). These results suggest
that the activation of the SUR subunit of the KATP
channel could account for the vasorelaxing properties
of these compounds.
Resu lts a n d Discu ssion
The KATP-opening activity was evaluated in vitro as
the vasorelaxing effect evoked by the test compounds
on endothelium-denuded rat aortic rings precontracted

3
674 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Cecchetti et al.
The biological data indicate that the 1,4-benzothiazine
substituent, afforded compounds with good vasorelaxant
potency that was comparable or superior to that of
LCRK. From the limited structure-activity data as-
sociated with the presence of a trifluoromethyl or cyano
group at the C-6 position, it appears that these substit-
uents, such as the nitro group, confer high activity levels
as in compounds 5c, 6a , and 6c, which are 100 to
100 000 times more potent than LCRK.
nucleus is a suitable replacement for the benzopyran
nucleus since most of the 1,4-benzothiazine derivatives
reported in this study showed high vasorelaxant potency
which, in some cases, was considerably higher than that
of LCRK. The increase in KATP channel opening activity
is strongly evident in a head-to-head comparison be-
tween LCRK and its closed 1,4-benzothiazine analogue
6
a , which was 100 times more potent. It should be noted
The oxidation of a sulfur atom to sulfoxide as in 3a a
caused a 40-fold decrease in potency, but a further
oxidation to sulfur dioxide, as in 3a b, determined a
slight recovery of potency. This is in agreement with
that compound 6a , as well as the other 1,4-benzothiaz-
ine derivatives, can be considered structural simplifica-
tion of LCRK, due to the absence of chiral centers.
2
6,27
As expected, the C-6 and especially the N-4 substit-
uents play a key role in activity modulation. Examining
the effect of the N-4 substituent, the highest level of
activity was achieved with compounds 4c, 5c, and 6c
which had a cyclopentenone moiety. The vasorelaxant
potency of these three compounds was at least 10 000
times greater than that of LCRK making them the most
potent KCOs reported to date. A comparison with the
cyclopentenone derivative in benzopyran series cannot
be made because it has never been reported in the
literature. The high potency of cyclopentenone deriva-
that already observed by Matsumoto et al.
In our screening test, the backward analysis on
Prasad’s compounds 2A-F (Table 2) only showed a
weak vasorelaxant activity for compounds 2B and 2D,
both of which have a C-6 trifluoromethyl group. How-
ever, the vasorelaxant activity did not seem to involve
the activation of KATP channels since it was not antago-
nized by glibenclamide. The introduction of a gem-
dimethyl group at the C-2 position did not increase the
vasorelaxant activity as it had in compound 7i which
was less effective than 2D. The efficacy was however
recovered by excluding the C-3 oxo function, as in
compound 5i which had the same potency as 2D.
The main observation which has emerged from this
study is that highly potent compounds can be obtained
by replacing the benzopyran ring with a 1,4-benzothi-
azine nucleus. The potency increases dramatically when
a suitable substitution pattern is present on the 1,4-
benzothiazine nucleus, such as in compounds 4c, 5c,
and 6c which had a cyclopentenone as the N-4 substitu-
ent coupled with a nitro, trifluomethyl, or cyano group
at the C-6 position, respectively. Indeed, these com-
pounds have a vasorelaxant potency that is at least
2
tives could be due to the sp carbon as the attachment
point to the 1,4-benzothiazine skeleton which forces the
N-4 substituent to adopt, with respect to the benzothi-
azine ring, a predominantly orthogonal conformation
that is the known active conformation for this class of
KCOs.32 However, it seems that this strong influence
on activity given by cyclopentenone moiety is peculiar
to the 1,4-benzothiazine series. In fact the same moiety
in the closed 1,4-benzoxazine series increases the activ-
ity but not to the same extent.24b
The expansion of a five-membered lactam substituent
to a six-membered lactam slightly decreased the activity
1
0 000 times greater than that of LCRK which makes
(
3
compare the 6-bromo derivative 3a with its homologue
b).
As in the benzopyran series, the classical lactam ring
them the most potent KCOs reported to date.
Exp er im en ta l Section
can be effectively replaced by acyclic amido groups to
afford compounds with a vasorelaxant potency that is
comparable or superior to that of LCRK, as in the case
of acetamido derivative 4d and m-fluorobenzoylamide
derivatives 3f and 4f. On the contrary, replacing the
acyclic amido group with a keto function, as in 4-acyl
derivatives 3e, 3g, 4e, and 4g, drastically reduces the
activity (compare vs 3d , 3f, 4d , and 4f, respectively).
This could be due, in part, to the shortening of the
distance between the carbonyl function and the 1,4-
benzothiazine nucleus. If so, it would indicate the
importance of a set distance between an electron-rich
hydrogen bond-accepting group and the N-4 of the
benzothiazine ring. This is in keeping with what has
already been found in the benzopyran series in which
All reactions were routinely checked by thin-layer chroma-
tography (TLC) on silica gel 60F254 (Merck) and visualized by
using UV. Column chromatography separations were carried
out on Merck silica gel 60 (mesh 70-230) and flash chroma-
tography on Merck silica gel 60 (mesh 230-400). Melting
points were determined in capillary tubes (B u¨ chi Electrotermal
Mod. 9100) and are uncorrected. Elemental analyses were
performed on a Carlo Erba elemental analyzer, Model 1106,
and the data for C, H, and N are within (0.4% of the
1
13
theoretical values. H NMR and C NMR spectra were
recorded at 200 MHz (Bruker AC-200), with CDCl as solvent,
unless otherwise indicated, and with Me Si as internal stan-
3
4
dard. Chemical shifts are given in ppm (d). 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. Reagents and solvents were pur-
chased from common commercial suppliers and were used as
received. Organic solutions were dried over anhydrous
this distance requirement is usually met by a three-bond
connection at the C-4 benzopyran nucleus.33 The inac-
tivity of chloroacetyl derivatives 3h and 4h can also be
explained in the same manner.
2 4
Na SO and concentrated with a B u¨ chi rotary evaporator at
low pressure. Yields were of purified product and were not
optimized. All starting materials were commercially available,
unless otherwise indicated.
Regarding the C-6 position, a limited number of
electron-withdrawing groups were inserted with a par-
ticular focus on 6-bromo and 6-nitro derivatives. In
general, the 6-nitro derivatives had a higher activity
than their 6-bromo counterparts (compare 4c vs 3c, 4d
vs 3d , and 4f vs 3f). However, the presence of a bromine
atom at the C-6 position, coupled with a suitable N-4
6
-Br om o-2,2-d im eth yl-2H-1,4-ben zoth ia zin -3(4H)-on e
9). A mixture of 2,5-dibromonitrobenzene (8.4 g, 30 mmol)
-mercapto-2-methylpropanoic acid30 (3.6 g, 30 mmol), and dry
CO (10 g, 72 mmol) in dry DMF (100 mL) was heated at
(
2
K
2
3
100 °C for 7 h under mechanical stirring and nitrogen
atmosphere. The cooled mixture was diluted with water,

1
,4-Benzothiazines as KATP-Channel Openers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3675
1
washed with EtOAc, acidified with 12 N HCl, and then
extracted with EtOAc. The combined organic layers were
washed with water, dried, and evaporated to dryness to give
mp 115-116 °C. H NMR δ 1.48 (6H, s, CH
3
), 3.35 (2H, d, J )
3 Hz, CH ), 4.5 (1H, bs, NH), 7.07 (1H, d, J ) 8.5 Hz, H-8),
7.42 (1H, d, J ) 2.3 Hz, H-5), 7.51 (1H, dd, J ) 2.3 and 8.5
2
a residue which was recrystallized from H
-[(4-br om o-2-n itr op h en yl)th io]-2-m eth ylp r op a n oic a cid
as a crystalline yellowish solid (6.24 g, 65%); mp 137-140 °C.
2
O/AcOH to give
Hz, H-7).
2
6-Cyan o-2,2-dim eth yl-3,4-dih ydr o-2H-1,4-ben zoth i-azin e
(15). A mixture of 6-bromobenzothiazine 12 (1 g, 3.88 mmol)
and CuCN (0.69 g, 7.76 mmol) in N-methylpyrrolidinone (50
mL) was refluxed for 10 h. The reaction mixture was cooled,
diluted with a 10% aqueous ethylendiamine (50 mL), and then
extracted with EtOAc. The combined organic layers were
washed with brine, dried, and evaporated to dryness. The
obtained residue was recrystallized from Et
anobenzothiazine 15 (0.32 g, 41%) as a yellowish solid; mp
3 2
120-122 °C. H NMR δ 1.40 (6H, s, CH ), 3.26 (2H, s, CH ),
6.74 (1H, d, J ) 1.5 Hz, H-5), 6.80 (1H, dd, J ) 8.0 and 1.5
Hz, H-7), 7.00 (1H, d, J ) 8.0 Hz, H-8). 13C NMR 140.6, 128.0,
123.6, 120.7, 118.5, 116.9, 107.8, 53.7, 40.1, 27.8. MS m/z (rel
int) 204 (86), 189 (29), 174 (9), 161 (100).
6-Br om o-2,2-dim eth yl-4-n itr oso-3,4-dih ydr o-2H-1,4-ben -
zoth ia zin e (16). An aqueous solution of NaNO2 (2 g, 29
mmol), dissolved in a minimum amount of water, was added
dropwise to a solution of 6-bromobenzothiazine 12 (5 g, 19
mmol) in MeOH (70 mL) and AcOH (2.7 mL), cooled to 0 °C.
The mixture was stirred overnight at room temperature,
1
3 2
H NMR δ 1.65 (6H, s, CH ), 6.65 (1H, bs CO H), 7.50 (1H, d,
J ) 8.5 Hz, H-6), 7.65 (1H, dd, J ) 2.4 e 8.5 Hz, H-5), 7.95
(
1H, d, J ) 2.4 Hz, H-3).
An aqueous solution of FeSO
4
2
‚7H O (36.5 g, 131 mmol
dissolved in 60 mL of hot water) was added portionwise to a
solution of the above nitro acid (6 g, 18.7 mmol) in NH OH
60 mL). The mixture was stirred at room temperature for 3
4
2
O to give 6-cy-
(
1
h then filtered, washed with diluted NH
4
OH, and then with
water. The filtrate was acidified with 12 N HCl, and the
precipitate obtained was filtered off and recrystallized from
EtOH to give the 6-bromobenzothiazinone 9 (3.47 g, 68%) as
1
a white solid; mp 189-191 °C. H NMR δ 1.60 (6H, s, CH
3
),
7
.00 (1H, d, J ) 8.5 Hz, H-8), 7.15 (1H, dd, J ) 2.4 e 8.5 Hz,
H-7), 7.25 (1H, d, J ) 2.4 Hz, H-5), 8.30 (1H, bs, NH).
,2-Dim et h yl-6-n it r o-2H -1,4-b en zot h ia zin -3(4H )-on e
2
2
6
(
2
10). A mixture of 2-fluoro-5-nitroaniline (13 g, 83 mmol),
3
0
-mercapto-2-methylpropanoic acid (10 g, 83 mmol), and dry
CO (27.5 g, 0.2 mol) in dry DMF (140 mL) was heated at
00 °C for 18 h under nitrogen atmosphere and mechanical
K
2
3
1
3
neutralized with aqueous saturated solution of NaHCO , and
stirring. The mixture was poured into ice-water and the
obtained precipitate filtered off, washed with water, dried, and
purified by column chromatography eluting with EtOAc/
petroleum ether (2:8) to give the 6-nitrobenzothiazinone 10
extracted with EtOAc. The combined organic layers were
washed with water, dried, and evaporated to dryness to give
the 4-nitroso derivative 16 (5.2 g, 96%) as a dark yellow solid
which was used in the next step without further purification;
1
(
2
7.9 g, 40%) as an orange solid; mp 221-222 °C (litt. mp 228-
mp 48 °C. H NMR δ 1.35 (6H, s, CH
3
), 3.90 (2H, s, CH
2
), 7.05
1
30 °C). H NMR δ 1.55 (6H, s, CH
3
), 7.48 (1H, d, J ) 8.6 Hz,
(1H, d, J ) 8.5 Hz, H-8), 7.25 (1H, dd, J ) 2.4 and 8.5 Hz,
H-7), 8.10 (1H, d, J ) 2.4 Hz, H-5).
H-8), 7.80 (1H, d, J ) 2.3 Hz, H-5), 7.90 (1H, dd, J ) 2.3 e 8.
6
Hz; H-7), 9.00 (1H, bs, NH).
,2-Dim et h yl-6-t r iflu or om et h yl-2H -1,4-b en zot h ia zin -
(4H)-on e (11). A solution of ethyl 2-bromo-2-methylpro-
In an analogous procedure, 6-nitro-4-nitroso derivative 17
was prepared from the corresponding 6-nitrobenzothiazine 13
as a yellow solid in 78% yield; mp 119-121 °C.
4-Am in o-6-br om o-2,2-d im eth yl-3,4-d ih yd r o-2H-1,4-ben -
zoth ia zin e (18). A solution of 4-nitroso derivative 16 (4.8 g,
17 mmol) in MeOH (30 mL) and AcOH (5 mL) was added to a
suspension of Zn (5.4 g, 83 mmol) in water (10 mL), cooled to
0 °C. The mixture was stirred for 15 min, filtered, basified
2
3
panoate (6.8 g, 35 mmol) in dry DMF (20 mL) was added to a
mixture of 2-amino-4-(trifluoromethyl)benzenethiol hydrochlo-
ride (8 g, 35 mmol), and dry K CO (9.6 g, 70 mmol) in dry
2 3
DMF (70 mL), under stirring and nitrogen atmosphere. The
mixture was heated at 90 °C for 8 h and then poured into ice-
water. The precipitated solid was filtered off, washed with
water, dried, and recrystallized from cyclohexane to give the
with NH OH, and extracted with EtOAc. The combined
4
organic layers were washed with water, dried, and evaporated
to dryness. The residue was purified by column chromatog-
raphy eluting with cyclohexane to give the 4-amino derivative
6
-trifluoromethylbenzothiazinone 11 (8.7 g, 95%) as white
1
solid; mp 145-146 °C. H NMR δ 1.40 (6H, s, CH
7
8
3
), 7.20-
.25 (1H, m, H-5), 7.25-7.35 (1H, m, H-7), 7.42 (1H, d, J )
.2 Hz; H-8), 8.80 (1H, bs, NH).
-Br om o-2,2-d im e t h yl-3,4-d ih yd r o-2H -1,4-b e n zot h i-
1
18 (2 g, 43%) as whitish solid; mp 83-85 °C.
H NMR δ 1.40
(6H, s, CH ), 3.40 (2H, s, CH ), 3.80 (2H, bs, NH ), 6.70 (1H,
3
2
2
6
d, J ) 2.4 Hz, H-5), 6.80 (1H, dd, J ) 2.4 e 8.5 Hz, H-7), 7.40
(1H, d, J ) 8.5 Hz, H-8).
a zin e (12). A solution of 6-bromobenzothiazinone 9 (3 g, 11
mmol) in dry THF (100 mL) was added dropwise and under
nitrogen atmosphere to a suspension of LiAlH (0.98 g, 25.8
4
mmol) in THF (30 mL) cooled to 0 °C. After the addition was
complete, the mixture was refluxed for 4 h and then cooled,
4-Am in o-2,2-d im eth yl-6-n itr o-3,4-d ih yd r o-2H-1,4-ben -
zoth ia zin e (19). A solution of nitroso derivative 17 (2.0 g, 7.9
mmol) in MeOH (70 mL) was cooled to 0 °C, and then an
aqueous solution of 3.6 N NaOH (7 mL) was added. Forma-
midinesulfinic acid (2.56 g, 23.7 mmol) was then added
gradually. The resulting mixture was stirred at room temper-
ature for 22 h and then evaporated to dryness. The residue
was purified by flash chromatography, eluting with cyclohex-
ane/EtOAc (7:3) to give the 4-amino derivative 19 (0.72 g, 38%)
4
and EtOAc was carefully added to destroy the excess of LiAlH .
The mixture was diluted with water and extracted with EtOAc.
The combined organic layers were dried and evaporated to
dryness, yielding a residue which was recrystallized from
cyclohexane to give the 6-bromobenzothiazine 12 (2.4 g, 86%)
1
1
as a white solid; mp 184-188 °C. H NMR δ 1.40 (6H, s, CH
3
),
as a semisolid that decomposes in air. H NMR δ 1.45 (6H, s,
3
.27 (2H, s, CH
2
), 6.65 (1H, d, J ) 2.4 Hz, H-5), 6.75 (1H, dd,
J ) 2.4 e 8.5 Hz, H-7), 6.80 (1H, d, J ) 8.5 Hz, H-8).
CH ), 3.45 (2H, s, CH ), 3.90 (2H, bs, NH ), 7.05 (1H, d, J )
3
2
2
8.5 Hz, H-8), 7.54 (1H, dd, J ) 2.4 and 8.5 Hz, H-7), 8.2 (1H,
d, J ) 2.4 Hz, H-5).
In an analogous procedure, 6-trifluomethylbenzothiazine 14
was prepared from the corresponding 6-trifluomethylbenzothi-
azinone 11 as a white solid in 95% yield; mp 97.5-98 °C.
6-Br om o-2,2-d im et h yl-4-(2-oxo-1-p yr r olid in yl)-3,4-d i-
h yd r o-2H-1,4-ben zoth ia zin e (3a ). A solution of 4-chlorobu-
2
,2-Dim e t h yl-6-n it r o-3,4-d ih yd r o-2H -1,4-b e n zot h i-
tyryl chloride (0.3 g, 2.19 mmol) in CH
dropwise to a solution of 4-amino derivative 18 (0.6 g, 2.19
mmol) and Et N (0.3 mL, 2.19 mmol) in CH Cl (20 mL), cooled
to 0 °C. The mixture was stirred at 0 °C for 1 h, diluted with
water, and extracted with CH Cl . The combined organic layers
were washed with water, dried, and evaporated to dryness.
The residue was washed with Et O to give 4-ch lor o-N-(6-
2 2
Cl (10 mL) was added
a zin e (13). A solution of 6-nitrobenzothiazinone 10 (3.5 g, 15
mmol) in THF (30 mL) was added dropwise to a 1 M solution
of BH ‚THF (34 mL, 34 mmol) in THF at 0 °C. The solution
was refluxed under stirring for 2 h, cooled, carefully diluted
with MeOH (4 mL), and again refluxed for an additional 45
min. After cooling, the reaction was acidified with 12 N HCl
3
2
2
3
2
2
2
(
4 mL), refluxed for 1 h, cooled, basified with 10% NaOH, and
br om o-2,2-d im eth yl-3,4-d ih yd r o-2H-1,4-ben zoth ia zin -4-
yl)bu tyr yla m id e (0.6 g, 70%) as a white solid. NMR δ 1.40
1
finally extracted with EtOAc. The combined organic layers
were washed with water, dried, and evaporated to dryness to
give 6-nitrobenzothiazine 13 (2.8 g, 83%) as red coral solid;
and 1.60 (each 3H, s, CH
3
), 2.00-2.38 (2H, m, CH
2
CH
2
Cl),
),
2.40-2.80 (2H, m, CH
2
CO), 3.35 (1H, d, J ) 11.3 Hz, CH
2

3
676 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
.50-3.70 (3H, m, CH Cl and CH ), 6.80-7.10 (3H, m,
Cecchetti et al.
3
2
2
ane/EtOAc 9:1 to cyclohexane/EtOAc 7:3; yield 36%; mp 127-
aromatic H), 7.40 (1H, bs, NH).
129 °C. 1H NMR δ 1.45 (6H, s, CH
3
), 2.60-2.70 and 2.75-
The solution of the above amide (0.6 g, 1.59 mmol) in DMF
2.85 (each 2H, m, CH CH ), 3.55 (2H, s, CH ), 6.90 (1H, d, J
2
2
2
(
10 mL) was cooled to 0 °C, and then t-BuOK (0.214 g, 1.91
) 1.6 Hz, H-5), 7.10 (1H, dd, J ) 1.6 and 8.1 Hz, H-7), 7.15
(1H, d, J ) 8.1 Hz, H-8), 7.30 (1H, t, J ) 3.1 Hz, CH). Anal.
mmol) was added. After stirring for 5 min, the reaction mixture
was diluted with water, and the precipitate was filtered off,
washed with water, dried, and recrystallized from EtOAc to
give the pyrrolidinone derivative 3a (0.4 g, 74%) as a white
solid; mp 196-197 °C. NMR δ 1.40 and 1.50 (each 3H, s, CH
2
and 3.70 (each 1H, d, J ) 11.3 Hz, CH
3
6
(C16
16 2
H N OS), C, H, N.
N-(6-Br om o-2,2-d im eth yl-3,4-d ih yd r o-2H-1,4-ben zoth i-
a zin -4-yl)a ceta m id e (3d ). A solution of acetyl chloride (0.124
mL, 1.76 mmol) in dry CH Cl (10 mL) was added dropwise to
1
3
),
CO), 3.30
), 3.45-3.55 and 3.60-
N), 6.70 (1H, d, J ) 1.9 Hz, H-5),
.85 (1H, dd, J ) 1.9 and 8.2 Hz, H-7), 6.90 (1H, d, J ) 8.2
OS), C, H, N.
2
2
.10-2.34 (2H, m, CH
2
CH
2
N), 2.38-2.60 (2H, m, CH
2
a solution of 4-amino derivative 18 (0.4 g, 1.5 mmol) and Et N
3
2
(0.24 mL, 1.76 mmol) in CH Cl (20 mL), cooled to 0 °C. The
2
2
.70 (each 1H, m, CH
2
CH
2
mixture was stirred at 0 °C for 30 min, diluted with water,
and extracted with CH Cl . The combined organic layers were
2
2
Hz, H-8). Anal. (C14
H
17BrN
2
washed with water, dried, and evaporated to dryness. The
residue was crystallized from EtOAc to give the acetamido
In an analogous procedure, compound 4a was prepared from
the corresponding 4-amino-6-nitrobenzothiazine 19, as well as
compound 3b, by reacting 4-amino-6-bromobenzothiazine 18
with 5-chlorovaleryl chloride instead of 4-chlorobutyryl chlo-
ride.
derivative 3d (0.4 g, 87%) as a white solid; mp 175-176 °C.
1
H NMR δ 1.41 and 1.43 (each 3H, s, CH
3
), 2.10 (3H, s,
), 6.85
COCH
3
), 3.35 and 3.55 (each 1H, d, J ) 11.3 Hz, CH
2
(1H, d, J ) 2.4 Hz, H-5), 6.90 (1H, dd, J ) 2.4 and 8.5 Hz,
2
,2-Dim eth yl-6-n itr o-4-(2-oxo-1-p yr r olid in yl)-3,4-d ih y-
d r o-2H-1,4-ben zoth ia zin e (4a ). It was purified by column
chromatography eluting with CH Cl ; yield 60%; mp 105-106
), 2.23 (2H, tt, J ) 7 and 7.6
N), 2.64 (2H, t, J ) 7.6 Hz, CH CO), 3.90 (2H, t,
J ) 7 Hz, CH CH N), 4.00 (2H, s, CH ), 7.25 (1H, d, J ) 8.7
H-7), 7.10 (d, 1H, J ) 8.5 Hz, H-8), 7.40 (1H, bs, NH). Anal.
(C12
2
H15BrN OS), C, H, N.
2
2
In an analogous procedure, compounds 3f, 4d , and 4f were
prepared from the corresponding 4-aminobenzothiazines 18
and 19, while compounds 3e, 3g, 3h , 4e, 4g, and 4h were
prepared by reacting the corresponding 4-aminobenzothiazines
12 and 13, with the appropriate acyl chloride. When the
temperature and reaction times were different from that
described for compound 3d , they are reported below along with
the other chemical data.
1
°
C. H NMR δ 1.38 (6H, s, CH
3
Hz, CH CH
2
2
2
2
2
2
Hz, H-8), 7.80 (1H, dd, J ) 2.3 and 8.7 Hz, H-7), 8.10 (1H, d,
J ) 8.7 Hz, H-5). Anal. (C14
found, 5.01.
17 3 3
H N O S), C, H, N; H: calcd, 5.57;
6
-Br om o-2,2-d im eth yl-4-(2-oxo-1-p ip er id in yl)-3,4-d ih y-
d r o-2H-1,4-ben zoth ia zin e (3b). It was crystallized from
cyclohexane; yield 80%; mp 146-148 °C. H NMR δ 1.40 and
N-(2,2-Dim et h yl-6-n it r o-3,4-d ih yd r o-2H -1,4-b en zot h i-
a zin -4-yl)a ceta m id e (4d ): purified by column chromatogra-
1
1
2
1
.60 (each 3H, s, CH
.50-2.65 (2H, m, CH
1.5 Hz, CH ), 3.50-3.60 (2H, m, CH
3
2 2 2
), 1.70-2.10 (4H, m, CH CH CH N),
phy eluting cyclohexane/EtOAc 9:1; yield 27%; mp 188-189
2
CO), 3.20 and 3.90 (each 1H, d, J )
CH CH N), 6.60 (1H, d,
°
(
2
C. H NMR δ 1.50 (6H, s, CH
1
), 2.20 (3H, s, COCH
), 4.05
3
3
2
2
2
2
2H, s CH
2
), 7.15 (1H, d, J ) 8.2 Hz, H-8), 7.65 (1H, dd, J )
.3 and 8.2 Hz, H-7), 7.85 (1H, bs, NH), 8.00 (d, 1H, J ) 2.3
S), C, H, N.
J ) 1.8 Hz, H-5), 6.80-6.95 (2H, m, H-7 and H-8). Anal.
OS), C, H, N.
-Br om o-2,2-d im et h yl-4-(1-oxo-2-cyclop en t en e-2-yl)-
,4-d ih yd r o-2H-1,4-ben zoth ia zin e (3c). A mixture of freshly
15 2
(C H19BrN
Hz, H-5). Anal. (C12
15 3 3
H N O
6
N-(6-Br om o-2,2-d im eth yl-3,4-d ih yd r o-2H-1,4-ben zoth i-
a zin -4-yl)-3-flu or oben za m id e (3f): 1.5 h; crystallized from
3
distilled cyclopentanone (5,1 mL, 58 mmol), NBS (10.34 g, 58
mmol), and a catalytic amount of dibenzoyl peroxide in dry
CCl (80 mL) was refluxed for 3 h under nitrogen atmosphere,
4
cooled, filtered, and evaporated to dryness. The obtained
residue was cooled to - 70 °C, and a solution of 6-bromoben-
zothiazine 12 (1.0 g, 3.87 mmol) and Et N (6.5 mL, 46.5 mmol)
3
1
cyclohexane; yield 30%; mp 150-151 °C. H NMR δ 1.55 (6H,
s, CH
1H, bs, NH). Anal. (C17
N-(2,2-Dim et h yl-3,4-d ih yd r o-6-n it r o-2H -1,4-b en zot h i-
3 2
), 3.70 (2H, s CH ), 6.90-7.70 (7H, m, aromatic H), 7.90
(
2
H16BrFN OS), C, H, N.
a zin -4-yl)-3-flu or oben za m id e (4f): 1.5 h; purified by column
chromatography eluting with petroleum ether/EtOAc 9:1; yield
in dry THF (15 mL) was added. The resulting mixture was
stirred at room temperature for 48 h and then poured into
water and extracted with EtOAc. The combined organic layers
were dried and evaporated to dryness. The obtained residue
was purified by column chromatography eluting with cyclo-
hexane/EtOAc (9:1) to cyclohexane/EtOAc (1:1) to give the
cyclopentenyl derivative 3c (0.46 g, 35%) as a light yellow
1
3
5%; mp 181-184 °C. H NMR δ 1.45 (6H, s, CH
3
), 3.60 (2H,
s CH ), 7.00 (1H, d, J ) 8.5 Hz, H-8), 7.15-7.60 (6H, m,
aromatic H), 8.50 (1H, bs, NH). Anal. (C17
N.
2
3 3
H16FN O S), C, H,
4
-Acetyl-6-br om o-2,2-d im eth yl-3,4-d ih yd r o-2H-1,4-ben -
zoth ia zin e (3e): room temperature, 14 h; column chromatog-
raphy eluting with cyclohexane/EtOAc 9:1; yield 35%; mp 67-
1
solid: mp 128-129 °C. H NMR δ 1.40 (6H, s,CH
4H, m, CH CH ), 3.60 (2H, s, CH ), 6.85 (3H, bs, aromatic
H), 7.18 (1H, t, J ) 3.0 Hz, CH). Anal. C15 16BrNOS.
3
), 2.50-2.80
6
9 °C. 1H NMR (CD
3
OD) δ 1.50 (6H, s, CH
), 3.75 (2H, s, CH ), 7.00 (1H, d, J ) 8.5 Hz, H-8), 7.20
1H, dd, J ) 2.0 and 8.5 Hz, H-7), 7.30 (1H, bs, H-5). Anal.
14BrNOS), C, H, N.
-Acetyl-2,2-d im eth yl-6-n itr o-3,4-d ih yd r o-2H-1,4-ben -
3
), 2.25 (3H, s,
(
2
2
2
COCH
3
2
H
(
(
Compounds 4c, 5c, and 6c were prepared in a similar way
starting from benzothiazines 13, 14, and 15, respectively.
12
C H
4
2
,2-Dim et h yl-6-n it r o-4-(1-oxo-2-cyclop en t en -2-yl)-3,4-
zoth ia zin e (4e): room temperature, 17 h; column chromatog-
d ih yd r o-2H-1,4-ben zoth ia zin e (4c). It was crystallized from
1
raphy eluting with cyclohexane/EtOAc 9:1; yield 61%; mp 134-
cyclohexane/EtOAc; yield 35%; mp 115-116 °C. H NMR δ 1.40
6H, s, CH CH ),
), 7.05 (1H, d, J ) 8.6 Hz, H-8), 7.24 (1H, t, J
3.0 Hz, CH), 7.52 (1H, d, J ) 2.3 Hz, H-5), 7.61 (1H, dd, J
2.3 and 8.6 Hz, H-7). Anal. (C15 S), C, H, N.
,2-Dim eth yl-4-(1-oxo-2-cyclopen ten e-2-yl)-6-tr ifu or om -
1
1
(
2
35 °C. H NMR δ 1.50 (6H, s, CH
3
), 2.30 (3H, s, COCH
3
), 3.90
(
3
3
), 2.50-2.60 and 2.65-2.75 (each 2H, m, CH
.55 (2H, s, CH
2
2
2H, s, CH
2
), 7.25 (1H, d, J ) 8.5 Hz, H-8), 7.90 (1H, dd, J )
2
.3 and 8.5 Hz, H-7), 8.10 (1H, bs, H-5). Anal. (C12 S),
14 2 3
H N O
)
)
C, H, N.
16 2 3
H N O
2
6-Br om o-4-(3-flu or oben zoyl)-2,2-d im eth yl-3,4-d ih yd r o-
eth yl-3,4-d ih yd r o-2H-1,4-ben zoth ia zin e (5c). It was puri-
fied by column chromatography eluting with a gradient of
2H-1,4-ben zoth ia zin e (3g): room temperature, 12 h; crystal-
1
lized from cyclohexane; yield 74%; mp134-135 °C. H NMR δ
cyclohexane to cyclohexane/EtOAc 9:1; yield 33%; mp 89-91
3 2
1.50 (6H, s, CH ), 3.95 (2H, s CH ), 6.70 (1H, d, J ) 1.9 Hz,
1
°
(
C. H NMR δ 1.45 (6H, s, CH
3
), 2.50-2.60 and 2.65-2.75
), 6.90-7.00 (2H, m,
H-5, H-7), 7.15 (1H, d, J ) 8.0 Hz, H-8), 7.20 (1H, t, J ) 3.0
Hz, CH). Anal. (C16 NOS), C, H, N.
-Cya n o-2,2-Dim et h yl-4-(1-oxo-2-cyclop en t en e-2-yl)-
,4-d ih yd r o-2H-1,4-ben zoth ia zin e (6c). It was purified by
column chromatography eluting with a gradient of cyclohex-
H-5), 7.00 (1H, d, J ) 8.5 Hz, H-8), 7.10-7.20 (5H, m, aromatic
H). Anal. (C17 15BrFNOS), C, H, N.
each 2H, m, CH
2
CH
2
), 3.60 (2H, s, CH
2
H
4-(3-F lu or oben zoyl)-6-n itr o-2,2-d im eth yl-3,4-d ih yd r o-
2H-1,4-ben zoth ia zin e (4g): room temperature, 48 h; flash
H
16
F
3
6
chromatography eluting with cyclohexane/EtOAc 9:1; yield
1
3
29%; mp 178-179 °C. H NMR δ 1.60 (6H, s, CH
3
), 4.05 (2H,
s CH ), 7.10-7.35 (5H, m, aromatic H), 7.55 (1H, d, J ) 2.2
2

1
,4-Benzothiazines as KATP-Channel Openers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3677
Hz, H-5), 7.85 (1H, dd, J ) 2.2 and 8.8 Hz, H-7). Anal. (C17
FN S), C, H, N.
-Br om o-4-ch lor oa cetyl-2,2-d im eth yl-3,4-d ih yd r o-2H-
,4-ben zoth ia zin e (3h ): reflux, 4 h; column chromatography
eluting with cyclohexane/EtOAc 9:1; yield 65%; mp 84-85 °C.
H
15
-
tive 7i except that the reaction time was 1 h. The crude product
was purified by column chromatography eluting with petro-
leum ether/EtOAc (95:5) to give the allyl derivative 5i (53%)
2
O
3
6
1
1
as a light yellow foam; mp 67-69 °C. H NMR δ 1.25 and 1.40
(each 3H, s, CH ), 2.50 and 4.45 (each 1H, d, J ) 12.2 Hz,
3
1
CH ), 3.90-4.35 (2H, m, CH dCHCH N), 5.20-5.35 (2H, m,
H NMR δ 1.50 (6H, s, CH
CH Cl), 7.05 (1H, d, J ) 8.5 Hz, H-8), 7.25 (1H, dd, J ) 2 and
.5 Hz, H-7), 7.40 (1H, bs, H-5). Anal. (C12 13BrClNOS), C,
H, N.
-Ch lor oa cet yl-6-n it r o-2,2-d im et h yl-3,4-d ih yd r o-2H -
,4-ben zoth ia zin e (4h ): reflux, 13 h. column chromatography
eluting with cyclohexane/EtOAc 9:1; yield 29%; mp 117-118
3
), 3.90 (2H, bs, CH
2
), 4.25 (2H, s,
2
2
2
CH
2
dCHCH
2
2 2
N), 5.80-6.00 (1H, m, CH dCHCH N), 6.80-6.90
2
8
H
(2H, m, H-5 and H-7), 7.20 (1H, d, J ) 8.5 Hz, H-8). Anal.
(C14
16 3
H F NS), C, H, N.
4
Va sor ela xa n t Activity. All the experimental procedures
were carried out following the guidelines of the European
Community Council Directive 86-609.
1
1
°
C. H NMR δ 1.50 (6H, s, CH
3
), 3.90 (2H, s, CH
2
), 4.30 (2H,
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 anaesthesia
and bled. The aortae were immediately excised and freed of
extraneous tissues. The endothelial layer was removed by
gently rubbing the intimal surface of the vessels with a
hypodermic needle. Five mm wide aortic rings were suspended,
under a preload of 2 g, in 10 mL organ baths, containing
Tyrode solution (composition of saline in mM: NaCl 136.8; KCl
s, CH Cl), 7.25 (1H, d, J ) 8.8 Hz, H-8), 7.95 (1H, dd, J ) 2.3
2
and 8.8 Hz, H-7), 8.20 (1H, d, J ) 2.3 Hz, H-5). Anal. (C12
13
H -
ClN
2
O
3
S), C, H, N.
6
-Br om o-2,2-d im et h yl-4-(2-oxo-1-p yr r olid in yl)-3,4-d i-
h yd r o-2H-1,4-ben zoth ia zin e 1-Oxid e (3a a ). MCPBA 55%
0.18 g, 0.6 mmol) was added to a solution of pyrrolidone
derivative 3a (0.2 g, 0.6 mmol) in CH Cl (5 mL) cooled on ice.
After 5 min, an acqueous solution of NaHCO was added, and
(
2
2
3
the organic layer was washed with brine, dried, and evapo-
rated to dryness. The residue was purified by flash chroma-
tography eluting with CH Cl to give a solfoxide derivative 3a a
2 2
2.95; CaCl
11.9; glucose 5.5), thermostated at 37 °C and continuously
gassed with a mixture of O (95%) and CO (5%). Changes in
2 4 2 2 4 3
1.80; MgSO 7H O 1.05; NaH PO 0.41; NaHCO
1
(
0.19 g, 90%) as white solid; mp 152-154 °C. H NMR δ 1.60
2
2
and 1.70 (each 3H, s, CH
3
), 2.20-2.30 (2H, m, CH
CO), 3.10 and 4.00 (each 1H, d, J )
), 3.55-3.65 (2H, m, CH CH N), 6.85 (1H, d, J )
.8 Hz, H-5), 7.10 (1H, dd, J ) 1.8 and 8.2 Hz, H-7), 7.50 (1H,
S), C, H, N.
-Br om o-2,2-d im et h yl-4-(2-oxo-1-p yr r olid in yl)-3,4-d i-
2
CH
2
N), and
tension were recorded by means of an isometric transducer
(Basile mod. 7005), connected to an unirecord microdynamom-
eter (Basile mod. 7050).
2
1
1
.40-2.65 (2H, m, CH
2
2.3 Hz, CH
2
2
2
After an equilibration period of 60 min, the endothelial
integrity was confirmed by administrating acetylcholine (ACh)
(10 µM) to norepinephrine (NE, 1 µM)-precontracted vascular
rings. A <10% relaxation of the NE-induced contraction was
indicative of an acceptable lack of the endothelial layer, while
the organs, showing a g10% relaxation (i.e., significant pres-
ence 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
concentration of KCl (20 mM). When the contraction reached
a stable plateau, 3-fold increasing concentrations of the tested
compounds or of the reference compound levcromakalim were
added cumulatively. In parallel experimental procedures, to
investigate the influence of higher depolarization levels on the
vasorelaxing activity, the aortic preparations were contracted
by 60 mM KCl, and then 3-fold increasing concentrations of
some selected compounds 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 potassium
channel blocker glibenclamide (1 µM) was added, before the
KCl (20 mM)-induced contraction, and then selected com-
pounds were administered.
Norepinephrine hydrochloride (Sigma), acetylcholine chlo-
ride (Sigma), and KCl were dissolved in bidistilled water.
Glibenclamide (Sigma) was dissolved by sonication in aqueous
NaOH (0.1 N). Levcromakalim (Sigma) 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 pharmacological
experimental procedures. Previous experiments showed a
complete ineffectiveness of the administration of the vehicle.
2 2
d, J ) 8.2 Hz, H-8). Anal. (C14H17BrN O
6
h yd r o-2H-1,4-ben zot h ia zin e 1,1-d ioxid e (3a b). The title
compound was prepared using the procedure as described for
3
a a employing 2 equiv of MCPBA. It was obtained in 92%
1
yield; mp 92-94 °C. H NMR δ 1.40 and 1.60 (each 3H, s, CH
3
),
CO),
N), 3.65 e 3.80 (each 1H, d, J )
), 6.70 (1H, d, J ) 2.4 Hz, H-5), 6.95 (1H, dd, J )
.4 e 8.5 Hz, H-7), 7.60 (1H, d, J ) 8.5 Hz, H-8). Anal. (C14
S), C, H, N.
-Cya n o-2,2-d im et h yl-4-(2-oxo-1-p yr r olid in yl)-3,4-d i-
2
3
1
1
.10-2.30 (2H, m, CH
.35-3.60 (2H, m, CH
2.6 Hz, CH
2
CH
CH
2
N), 2.35-2.60 (2H, m, CH
2
2
2
2
17
H -
2 3
BrN O
6
h yd r o-2H-1,4-ben zoth ia zin e (6a ). The title compound was
prepared using the procedure as described for 15 starting from
6
-bromo counterpart 3a . It was obtained in 79% yield as a
1
whitish solid; mp 131-132 °C. H NMR δ 1.40 e 1.44 (each
H, s, CH ), 2.05-2.30 and 2.32-2.55 (each 2H, m, pyrrolidine-
CH ), 3.25 e 3.67 (each 1H, d, J ) 12 Hz, thiazine-CH ), 3.38-
.70 (2H, m, CH CO), 6.80 (1H, d, J ) 1.5 Hz, H-5), 6.90 (1H,
dd, J ) 1.5 and 8.0 Hz, H-7), 7.05 (1H, d, J ) 8.0 Hz, H-8).
NMR δ 173.3, 139.8, 128.0, 125.5, 122.4, 118.9, 113.9, 108.5,
0.2, 43.2, 40.8, 28.8, 28.6, 27.7, 16.5. MS m/z(rel, int.) 287
39), 244 (5), 202 (34), 187 (100), 174 (6), 161 (19), 134 (8).
Anal. (C15 OS), C, H, N.
-Allyl-2,2-d im et h yl-6-t r iflu om et h yl-2H-1,4-ben zot h i-
3
3
2
2
3
2
1
3
C
6
(
17 3
H N
4
a zin -3(4H)-on e (7i). A solution of 6-trifluomethylbenzothi-
azinone 11 (0.30 g, 1.15 mmol) in dry DMF (4 mL) was added
dropwise to a suspension of 60% NaH (0.055 g, 1.38 mmol) in
dry DMF (3 mL). After stirring for 1 h, allyl iodide (0.16 mL,
1
.75 mmol) was added dropwise. The resulting mixture was
stirred for an additional 2 h and then poured into ice-water
and extracted with EtOAc. The combined organic layers were
washed with water, dried, and evaporated to dryness to give
a residue which was purified by column chromatography
eluting with cyclohexane to cyclohexane/EtOAc (9:1) to give
allyl derivative 7i (0.3 g, 87%) as a yellowish oil. 1H NMR
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 0.1 mM (the highest concentration, which
could be administered) of the tested compounds did not reach
the maximal effect, the efficacy parameter represented the
vasorelaxing 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 pIC ,
(
DMSO-d
CHCH N), 5.00 (1H, dd, J ) 1.2, 17.3, CH
1H, dd, J ) 1.2, 10.6, CH dCHCH N), 5.75-6.00 (1H, m,
CH dCHCH N), 7.40-7.50 (2H, m, H-5 and H-7), 7.70 (1H,
NOS), C, H, N; C: calcd,
6
) δ 1.40 (6H, s, CH
3
), 4.65-4.75 (2H, m, CH
2
d
2
2
dCHCH N), 5.20
2
(
2
2
2
2
5
0
d, J ) 8.5 Hz, H-8). Anal. (C14
5
H
14
F
3
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 lower than 50%. The efficacy and potency
5.80; found 55,36.
-Allyl-2,2-d im et h yl-6-t r iflu om et h yl-3,4-d ih yd r o-2H -
,4-ben zoth ia zin e (5i). The title compound was prepared
4
1
using the procedure as described for 12 starting from deriva-

3
678 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Cecchetti et al.
parameters were expressed as the mean ( standard error, for
-10 experiments. The student t test was selected as statisti-
cal analysis; P < 0.05 was considered a significant statistical
difference. Experimental data were analyzed by a computer
fitting procedure (software GraphPad Prism 3.0).
(15) Evans, J . M.; Stemp, G. Structure-Activity Relationships of
Benzopyran Based Potassium Channel Activators. In Potassium
Channels and Their Modulators. From Synthesis to Clinical
Experience; Evans, J . M., Hamilton, T. C., Longman S. D.,
Stemp, G., Eds.; Taylor & Francis Ltd: London, 1996; pp 27-
5
5
5.
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Ack n ow led gm en t. The technical assistance of R.
Bianconi is acknowledged.
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,4-Benzothiazines as KATP-Channel Openers
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