Synthesis, antimalarial activity, structure-activity relationship analysis of thieno-[3,2-b]benzothiazine S,S-dioxide analogs

Article abstract of DOI:10.1016/j.bmc.2008.02.011

An improved procedure for the synthesis of 3-amino-9-arylsubstituted-thieno[3,2-b]benzothiazine S,S-dioxide 2-decarboxylated is reported. Thieno-[3,2-b]benzothiazine S,S-dioxide derivatives were investigated for their abilities to inhibit β-hematin formation, hemoglobin hydrolysis and in vivo for their efficacy in rodent Plasmodium berghei. Compounds 5j-o were the most promising as inhibitors of hemoglobin hydrolysis, however, the compounds are not as efficient as chloroquine. A structure-activity relationship (SAR) study was carried out in this series. Our results allow us to determine the minimal structural requirements to produce the biological response.

Full text of DOI:10.1016/j.bmc.2008.02.011

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3661–3674
Synthesis, antimalarial activity, structure–activity relationship
analysis of thieno-[3,2-b]benzothiazine S,S-dioxide analogs
a
a
a
a
b
´
´
´
Arthur Barazarte, Jose Camacho, Jose Domınguez, Gricela Lobo, Neira Gamboa,
b
c
d
e
´
´
Juan Rodrigues, Mario V. Capparelli, Angel Alvarez-Larena, Sebastian Andujar,
Daniel Enrize and Jaime Charrisa,*
aLaboratorio de Sı´ntesis Orga´nica, Universidad Central de Venezuela, Apartado 47206, Los Chaguaramos, 1041-A Caracas, Venezuela
bUnidad de Bioquı´mica, Facultad de Farmacia, Universidad Central de Venezuela,
Apartado 47206, Los Chaguaramos, 1041-A Caracas, Venezuela
cEscuela de Quı´mica, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela
dServicio de Difraccio´n de Rayos X, Universidad Auto´noma de Barcelona, Bellaterra, Spain
eDepartamento De Quı´mica, Universidad Nacional de San Luı´s, Argentina
Received 12 December 2007; revised 26 January 2008; accepted 4 February 2008
Available online 8 February 2008
Abstract—An improved procedure for the synthesis of 3-amino-9-arylsubstituted-thieno[3,2-b]benzothiazine S,S-dioxide 2-decar-
boxylated is reported. Thieno-[3,2-b]benzothiazine S,S-dioxide derivatives were investigated for their abilities to inhibit b-hematin
formation, hemoglobin hydrolysis and in vivo for their efficacy in rodent Plasmodium berghei. Compounds 5j–o were the most prom-
ising as inhibitors of hemoglobin hydrolysis, however, the compounds are not as efficient as chloroquine. A structure–activity rela-
tionship (SAR) study was carried out in this series. Our results allow us to determine the minimal structural requirements to produce
the biological response.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ization of heme,6 but it has now been demonstrated to
be a crystalline cyclic dimer of ferriprotoporphyrin
IX.7 Thus, hemozoin synthesis, a process unique to the
malaria parasite, offers a logical and valuable potential
target for new antimalarial drug development. New
drugs that attack the same vital target of chloroquine,
but that are not subject to the same resistance mecha-
nism, would be highly desirable. Fluoroquinolones, such
as ciprofloxacin, gatifloxacin, moxifloxacin, and trova-
floxacin, have been reported for their antimalarial activ-
ities.8 We have recently described the preparation and
antimalarial activities of several tricyclic quinolone
analogs.9
Malaria is one of the most important infectious disease
problems for humans, particularly in tropical regions of
the world. As per WHO reports, annually there are more
than 500 million new cases, resulting in at least 1–2 mil-
lion deaths.1,2 In addition, since resistance to currently
used antimalarials is spreading rapidly, there is a great
need for new effective drugs. Thus, there is a compelling
and urgent need for new antimalarials with mechanisms
of action different from those of existing ones in order to
replace those that are becoming obsolete and to identify
new drug targets.3 Chloroquine has recently been shown
to inhibit hemozoin formation within the parasite food
vacuole.4 This process is also thought to be the molecu-
lar target of other quinoline antimalarials.5 Hemozoin
was originally considered to be formed by the polymer-
The quinolone and benzothiazine nuclei are often found
in biologically active molecules, and a large variety of
methods have been employed for their synthesis.10
Generally, these synthetic routes were carried out in
solution. These methods have their merits, but all have
drawbacks such as using large amounts of volatile
and poisonous solvents. Moreover, the reaction times
are long and the yields are not high. In recent years,
microwave dielectric heating technology combined with
Keywords: Thieno; 1,4-Benzothiazines; Cyclization; Antimalarial; SAR
study.
*
Corresponding
charrisj@ucv.ve
author.
Fax:
+58
2126052707; e-mail:
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.011

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A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
solvent-free conditions has been used in many organic
reactions, leading to shorter reaction times, higher
yields, cleaner reaction products and environmentally
benign condition compared to the classical heating.11
sulfone S,N-acetals were mixed with potassium carbon-
ate and irradiated without solvent in a domestic micro-
wave oven to afford compounds 4a–r. The target
compounds were easily recovered by adding water to
the final reaction mixture, removing the solid product
by filtration, and recrystallization from ethanol/water
1:1 to give a pure sample. The identities of compounds
4a–r were established by comparing their physical and
spectroscopic properties with those in the literature
and were confirmed by X-ray diffraction.14 Products
5a–r were obtained when 4a–r were reacted with ethyl
2-mercaptoacetate, triethylamine in dry ethanol under
an inert atmosphere of nitrogen.
In continuation of our studies directed toward synthesis
of quinolones and benzothiazine annulated with various
five and six member heterocycles, we report here the syn-
thesis of thieno-[3,2-b]benzothiazine S,S-dioxide analogs
and their abilities to inhibit b-hematin formation and
hemoglobin hydrolysis in vitro and in vivo for their effi-
cacy in rodent P. berghei. A structure–activity relation-
ship (SAR) study using molecular electrostatic
potentials (MEPs) was performed in order to determine
the minimal structural requirements to produce the
inhibitory effect in this series.
The decarboxylation of the ethyl ester 5a–r to obtain
6a–r in a one-pot reaction was made using several con-
ditions and concentrations of NaOH, KOH, LiOH, or
HCl (Table 1). In some analog systems although the
reaction is straightforward, it suffers from several draw-
backs, such as long reaction times, poor atom economy
(requires extra mole of base as acid scavenger), use of
2. Results and discussion
2.1. Synthesis
We first prepared the sulfone S,N-acetals 3a–r by the
reaction of o-chlorobenzenesulfonylacetonitrile 1a–b
with the respective phenyl isothiocyanates 2a–i, methyl
iodide, and potassium hydroxide in 1,4-dioxane (Scheme
1).12 The stereochemistry around the olefinic carbon–
carbon bond was established using the NMR and
confirmed by X-ray diffraction studies.13 The resulting
Table 1. Different conditions used for decarboxylation
Base/acid
Refluxing/6 h
Room temperature/24 h
NaOH 20, 10, 5% Decarboxylation Starting material
KOH 20, 10, 5%
LiOH 20, 10, 5%
HCl 5%
Decarboxylation Starting material
Decarboxylation Starting material
Decarboxylation Starting material
O2
H
R2
S
CN
O2
S
N
NCS
i
+
R1
R2
SCH3
Cl
R1
CN
Cl
1a-b
2a-i
R2
3a-r
O2
S
CN
H
R1
O2
S
N
ii
N
SCH3
SCH3
R1
CN
R2
Cl
3a-r
4a-r
O2
O2
S
O2
S
NH2
CO2Et
NH2
S
CN
iii
iv
R1
R1
R1
S
S
N
N
N
SCH3
R2
R2
R2
4a-r
5a-r
6a-r
Scheme 1. Reagents and conditions: (i) KOH, 1,4-dioxane, MeI, rt; (ii) K2CO3, MW; (iii) HSCH2CO2Et, Et3N, EtOH, D; (iv) NaOH, KOH, LiOH,
or HCl, D.

A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
3663
Table 2. Inhibition of b-hematin synthesis (IbHS) and globin prote-
olysis (IPG) by benzothiazine derivatives
hazardous solvents, and high temperature which results
in the tar formation and reduces the yields of the desired
products.15
Compound
aR1, R2
%IbHS
%IPG
5a
5b
H
<5
<5
0
All new compounds were characterized by NMR (1H,
13C, HETCOR and FLOCK experiments) and IR. The
purity was established by TLC and microanalysis.
2-CH3
4-CH3
2,5-CH3
3-OCH3
4-OCH3
3-Cl
0
5c
5d
<5
<5
0
0
5e
<5
<5
0
5f
5g
0
In the X-ray crystal structures of compounds 4d and 5d
(Figs. 1 and 2) the bond distances are within the ex-
pected values.16
<5
<5
0
5h
5i
4-Cl
3,4-Cl
H
0
<5
<5
0
5j
5k
78.17 0.65b
74.36 0.43b
64.98 0.50b
76.81 0.69b
55.96 1.56b
37.01 1.52b
0
2.2. Biological assays
2-CH3
4-CH3
2,5-CH3
3-OCH3
4-OCH3
3-Cl
<5
<5
5l
5m
N-Phenylthieno-[3,2-b]benzothiazine S,S-dioxide ana-
logs 5a–r and 6a–r were tested in vitro for their activity
against cultured b-hematin formation and hemoglobin
hydrolysis and in vivo for their efficacy in rodent P.
berghei (Table 2). The in vitro assay was used to assess
the abilities of the N-phenylthieno[3,2-b]benzothiazine
derivatives to inhibit b-hematin formation. In that as-
say, hemin was allowed to form b-hematin under acidic
conditions. Among the 36 compounds tested, none
showed a measurable activity compared to chloroquine
(86.6 2.75%).
<5
<5
5n
5o
<5
<5
5p
5q
4-Cl
<5
<5
0
<5
5r
3,4-Cl
—
—
Leupeptin
Pepstatin
Chloroquine
—
—
89.06 0.69
92.94 0.67
24.12 1.16
—
86.60 2.75
The results are expressed by means standard error of the mean.
a R15a–i = 6-Cl; 5j–r = 7-Cl.
b p > 0.05 compared to leupeptin (LEP) and pepstatin (PEP).
Consequently, compounds 5a–r and 6a–r were tested for
inhibition of globin proteolysis, in an in vitro assay
which uses rich extract of trophozoyte to digest the
native hemoglobin of mice. Electrophoretic analyses
indicated that compounds 5j–o, were effective as inhibi-
tors of hemoglobin degradation 78.17 0.65%, 74.36
0.43%, 64.98 0.5%, 76.81 0.69%, 55.96 1.56%,
and 37.01 1.52%, respectively (band at MW 14.4 kDa)
(Fig. 3).
fourth post-infection, the parasitemia was determined;
the survival days were monitored and compared with
control mice receiving saline (untreated mice). Control
mice died within 12 days post-infection, compound 5k
increased the survival time for 16 days, while chloro-
quine prolonged the survival time of the infected mice
to 30 days. Compound 5k was able to reduce and delay
the progression of malaria 8.7% but did not eradicate
the infection (Table 3).
Compounds 5j–o, were tested in mice infected with P.
berghei ANKA, a chloroquine-susceptible strain of mur-
ine malaria. Mice were given the compound (chloro-
quine or 5j–o, in 20 mg kgꢀ1, ip once daily) for four
consecutive days (days 0–4 post-infection). At day
It should be emphasized that compounds 5j–o, which
bear methyl groups on the phenyl and Cl group on posi-
tion 7 of the benzothiazine, showed the highest activity.
It is interesting to note that the presence of Cl group at
Figure 1,2. Molecular structure of compounds 4d and 5d showing the atomic numbering. The displacement parameters are drawn at 50% probability.
Dashed lines indicate intramolecular hydrogen bonds.

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A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
Figure 3. Standard molecular weight (MW) is expressed in kilodaltons (14.4 kDa). A, under graded globin (control hemoglobin without
trophozoites); B, control hemoglobin with trophozoites of P. berghei; 5j–o, benzothiazine derivatives; LEP, hemoglobin with trophozoites and
leupeptin; PEP, hemoglobin with trophozoites and pepstatin.
Table 3. Effect of benzothiazine derivatives (20 mg/kg) on parasitemia
at fourth day post-infection (%P) and survival days (SD) of P. berghei
infected-mice
azine ring. Only compounds possessing the Cl group
at position 7 displayed inhibitory activity.
Treatment
%P
SD
The MEPs of all the active compounds exhibit two
clear minima values (deep red zones) in the vicinity
of the SO2 and the COOEt groups. The region of
the SO2 is symmetrical with respect to the sulfur atom
displaying two deep and extensive negative zones with
V(r) values of about ꢀ0.079 el/au3. The other mini-
mum is located in the zone near to the COOEt, (red
zone with V(r) values of about ꢀ0.078 el/au3). A much
smaller minimum, which is almost a neutral zone
(V(r) ꢁ ꢀ0.047 el/au3) was found near the Cl substitu-
ent at position 7 on the benzothiazine ring. There is
only one significant positive region located near to
the phenyl ring (blue areas with V(r) ꢁ +0.035 el/au3).
The different electronic distribution displayed for com-
pounds of series 5 in comparison to that of series 6
might be appreciated comparing Figure 4 (compounds
5k, 6k). It should be noted that compound type 6 dis-
played a clear second positive zone near to the NH2
group (blue zone with V(r) values of about +0.030 el/
au3). Thus, the lack of COOEt group in series 6 intro-
duces a profound change in the electronic distribution
which can explain the lack of biological activity ob-
tained for these compounds.
Saline solution
21.80 2.31
9.75 3.01*
8.70 2.43*
15.72 4.11
9.40 3.88*
11.60 2.69
11.21 3.21
1.31 0.30
11.66 1.66
13 1.26
5j
5k
15.5 2.13**
10.80 1.59
11.42 2.65
12.16 1.78
11.6 2.34
30
5l
5m
5n
5o
Chloroquine
The results are expressed by means standard error of the mean.
*p < 0.05 and **p < 0.01 compared to untreated mice (saline). n = 5.
position 7 appears to be necessary, but is not by itself
sufficient to produce the biological response. The lack
of inhibitory effect of compounds 5p–r illustrates this sit-
uation. Compounds that have a Cl substituent on posi-
tion 6 of the benzothiazine nucleus, the same ester on
position 2 or where the ester group has been removed,
and the same substitution pattern on the phenyl group
markedly decreases the activity. Thus, the poor inhibi-
tion of b-hematin formation appears to have close rela-
tion with this substitution pattern.
To further understand the above experimental results,
we performed a conformational and electronic study
on these compounds using theoretical calculations.
The electronic study of these compounds was carried
out using molecular electrostatic potential surfaces
(MEPs).17,18 The purpose was to obtain more precise
information as to how closely these compounds resem-
ble each other in terms of the electronic distribution
and hydrophobic properties. Once the low-energy con-
formations for the different compounds were obtained
(see Section 6 in experimental section) and in an attempt
to find potentially reactive sites for the ligands, we eval-
uated the electronic aspects of the molecules using
MEPs. The electrostatic potentials have long been ap-
plied as a guide to molecular reactive behavior;19,20 for
instance, the most negative values of MEP were inter-
preted as identifying and ranking sites for electrophilic
attack, while its overall pattern served as the basis for
qualitative analyses of biologic recognition interactions.
Compounds displaying different substituents in the phe-
nyl group showed different activities. Thus, compounds
without substituents (5j) or with CH3 groups (5k–5m)
having a large-size positive zone near to the phenyl
group were the most active molecules in this series.
Compounds possessing OCH3 group (5n and 5o) possess
a reduced positive zone by the presence of the oxygen
atom giving negative zones. These compounds display
only a mediocre activity. In contrast, compounds pos-
sessing electron-withdrawing substituents (compounds
5p–r) were devoid of any inhibitory activity. These re-
sults clearly indicate that the increase of lipophilic prop-
erty with appropriate groups on the phenyl substituent
allows for a good activity for inhibition of hemoglobin
degradation.
The fact that the activity is markedly affected by alter-
ing the substituents at the phenyl ring suggests that
this aromatic ring makes a specific contribution to
the binding via an aromatic ring orientation. In fact,
there are various ways in which these moieties may
be involved, on which we can only speculate. Thus,
we may assume that a flat portion of the receptor
could allow binding with this aromatic ring through
dispersion forces (van der Waals). Our results indicate
that a characteristic electronic distribution on the phe-
nyl ring might be important to produce the adequate
Figure 4 shows the MEPs obtained for compounds 5a,
5j–5q, and 6k. These results account for the general
characteristics of the electronic behavior of compounds
reported here. The general pattern is similar for all 5
derivatives. Comparing the MEPs of 5a with the rest,
it is clear that the different positions of Cl (6 or 7) pro-
duce a different electronic distribution at the benzothi-

A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
3665
Figure 4. Electrostatic potential-encoded electro´n density surfaces of the core structures of compounds 5a, 5j–5q, and 6k. The surfaces were generated
with Gaussian 03 after DFT (B3LYP) minimizations with 6-31G (d) basis set. The coloring represents electrostatic potential with red indicating the
strongest attraction, a positive point charge and blue indicating the strongest repulsion. The electrostatic potential is the energy of interaction of the
positive point charge with the nuclei and electron of a molecule. It provides a representative measure of overall molecular charge distribution.
interaction. Whereas methyl substituents give major
inhibitions, only mediocre activity was determined
for methoxy derivatives. The marginal efficacy results
in the Peter test may be a result of poor bioavailabil-
ity of these compounds.
and their thiene analogs carboxylated and their decar-
boxylation in a one-pot reaction have been developed.
We also report a new group of thieno-[3,2-b]benzothi-
azine S,S-dioxide derivatives acting as antimalarial
agents. Among them, compound 5j and some of its
congeners exhibited remarkable inhibitory activity.
Although compounds 5j–o were not tested as specific
protease inhibitors in vitro, the mechanism of action
of these compounds on hemoglobin degradation could
be related to the inhibition of some aspartic, cysteinic,
or metalloproteases due to the presence of a globin
3. Conclusions
Methods for synthesis of the benzothiazine nucleus
using a microwave proceed with a significant decrease
in reaction time and comparable high chemical yield,

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A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
Fig. 4 (continued)
band. Some new benzothiazine analogs showed only
marginal antimalarial activity in the Peter test against
P. berghei by intraperitoneal administration. The poor
solubility in organic solvents and water or an in vivo
enzymatic hydrolysis and decarboxylation to the corre-
sponding inactive 6j–o, may be partially responsible
for the poor in vivo activity observed.
4.1. General procedure for preparation of 6 or 7-chloro-3-
methylthio-4-phenyl-1,4-benzothiazine-2-carbonitrile 1,1-
dioxide (4a–r)
Compounds were prepared following methods devel-
oped by us.14
4.1.1. 6-Chloro-3-methylthio-4-(2-methylphenyl)-1,4-ben-
zothiazine-2-carbonitrile 1,1-dioxide (4b). Yield 76%, mp
1
4. Experimental
198–200 ꢁC; IR (KBr cmꢀ1): 2192 CN, 1453 SO2; H
NMR d CDCl3: 1.96 (s, 3H, CH3), 2.70 (s, 3H, SCH3),
0
6.63 (d, 1H, H5, J = 1.9 Hz), 7.26–7.29 (m, 1H, H6 ),
Melting points were determined with a Fischer–Johns
micro hot-stage apparatus and are uncorrected. IR spec-
tra were determined as KBr pellets on a Shimadzu mod-
0
0
0
0
7.57–7.61 (m, 3H, H2 ;3 ;5 ), 7.67 (d, 1H, H4 ,
J = 7.67 Hz), 7.71 (dd, 1H, H7, J = 8.4, 1.9 Hz), 8.11
(d, 1H, H8, J = 8.4 Hz). Anal. Calcd for
C17H13ClN2O2S2: C, 54.17; H, 3.48; N, 7.43. Found:
C, 53.95; H, 3.40; N, 7.23.
1
el 470 spectrophotometer. The H NMR spectra were
recorded using a Jeol Eclipse 270 MHz spectrometer
using CDCl3, and are reported in ppm downfield from
the CHCl3 residual. Elemental analyses were performed
on a Perkin-Elmer 2400CHN analyzer, results were
within 0.4% of predicted values for all compounds.
Compounds 4a, e, f, h, j, n, o, q were previously de-
4.1.2. 6-Chloro-3-methylthio-4-(4-methylphenyl)-1,4-ben-
zothiazine-2-carbonitrile 1,1-dioxide (4c). Yield 83%, mp
1
226–228 ꢁC; IR (KBr cmꢀ1): 2208 CN, 1453 SO2; H
1
scribed except the H NMR spectra of 4b–d, g, i, k, l,
NMR d CDCl3: 2.44 (s, 3H, CH3), 2.68 (s, 3H,
SCH3), 6.66 (d, 1H, H5, J = 1.8 Hz), 7.42 (d, 2H,
p, r.14 A white-Westinghouse microwave oven equipped
with a turntable and operating at 2450 MHz was used at
its full power, 750 W, for all the experiments. An alu-
mina batch (Aluminum oxide 60 G neutral, type E,
Merck: 50 g; batch 4.0 cm diameter) was used as a sink
inside the MW oven to irradiate the reaction mixture.
0
0
0
0
H2 ;6 , J = 8.3 Hz), 7.49 (d, 2H, H3 ;5 , J = 8.3 Hz),
7.68 (dd, 1H, H7, J = 8.5, 1.8 Hz), 8.09 (d, 1H, H8,
J = 8.5 Hz). Anal. Calcd for C17H13ClN2O2S2: C,
54.17; H, 3.48; N, 7.43. Found: C, 54.23; H, 3.27;
N, 7.37.

A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
3667
4.1.3. 6-Chloro-3-methylthio-4-(2,5-dimethylphenyl)-1,4-
benzothiazine-2-carbonitrile 1,1-dioxide (4d). Yield 92%,
mp 210–212 ꢁC; IR (KBr cmꢀ1): 2192 CN, 1456 SO2;
1H NMR d CDCl3: 1.92 (s, 3H, CH3), 2.49 (s, 3H,
CH3), 2.72 (s, 3H, SCH3), 6.66 (d, 1H, H5,
1H NMR d CDCl3: 2.69 (s, 3H, SCH3), 6.70 (d, 1H,
0
0
0
0
H5, J = 8.4 Hz), 7.80–7.85 (m, 5H, H2 ;4 ;5 ;6 ;6), 8.11 (d,
1H, H8, J = 2.2 Hz). Anal. Calcd for C16H10Cl2N2O2S2:
C, 48.37; H, 2.54; N, 7.05. Found: C, 48.31; H, 2.78; N,
6.94.
0
0
0
J = 1.7 Hz), 7.46–7.49 (m, 3H, H3 ;4 ;6 ), 7.71 (dd, 1H,
H7, J = 8.5, 1.7 Hz), 8.11 (d, 1H, H8, J = 8.5 Hz). Anal.
Calcd for C18H15Cl2N2O2S2: C, 55.31; H, 3.86; N, 7.16.
Found: C, 54.99; H, 3.76; N, 7.23.
4.1.10. 7-Chloro-4-methylthio-4-(3,4-dichlorophenyl)-1,4-
benzothiazine-2-carbonitrile 1,1-dioxide (4r). Yield 83%,
1
mp 234 ꢁC d; IR (KBr cmꢀ1): 2192 CN, 1483 SO2; H
NMR d CDCl3: 2.07 (s, 3H, SCH3), 7.03 (d, 1H, H5,
0
J = 8.7 Hz), 6.43 (dd, 1H, H6 , J = 8.6, 2.2 Hz), 6.50
4.1.4. 6-Chloro-3-methylthio-4-(3-chlorophenyl)-1,4-ben-
zothiazine-2-carbonitrile 1,1-dioxide (4g). Yield 77%,
mp 200–202 ꢁC; IR (KBr cmꢀ1): 2192 CN, 1449 SO2;
1H NMR d CDCl3: 2.69 (s, 3H, SCH3), 6.70 (d, 1H,
0
0
(d, 1H, H2 ; J = 2.2 Hz), 7.24 (d, 1H, H5 , J = 8.6 Hz),
7.53 (d, 1H, H6, J = 8.7 Hz), 7.99 (d, 1H, H8,
J = 2.2 Hz). Anal. Calcd for C16H9Cl3N2O2S2: C,
44.51; H, 2.10; N, 6.49. Found: C, 44.63; H, 2.25; N,
6.57.
0
0
0
0
H5, J = 1.7Hz), 7.79–7.84 (m, 5H, H2 ;4 ;5 ;6 ;7), 8.11 (d,
1H, H8, J = 8.4 Hz). Anal. Calcd for C16H10Cl2N2O2S2:
C, 48.37; H, 2.54; N, 7.05. Found: C, 48.35; H, 2.71; N,
6.93.
4.2. General procedure for preparation of ethyl 3-amino-
9-phenylsubstituted-thieno-[3,2-b]-benzothiazine 4,4-diox-
ide 2-carboxylate (5a–r)
4.1.5. 6-Chloro-4-methylthio-4-(3,4-dichlorophenyl)-1,4-
benzothiazine-2-carbonitrile 1,1-dioxide (4i). Yield 76%,
mp 224–226 ꢁC; IR (KBr cmꢀ1): 2192 CN, 1459 SO2;
1H NMR d CDCl3: 2.69 (s, 3H, SCH3), 6.89 (d, 1H,
A mixture of appropriate benzothiazine 1,1-dioxide 4a–r
(1 mmol), ethyl mercaptoacetate (1 mmol), triethyl-
amine (3 mmol) in dry ethanol (5 mL) was refluxed for
5 h under an inert atmosphere. The solvent was evapo-
rated to dryness under reduced pressure, water was
added (8 mL) and the solid obtained was collected by fil-
tration. Further purification was accomplished by
recrystallization from ethanol/water (4:1).
0
H5, J = 1.7 Hz), 7.40 (dd, 1H, H6 , J = 8.5, 2.5 Hz),
0
7.70 (dd, 1H, H7, J = 8.7, 1.7 Hz), 7.96 (d, 1H, H5 ,
0
J = 8.5 Hz), 8.04 (d, 1H, H2 , J = 2.5 Hz), 8.10 (d, 1H,
H8, J = 8.7 Hz). Anal. Calcd for C16H9Cl3N2O2S2: C,
44.51; H, 2.10; N, 6.49. Found: C, 44.57; H, 2.07; N,
6.63.
4.1.6. 7-Chloro-3-methylthio-4-(2-methylphenyl)-1,4-ben-
zothiazine-2-carbonitrile 1,1-dioxide (4k). Yield 81%, mp
4.2.1. Ethyl 3-amino-6-chloro-9-phenylthieno-[3,2-b]ben-
zothiazine 4,4-dioxide-2-carboxylate (5a). Yield 83%, mp
242–244 ꢁC; IR (KBr cmꢀ1): 3480 NH2, 1662 CO, 1495
1
196–198 ꢁC; IR (KBr cmꢀ1): 2192 CN, 1485 SO2; H
1
NMR d CDCl3: 2.05 (s, 3H, CH3), 2.77 (s, 3H, SCH3),
SO2; H NMR d CDCl3: 1.24 (t, 3H, CH3, J = 7.2 Hz),
0
6.65 (d, 1H, H5, J = 9.2 Hz), 7.23–7.27 (m, 1H, H6 ),
7.37 (dd, 1H, H6, J = 9.2, 2.5 Hz), 7.54–7.58 (m, 3H,
4.21 (q, 2H, CH2, J = 7.2 Hz), 6.23 (br s, 2H, NH2), 6.52
(d, 1H, H8, J = 9.2 Hz), 7.32 (dd, 1H, H7, J = 9.2,
0
0
0
0 0
2.3 Hz), 7.39 (m, 2H, H2 ;6 ), 7.66–7.71 (m, 3H,
H3 ;4 ;5 ), 8.04 (d, 1H, H8, J = 2.5 Hz). Anal. Calcd for
C17H13ClN2O2S2: C, 54.17; H, 3.48; N, 7.43. Found:
C, 54.33; H, 3.55; N, 7.62.
H3 ;4 ;5 ), 8.07 (d, 1H, H5, J = 2.3 Hz); 13C NMR 14.6,
60.4, 105.9, 118.2, 122.9, 126.3, 128.9, 129.3, 131.3,
131.6, 132.9, 137.5, 138.3, 149.2, 153.4, 163.8. Anal.
Calcd for C19H15ClN2O4S2: C, 52.47; H, 3.47; N, 6.44.
Found: C, 52.71; H, 3.40; N, 6.23.
0
0
0
4.1.7. 7-Chloro-3-methylthio-4-(4-methylphenyl)-1,4-ben-
zothiazine-2-carbonitrile 1,1-dioxide (4l). Yield 87%, mp
223–225 ꢁC; IR (KBr cmꢀ1): 2192 CN, 1481 SO2; H
1
NMR d CDCl3: 2.48 (s, 3H, CH3), 2.68 (s, 3H, SCH3),
4.2.2. Ethyl 3-amino-6-chloro-9-(20-methylphenyl)thieno-
[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5b). Yield
47%, mp 241–243 ꢁC; IR (KBr cmꢀ1): 3480 NH2,
1668 CO, 1490 SO2; 1H NMR d CDCl3: 1.24 (t,
3H, CH3, J = 7.0 Hz), 2.06 (s, 3H, CH3), 4.20 (q,
2H, CH2, J = 7.0 Hz), 6.25 (br s, 2H, NH2), 6.45
0
6.69 (d, 1H, H5, J = 8.9 Hz), 7.16 (d, 2H, H2 ;6
0
,
0
0
J = 7.7 Hz), 7.53–7.59 (m, 3H, H3 ;5 ;6), 8.00 (d, 1H, H8,
J = 2.3 Hz). Anal. Calcd for C17H13ClN2O2S2: C,
54.17; H, 3.48; N, 7.43. Found: C, 54.20; H, 3.51; N,
7.17.
0
H6 ,
(d, 1H, H8, J = 9.2 Hz), 7.28 (d, 1H,
J = 7.7 Hz), 7.34 (dd, 1H, H7, J = 9.2, 2.2 Hz), 7.55–
4.1.8. 7-Chloro-3-methylthio-4-(2,5-dimethylphenyl)-1,4-
benzothiazine-2-carbonitrile 1,1-dioxide (4m). Yield
77%, mp 210 ꢁC d; IR (KBr cmꢀ1): 2192 CN, 1491
0
0
0
7.59 (m, 3H, H3 ;4 ;5 ), 8.09 (d, 1H, H5, J = 2.2 Hz);
13C NMR 14.6, 17.1, 60.4, 105.9, 117.6, 123.1,
126.4, 129.0, 129.1, 129.5, 131.5, 133.1, 133.1, 136.5,
136.8, 137.2, 149.5, 152.8, 163.8. Anal. Calcd for
C20H17ClN2O4S2: C, 53.51; H, 3.82; N, 6.24. Found:
C, 53.51; H, 3.79; N, 6.33.
1
SO2; H NMR d CDCl3: 2.00 (s, 3H, CH3), 2.40 (s,
3H, CH3), 2.77 (s, 3H, SCH3), 6.65 (d, 1H, H5,
0
0
0
J = 9.1 Hz), 7.29–7.35 (m, 3H, H3 ;4 ;6 ), 7.40 (dd, 1H,
H6, J = 9.1, 2.5 Hz), 8.03 (d, 1H, H8, J = 2.5 Hz). Anal.
Calcd for C18H15ClN2O2S2: C, 55.31; H, 3.86; N, 7.16.
Found: C, 55.22; H, 3.91; N, 7.05.
4.2.3. Ethyl 3-amino-6-chloro-9-(40-methylphenyl)thieno-
[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5c). Yield
67%, mp 240–242 ꢁC; IR (KBr cmꢀ1): 3456 NH2,
1661 CO, 1459 SO2; 1H NMR d CDCl3: 1.24 (t,
3H, CH3, J = 7.0 Hz), 2.50 (s, 3H, CH3), 4.19 (q,
4.1.9. 7-Chloro-3-methylthio-4-(3-chlorophenyl)-1,4-ben-
zothiazine-2-carbonitrile 1,1-dioxide (4p). Yield 71%,
mp 230–232 ꢁC; IR (KBr cmꢀ1): 2192 CN, 1483 SO2;

3668
A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
0
0
0
2H, CH2, J = 7.0 Hz), 6.22 (br s, 2H, NH2), 6.53 (d,
0
1H, H2 ), 7.63–7.67 (m, 2H, H4 ;5 ), 8.08 (d, 1H, H5,
J = 2.4 Hz); 13C NMR 14.6, 60.5, 106.3, 118.0, 123.1,
126.5, 127.3, 129.4, 129.4, 129.6, 131.7, 132.6, 133.0,
137.1, 137.1, 149.1, 152.7, 163.7. Anal. Calcd for
C19H14Cl2N2O4S2: C, 48.62; H, 3.01; N, 5.97. Found:
C, 48.39; H, 3.25; N, 6.29.
0
1H, H8, J = 8.9 Hz), 7.23 (d, 2H, H2 ;6 , J = 7.9 Hz),
7.29 (dd, 1H, H7, J = 8.9, 1.5 Hz), 7.45 (d, 1H,
H3 ;5 , J = 7.9 Hz), 8.05 (d, 1H, H5, J = 1.5 Hz); 13C
NMR 14.6, 21.6, 60.3, 105.7, 118.3, 122.9, 126.3,
128.5, 129.6, 132.2, 132.8, 135.7, 137.1, 141.7, 149.5,
153.7, 163.8. Anal. Calcd for C20H17ClN2O4S2: C,
53.51; H, 3.82; N, 6.24. Found: C, 53.49; H, 3.67;
N, 6.43.
0
0
4.2.8. Ethyl 3-amino-6-chloro-9-(40-chlorophenyl)thieno-
[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5h). Yield
61%, mp 278–280 ꢁC; IR (KBr cmꢀ1): 3460 NH2, 1666
1
4.2.4. Ethyl 3-amino-6-chloro-9-(20,50-dimethylphenyl)thi-
eno-[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5d).
Yield 43%, mp 244–246 ꢁC; IR (KBr cmꢀ1): 3482 NH2,
CO, 1490 SO2; H NMR d CDCl3: 1.26 (t, 3H, CH3,
J = 7.1 Hz), 4.22 (q, 2H, CH2, J = 7.1 Hz), 6.22 (br s,
2H, NH2), 6.52 (d, 1H, H8, J = 9.1 Hz), 7.34 (d, 2H,
0 0
H2 ;6 , J = 8.9 Hz), 7.36 (dd, 1H, H7, J = 9.1, 2.2 Hz),
7.65 (d, 2H, H3 ;5 , J = 8.9 Hz), 8.08 (d, 1H, H5,
J = 2.2 Hz); 13C NMR 14.5, 60.5, 106.2, 117.9, 123.2,
126.6, 129.6, 130.5, 131.9, 132.9, 136.7, 137.3, 137.6,
149.2, 152.8, 163.7. Anal. Calcd for C19H14Cl2N2O4S2:
C, 48.62; H, 3.01; N, 5.97. Found: C, 48.63; H, 3.12;
N, 5.93.
1
1665 CO, 1490 SO2; H NMR d CDCl3: 1.25 (t, 3H,
0
0
CH3, J = 7.2 Hz), 1.99 (s, 3H, CH3), 2.40 (s, 3H,
CH3), 4.21 (q, 2H, CH2, J = 7.2 Hz), 6.24 (br s, 2H,
0
NH2), 6.46 (d, 1H, H8, J = 9.2 Hz), 7.07 (s, 1H, H6 ),
7.33 (dd, 1H, H7, J = 9.2, 2.5 Hz), 7.36–7.40 (m, 2H,
H3 ;4 ), 8.09 (d, 1H, H5, J = 2.5 Hz); 13C NMR 14.6,
16.9, 20.9, 60.4, 105.7, 117.3, 123.1, 126.3, 129.2,
129.3, 132.3, 132.8, 133.1, 133.7, 136.6, 136.6, 139.3,
149.4, 152.8, 163.8. Anal. Calcd for C21H19ClN2O4S2:
C, 54.48; H, 4.14; N, 6.05. Found: C, 54.66; H, 4.30;
N, 6.17.
0
0
4.2.9. Ethyl 3-amino-6-chloro-9-(30,40-dichlorophenyl)thi-
eno-[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5i).
Yield 55%, mp 180–182 ꢁC; IR (KBr cmꢀ1): 3475
1
NH2, 1662 CO, 1490 SO2; H NMR d CDCl3: 1.27 (t,
4.2.5. Ethyl 3-amino-6-chloro-9-(30-methoxylphenyl)thie-
no-[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5e).
Yield 47%, mp 227–229 ꢁC; IR (KBr cmꢀ1): 3471
3H, CH3, J = 6.9 Hz), 4.20 (q, 2H, CH2, J = 6.9 Hz),
6.19 (br s, 2H, NH2), 6.54 (d, 1H, H8, J = 9.2 Hz),
0
7.27 (dd, 1H, H6 , J = 8.4, 2.5 Hz), 7.37 (dd, 1H, H7,
1
0
NH2, 1672 CO, 1443 SO2; H NMR d CDCl3: 1.24 (t,
J = 9.2, 2.4 Hz), 7.54 (d, 1H, H2 , J = 2.5 Hz), 7.77 (d,
0
3H, CH3, J = 6.8 Hz), 3.79 (s, 3H, OCH3), 4.26 (q,
2H, CH2, J = 6.8 Hz), 6.19 (br s, 2H, NH2), 6.59 (d,
0
1H, H8, J = 9.2 Hz), 6.71 (d, 1H, H2 J = 2.4 Hz), 6.84
1H, H5 , J = 8.4 Hz), 8.08 (d, 1H, H5, J = 2.4 Hz); 13C
NMR 14.5, 60.6, 106.8, 117.8, 123.3, 126.7, 128.5,
129.9, 131.2, 131.5, 133.1, 137.4, 136.9, 137.2, 149.2,
153.2, 163.7. Anal. Calcd for C19H13Cl3N2O4S2: C,
45.30; H, 2.60; N, 5.56. Found: C, 45.37; H, 2.73; N,
5.81.
0
0
(d, 1H, H4 ; J = 7.8 Hz), 7.10 (dd, 1H, H6 , J = 7.8,
2.3 Hz), 7.33 (dd, 1H, H7, J = 9.2, 1.7 Hz), 7.60 (t, 1H,
H5 , J = 7.6 Hz), 8.05 (d, 1H, H5, J = 1.7 Hz); 13C
0
NMR 13C NMR 14.6, 55.8, 60.4, 105.8, 116.4, 118.3,
123.1, 126.4, 129.1, 130.2, 130.8, 132.8, 136.9, 149.7,
154.2, 161.3, 163.8. Anal. Calcd for C20H17ClN2O5S2:
C, 51.67; H, 3.68; N, 6.02. Found: C, 51.73; H, 3.59;
N, 6.03.
4.2.10. Ethyl 3-amino-7-chloro-9-phenylthieno-[3,2-b]ben-
zothiazine 4,4-dioxide-2-carboxylate (5j). Yield 71%, mp
220–222 ꢁC; IR (KBr cmꢀ1): 3480 NH2, 1662 CO,
1495 SO2; 1H NMR d CDCl3: 1.24 (t, 3H, CH3,
J = 7.3 Hz), 4.21 (q, 2H, CH2, J = 7.3 Hz), 6.22 (br
s, 2H, NH2), 6.53 (d, 1H, H8, J = 1.7 Hz), 7.27 (dd,
4.2.6. Ethyl 3-amino-6-chloro-9-(40-methoxylphenyl)thie-
no-[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5f).
Yield 63%, mp 218–220 ꢁC; IR (KBr cmꢀ1): 3456
0
1H, H6, J = 8.7, 1.7 Hz), 7.38–7.42 (m, 2H, H2 ),
7.69–7.64 (m, 4H, Ar), 8.05 (d, 1H, H5, J = 8.7 Hz);
13C NMR 14.6, 60.4, 90.7, 106.4, 116.4, 123.9, 124.1,
124.9, 128.9, 131.7, 131.9, 138.1, 138.1, 139.9, 149.3,
153.4, 163.8. Anal. Calcd for C19H15ClN2O4S2: C,
52.47; H, 3.47; N, 6.44. Found: C, 52.86; H, 3.57;
N, 6.68.
1
NH2, 1674 CO, 1459 SO2; H NMR d CDCl3: 1.25 (t,
3H, CH3, J = 6.2 Hz), 3.85 (s, 3H, OCH3), 4.21 (q,
2H, CH2, J = 6.2 Hz), 6.21 (br s, 2H, NH2), 6.57 (d,
0
1H, H8, J = 9.1 Hz), 7.13 (d, 2H, H2 ;6 , J = 8.9 Hz),
0
0
0
7.28 (d, 2H, H3 ;5 , J = 8.9 Hz), 7.32 (dd, 1H, H7,
J = 9.1, 1.4 Hz), 8.07 (d, 1H, H5, J = 1.4 Hz); 13C
NMR 14.6, 55.8, 60.3, 105.7, 116.6, 118.3, 122.9,
126.3, 129.2, 130.1, 130.8, 132.8, 137.8, 149.6, 154.1,
161.3, 163.8. Anal. Calcd for C20H17ClN2O5S2: C,
51.67; H, 3.68; N, 6.02. Found: C, 51.88; H, 4.01; N,
5.87.
4.2.11. Ethyl 3-amino-7-chloro-9-(20-methylphenyl)thie-
no-[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5k).
Yield 67%, mp 180–182 ꢁC; IR (KBr cmꢀ1): 3480
1
NH2, 1668 CO, 1490 SO2; H NMR d CDCl3: 1.24 (t,
3H, CH3, J = 6.8 Hz), 2.07 (s, 3H, CH3), 4.21 (q, 2H,
CH2, J = 6.8 Hz), 6.24 (br s, 2H, NH2), 6.46 (d, 1H,
H8, J = 1.5 Hz), 7.28 (d, 1H, H6, J = 8.4 Hz), 7.51–7.55
(m, 4H, Ar), 8.06 (d, 1H, H5, J = 8.4 Hz); 13C NMR
14.6, 17.1, 60.4, 90.8, 106.3, 115.7, 124.0, 124.1, 125.1,
129.1, 129.1, 131.6, 133.2, 136.6, 137.2, 139.5, 149.4,
152.8, 163.8. Anal. Calcd for C20H17ClN2O4S2: C,
53.51; H, 3.82; N, 6.24. Found: C, 53.27; H, 3.81; N,
6.18.
4.2.7. Ethyl 3-amino-6-chloro-9-(30-chlorophenyl)thieno-
[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5g). Yield
43%, mp 200 ꢁC dec; IR (KBr cmꢀ1): 3488 NH2, 1676
1
CO, 1459 SO2; H NMR d CDCl3: 1.26 (t, 3H, CH3,
J = 7.1 Hz), 4.19 (q, 2H, CH2, J = 7.1 Hz), 6.24 (br s,
2H, NH2), 6.53 (d, 1H, H8, J = 9.2 Hz), 7.31–7.35 (m,
0
1H, H6 ), 7.36 (dd, 1H, H7, J = 9.2, 2.4 Hz), 7.41 (s,

A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
3669
4.2.12. Ethyl 3-amino-7-chloro-9-(40-methylphenyl)thie-
no-[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5l).
Yield 78%, mp 192–194 ꢁC; IR (KBr cmꢀ1): 3456
2H, NH2), 6.53 (d, 1H, H8, J = 1.7 Hz), 7.31–7.34 (m,
0
0
2H, H6;5 ), 7.41–7.46 (m, 1H, H6 ), 7.67–7.70 (m, 2H,
H2 ;4 ), 8.05 (d, 1H, H5, J = 8.5 Hz); 13C NMR 14.6,
60.5, 90.9, 106.8, 116.2, 124.1, 124.4, 125.1, 127.3,
129.4, 131.9, 132.7, 137.2, 138.9, 139.3, 139.5, 149.0,
152.8, 163.7. Anal. Calcd for C19H14Cl2N2O4S2: C,
48.62; H, 3.01; N, 5.97. Found: C, 48.54; H, 3.07; N,
6.39.
0
0
1
NH2, 1661 CO, 1459 SO2; H NMR d CDCl3: 1.25 (t,
3H, CH3, J = 7.1 Hz), 2.51 (s, 3H, CH3), 4.20 (q, 2H,
CH2, J = 7.1 Hz), 6.22 (br s, 2H, NH2), 6.53 (d, 1H,
0
H8, J = 1.7 Hz), 7.27–7.31 (m, 3H, H6;2 ;6 ), 7.47 (d,
0
0
0
2H, H3 ;5 , J = 8.2 Hz), 8.05 (d, 1H, H5, J = 8.4 Hz);
13C NMR 14.6, 21.6, 60.3, 91.2, 106.2, 116.4, 123.9,
124.9, 128.5, 132.3, 135.5, 139.1, 139.9, 141.8, 149.3,
153.7, 163.8. Anal. Calcd for C20H17ClN2O4S2: C,
53.51; H, 3.82; N, 6.24. Found: C, 53.62; H, 3.60; N,
6.43.
4.2.17. Ethyl 3-amino-7-chloro-9-(40-chlorophenyl)thieno-
[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5q). Yield
53%, mp 218–220 ꢁC; IR (KBr cmꢀ1): 3460 NH2, 1666
1
CO, 1490 SO2; H NMR d CDCl3: 1.26 (t, 3H, CH3,
J = 6.8 Hz), 4.22 (q, 2H, CH2, J = 6.8 Hz), 6.22 (br s,
2H, NH2), 6.53 (d, 1H, H8, J = 1.7 Hz), 7.29 (dd, 1H,
4.2.13.
Ethyl
3-amino-7-chloro-9-(20,50-dimethyl-
0 0
H6, J = 8.4, 1.7 Hz), 7.35 (d, 2H, H2 ;6 , J = 8.5 Hz),
phenyl)thieno-[3,2-b]benzothiazine 4,4-dioxide-2-carbox-
ylate (5m). Yield 62%, mp 218–220 ꢁC; IR (KBr
cmꢀ1): 3482 NH2, 1665 CO, 1490 SO2; 1H NMR d
CDCl3: 1.22 (t, 3H, CH3, J = 6.8 Hz), 2.02 (s, 3H,
CH3), 2.42 (s, 3H, CH3), 4.22 (q, 2H, CH2,
J = 6.8 Hz), 6.24 (br s, 2H, NH2), 6.46 (d, 1H, H8,
0
0
7.67 (d, 2H, H3 ;5 , J = 8.5 Hz), 8.04 (d, 1H, H5,
J = 8.4 Hz); 13C NMR 14.6, 60.5, 90.0, 106.7, 116.2,
124.1, 124.3, 125.1, 130.5, 132.1, 136.4, 137.7, 139.3,
139.7, 149.0, 152.9, 163.7. Anal. Calcd for
C19H14Cl2N2O4S2: C, 48.62; H, 3.01; N, 5.97. Found:
C, 48.72; H, 3.33; N, 5.87.
0
J = 1.9 Hz), 7.08 (s, 1H, H6 ), 7.27 (dd, 1H, H6,
0
0
J = 8.7, 1.9 Hz), 7.35–7.39 (m, 2H, H2 ;3 ), 8.06 (d, 1H,
H5, J = 8.7 Hz); 13C NMR 14.6, 16.7, 21.0, 60.0, 89.9,
107.0, 115.8, 123.9, 124.1, 125.1, 129.2, 132.9, 136.5,
139.0, 139.4, 149.2, 152.9, 163.6. Anal. Calcd for
C21H19ClN2O4S2: C, 54.48; H, 4.14; N, 6.05. Found:
C, 54.27; H, 3.95; N, 6.33.
4.2.18.
Ethyl
3-amino-7-chloro-9-(30,40-dichloro-
phenyl)thieno-[3,2-b]benzothiazine 4,4-dioxide-2-carbox-
ylate (5r). Yield 49%, mp 237–239 ꢁC; IR (KBr cmꢀ1):
3472 NH2, 1662 CO, 1495 SO2; 1H NMR d CDCl3:
1.27 (t, 3H, CH3, J = 6.8 Hz), 4.21 (q, 2H, CH2,
J = 6.8 Hz), 6.24 (br s, 2H, NH2), 6.54 (d, 1H, H8,
4.2.14. Ethyl 3-amino-7-chloro-9-(30-methoxylphenyl)thi-
eno-[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5n).
Yield 62%, mp 210–212 ꢁC; IR (KBr cmꢀ1): 3488 NH2,
0
J = 1.7 Hz), 7.30–7.34 (m, 2H, H6;6 ), 7.55 (d, 2H, H2
0
0
J = 2.5 Hz), 7.79 (d, 1H, H5 , J = 8.4 Hz), 8.05 (d, 1H,
H5, J = 8.4 Hz); 13C NMR 14.6, 60.6, 90.3, 107.0,
116.0, 124.5, 125.1, 128.5, 131.2, 133.4, 135.8, 136.4,
136.9, 139.4, 139.4, 163.6. Anal. Calcd for
C19H13Cl3N2O4S2: C, 45.30; H, 2.60; N, 5.56. Found:
C, 45.18; H, 2.40; N, 5.68.
1
1664 CO, 1440 SO2; H NMR d CDCl3: 1.26 (t, 3H,
CH3, J = 6.8 Hz), 3.86 (s, 3H, OCH3), 4.20 (q, 2H,
CH2, J = 6.8 Hz), 6.23 (br s, 2H, NH2), 6.59 (d, 1H,
0
H8, J = 1.7 Hz), 6.87 (d, 1H, H2 J = 2.4 Hz), 6.94 (d,
0
0
1H, H4 ; J = 7.8 Hz), 7.19 (dd, 1H, H6 , J = 7.8,
0
2.3 Hz), 7.28 (d, 1H, H6, J = 8.5 Hz), 7.58 (t, 1H, H5 ,
4.3. General procedure for preparation of 3-amino-9-
phenyl-thieno-[3,2-b]benzothiazine 4,4-dioxide (6a–r)
J = 7.6 Hz), 8.05 (d, 1H, H5, J = 8.5 Hz); 13C NMR
14.6, 55.8, 60.4, 90.1, 106.3, 113.9, 116.5, 117.3, 120.5,
123.9, 124.1, 124.8, 132.3, 138.9, 139.1, 139.8, 149.1,
152.2, 161.9, 163.8. Anal. Calcd for C20H17ClN2O5S2:
C, 51.67; H, 3.68; N, 6.02. Found: C, 51.64; H, 3.83;
N, 6.15.
Compounds 5a–r (0.1 mmol) were stirred with (NaOH,
KOH, LiOH, or HCl) in ethanol/water 4:1 at several
conditions Table 1. The solvent was then removed under
reduced pressure, dichloromethane was added (15 mL)
and washed with water (2· 20 mL). Organic phase was
dried over anhydrous sodium sulfate, filtered, and sol-
vent was removed under reduced pressure to obtain star-
ter material. Water was neutralized with hydrochloric
acid (10%), dichloromethane was added, organic phase
was separated, washed with water, brine, and dried with
sodium sulfate, filtered, and evaporated to dryness un-
der reduced pressure, and the solid thus obtained was
collected, the crude product was recrystallized from eth-
anol to give a pure sample. The physical and spectra
data of the compounds 6a–r are as follows.
4.2.15. Ethyl 3-amino-7-chloro-9-(40-methoxylphenyl)thi-
eno-[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5o).
Yield 71%, mp 250–252 ꢁC; IR (KBr cmꢀ1): 3456 NH2,
1
1674 CO, 1459 SO2; H NMR d CDCl3: 1.25 (t, 3H,
CH3, J = 6.8 Hz), 3.85 (s, 3H, OCH3), 4.22 (q, 2H, CH2,
J = 6.8 Hz), 6.21 (br s, 2H, NH2), 6.58 (d, 1H, H8,
0
0
J = 1.8 Hz), 7.14 (d, 2H, H2 ;6 , J = 8.9 Hz), 7.27–7.33
(m, 3H, H6;3 ;5 ), 8.04 (d, 1H, H5, J = 8.9 Hz); 13C NMR
14.6, 55.8, 60.4, 90.9, 106.2, 116.4, 116.7, 123.9, 124.5,
124.8, 130.1, 139.1, 139.5, 140.2, 149.4, 154.1, 161.3,
163.8. Anal. Calcd for C20H17ClN2O5S2: C, 51.67; H,
3.68; N, 6.02. Found: C, 51.61; H, 3.69; N, 5.98.
0
0
4.3.1. 3-Amino-6-chloro-9-phenylthieno-[3,2-b]benzothi-
azine 4,4-dioxide (6a). Yield 67%, mp 166–168 ꢁC; IR
1
4.2.16. Ethyl 3-amino-7-chloro-9-(30-chlorophenyl)thieno-
[3,2-b]benzothiazine 4,4-dioxide-2-carboxylate (5p). Yield
48%, mp 234–236 ꢁC; IR (KBr cmꢀ1): 3488 NH2, 1676
(KBr cmꢀ1): 3340 NH2, 1460 SO2; H NMR d CDCl3:
4.27 (br s, 2H, NH2), 5.56 (s, 1H, H2), 6.50 (d, 1H,
H8, J = 9.3 Hz), 7.29 (dd, 1H, H7, J = 9.3, 1.9 Hz),
0
1
0
0
0
0
CO, 1459 SO2; H NMR d CDCl3: 1.24 (t, 3H, CH3,
7.37–7.40 (m, 2H, H2 ;6 ), 7.66 (m, 3H, H3 ;4 ;5 ), 8.09 (d,
J = 6.8 Hz), 4.23 (q, 2H, CH2, J = 6.8 Hz), 6.22 (br s,
1H, H5, J = 1.9 Hz). 13C NMR 88.7, 107.9, 117.7,

3670
A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
H7;3 ;5 ), 8.06 (d, 1H, H5, J = 1.9 Hz). 13C NMR 55.8,
88.7, 107.4, 116.4, 117.8, 123.1, 125.7, 127.9, 130.4,
131.3, 132.5, 137.9, 140.2, 150.9, 160.9. Anal. Calcd
for C17H13ClN2O3S2: C, 51.97; H, 3.34; N, 7.13. Found:
C, 52.13; H, 3.42; N, 6.97.
0
0
123.2, 125.9, 129.2, 130.9, 131.5, 132.5, 137.6, 138.9,
139.3, 140.2, 150.3. Anal. Calcd for C16H11ClN2O2S2:
C, 52.96; H, 3.06; N, 7.72. Found: C, 53.09; H, 3.11;
N, 7.83.
4.3.2. 3-Amino-6-chloro-9(20-methylphenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6b). Yield 45%, mp 174–
176 ꢁC; IR (KBr cmꢀ1): 3430 NH2, 1462 SO2; 1H
NMR d CDCl3: 2.06 (s, 3H, CH3), 4.02 (br s, 2H,
NH2), 5.55 (s, 1H, H2), 6.41 (d, 1H, H8, J = 9.2 Hz),
4.3.7. 3-Amino-6-chloro-9-(30-chlorophenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6g). Yield 41%, mp 192–
194 ꢁC; IR (KBr cmꢀ1): 3360 NH2, 1455 SO2; 1H
NMR d CDCl3: 4.23 (br s, 2H, NH2), 5.57 (s, 1H,
H2), 6.50 (d, 1H, H8, J = 9.2 Hz), 7.31–7.34 (m, 2H,
0
0
0
0
7.30–7.34 (m, 2H, H7;6 ), 7.50–7.55 (m, 3H, H3 ;4 ;5 ),
8.11 (d, 1H, H5, J = 2.2 Hz). 13C NMR 17.1, 88.5,
107.6, 117.1, 123.4, 125.8, 128.1, 128.9, 129.4, 131.1,
132.8, 132.9, 136.7, 137.3, 137.5, 140.2, 149.7. Anal.
Calcd for C17H13ClN2O2S2: C, 54.18; H, 3.48; N, 7.43.
Found: C, 53.99; H, 3.67; N, 7.34.
0
0
0
0
H7;6 ), 7.50 (s, 1H, H2 ), 7.60–7.64 (m, 2H, H4 ;5 ), 7.50
0
(s, 1H, H2 ), 8.08 (d, 1H, H5, J = 2.3 Hz). 13C NMR
88.7, 107.9, 117.5, 123.3, 125.9, 127.7, 129.7, 131.7,
132.5, 132.7, 136.9, 137.3, 139.7, 140.3, 149.6. Anal.
Calcd for C16H10Cl2N2O2S2: C, 48.37; H, 2.54; N,
7.05. Found: C, 48.23; H, 2.67; N, 7.28.
4.3.3. 3-Amino-6-chloro-9-(40-methylphenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6c). Yield 59%, mp 208–
210 ꢁC; IR (KBr cmꢀ1): 3408 NH2, 1465 SO2; 1H
NMR d CDCl3: 2.48 (s, 3H, CH3), 3.90 (br s, 2H,
NH2), 5.54 (s, 1H, H2), 6.52 (d, 1H, H8, J = 9.2 Hz),
4.3.8. 3-Amino-6-chloro-9-(40-chlorophenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6h). Yield 48%, mp 242–
244 ꢁC; IR (KBr cmꢀ1): 3356 NH2, 1464 SO2; 1H
NMR d CDCl3: 4.10 (br s, 2H, NH2), 5.56 (s, 1H,
H2), 6.49 (d, 1H, H8, J = 7.7 Hz), 7.29–7.35 (m, 5H,
0
0
0
0
7.26–7.31 (m, 3H, H7;2 ;6 ), 7.43 (d, 2H, H3 ;5 ,
J = 7.7 Hz), 8.06 (d, 1H, H5, J = 2.2 Hz). 13C NMR
21.5, 88.7, 107.4, 117.8, 123.1, 125.7, 127.9, 128.9,
132.0, 132.5, 136.2, 137.7, 141.2, 140.1, 150.7. Anal.
Calcd for C17H13ClN2O2S2: C, 54.18; H, 3.48; N, 7.43.
Found: C, 54.31; H, 3.63; N, 7.45.
H7;2 ;3 ;5 ;6 ), 8.08 (d, 1H, H5, J = 2.3 Hz). 13C NMR
88.8, 107.9, 117.4, 123.3, 125.2, 130.0, 131.3, 132.5,
133.2, 137.2, 139.2, 139.6, 150.2. Anal. Calcd for
C16H10Cl2N2O2S2: C, 48.37; H, 2.54; N, 7.05. Found:
C, 48.19; H, 2.37; N, 6.93.
0
0
0
0
4.3.4. 3-Amino-6-chloro-9-(20,50-dimethylphenyl)-thieno-
[3,2-b]benzothiazine 4,4-dioxide (6d). Yield 53%, mp
4.3.9. 3-Amino-6-chloro-9-(30,40-dichlorophenyl)-thieno-
[3,2-b]benzothiazine 4,4-dioxide (6i). Yield 37%, mp
1
1
152–154 ꢁC; IR (KBr cmꢀ1): 3428 NH2, 1460 SO2; H
172–174 ꢁC; IR (KBr cmꢀ1): 3380 NH2, 1459 SO2; H
NMR d CDCl3: 2.00 (s, 3H, CH3), 2.50 (s, 3H, CH3),
4.25 (br s, 2H, NH2), 5.55 (s, 1H, H2), 6.45 (d, 1H,
0
H8, J = 8.7 Hz), 7.07 (s, 1H, H6 ), 7.22 (dd, 1H, H7,
NMR d CDCl3: 4.00 (br s, 2H, NH2), 5.58 (s, 1H,
0
H2), 6.51 (d, 1H, H8, J = 9.2 Hz), 7.27 (dd, 1H, H6 ,
J = 8.7, 1.9 Hz), 7.33 (dd, 1H, H7, J = 9.2, 1.9 Hz),
0
0
0
J = 8.7, 1.9 Hz), 7.32(d, 1H, H4 , J = 8.4 Hz), 7.37 (d,
0
7.53 (d, 1H, H2 , J = 1.9 Hz), 7.74 (d, 1H, H5 ;
J = 8.7 Hz), 8.08 (d, 1H, H5, J = 1.9 Hz). 13C NMR
88.7, 108.5, 117.3, 123.4, 126.2, 128.8, 131.5, 132.7,
133.2, 135.5, 135.8, 137.2, 137.8, 139.8, 140.4, 149.7.
Anal. Calcd for C16H9Cl3N2O2S2: C, 44.51; H, 2.10;
N, 6.49. Found: C, 44.61; H, 2.30; N, 6.73.
1H, H5 , J = 8.4 Hz), 8.08 (d, 1H, H5, J = 1.9 Hz). 13C
NMR 16.7, 21.0, 89.9, 106.9, 117.0, 123.1, 126.1,
129.1, 130.0, 131.3, 132.9, 133.1, 133.3, 136.9, 138.0,
138.5, 139.90, 151.0. Anal. Calcd for C18H15ClN2O2S2:
C, 53.31; H, 3.87; N, 7.16. Found: C, 53.40; H, 3.79;
N, 7.45.
4.3.10. 3-Amino-7-chloro-9-phenylthieno-[3,2-b]benzothi-
azine 4,4-dioxide (6j). Yield 63%, mp 230–232; IR (KBr
cmꢀ1): 3424 NH2, 1462 SO2; 1H NMR d CDCl3: 4.15 (br
s, 2H, NH2), 5.56 (s, 1H, H2), 6.51 (d, 1H, H8,
J = 1.7 Hz), 7.21 (dd, 1H, H6, J = 8.7, 1.7 Hz), 7.37–
4.3.5.
3-Amino-6-chloro-9-(30-methoxylphenyl)-thieno-
[3,2-b]benzothiazine 4,4-dioxide (6e). Yield 61%, mp
214–216 ꢁC; IR (KBr cmꢀ1): 3420 NH2, 1457 SO2; H
NMR d CDCl3: 3.88 (s, 3H, OCH3), 4.11 (br s, 2H,
NH2), 5.56 (s, 1H, H2), 6.57 (d, 1H, H8, J = 8.5 Hz),
0
6.88 (t, 1H, H2 J = 2.2 Hz), 6.95 (d, 1H, H4 ,
0
J = 7.9 Hz), 7.16 (dd, 2H, H6 , J = 7.9, 2.2 Hz), 7.26
1
0
0
0
0
0
7.40 (m, 2H, H2 ;6 ), 7.66–7.40 (m, 3H, H3 ;4 ;5 ), 8.07 (d,
1H, H5, J = 8.7 Hz). 13C NMR 88.8, 108.1, 115.7,
123.0, 123.4, 125.2, 129.2, 131.5, 138.6, 138.7, 139.9,
140.2, 150.2. Anal. Calcd for C16H11ClN2O2S2: C,
52.96; H, 3.06; N, 7.72. Found: C, 52.86; H, 3.07; N,
7.68.
0
0
(dd, 1H, H7, J = 8.6, 2.1 Hz), 7.56 (t, 1H, H5 ,
J = 7.9 Hz), 8.07 (d, 1H, H5, J = 2.1 Hz). 13C NMR
55.8, 88.8, 107.4, 116.3, 117.6, 123.0, 125.3, 127.1,
129.4, 131.1, 132.5, 137.7, 140.1, 150.9, 161.2. Anal.
Calcd for C17H13ClN2O3S2: C, 51.97; H, 3.34; N, 7.13.
Found: C, 51.97; H, 3.21; N, 6.92.
4.3.11. 3-Amino-7-chloro-9(20-methylphenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6k). Yield 60%, mp 206–
208 ꢁC; IR (KBr cmꢀ1): 3424 NH2, 1465 SO2; 1H
NMR d CDCl3: 2.06 (s, 3H, CH3), 4.02 (br s, 2H,
NH2), 5.55 (s, 1H, H2), 6.43 (d, 1H, H8, J = 1.5 Hz),
4.3.6.
3-Amino-6-chloro-9-(40-methoxylphenyl)-thieno-
[3,2-b]benzothiazine 4,4-dioxide (6f). Yield 73%, mp
224–226 ꢁC; IR (KBr cmꢀ1): 3440 NH2, 1465 SO2; H
7.23 (dd, 1H, H6, J = 8.4, 1.5 Hz), 7.28 (d, 1H, H6 ,
1
0
0
0
0
NMR d CDCl3: 3.90 (s, 3H, OCH3), 4.01 (br s, 2H,
NH2), 5.55 (s, 1H, H2), 6.53 (d, 1H, H8, J = 8.9 Hz),
J = 7.2 Hz), 7.52–7.57 (m, 3H, H3 ;4 ;5 ), 8.09 (d, 1H,
H5, J = 8.4 Hz). 13C NMR 17.1, 88.6, 107.9, 115.1,
123.1, 123.4, 125.4, 128.9, 129.4, 131.3, 133.0, 137.4,
0
7.11 (d, 2H, H2 ;6 , J = 8.7 Hz), 7.26–7.30 (m, 3H,
0

A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
3671
138.9, 139.1, 143.0, 140.2, 151.0. Anal. Calcd for
C17H13ClN2O2S2: C, 54.18; H, 3.48; N, 7.43. Found:
C, 54.26; H, 3.47; N, 7.38.
237 ꢁC; IR (KBr cmꢀ1): 3388 NH2, 1459 SO2; 1H NMR
d CDCl3: 4.24 (br s, 2H, NH2), 5.58 (s, 1H, H2), 6.50 (d,
1H, H8, J = 1.5 Hz), 7.23 (dd, 1H, H6, J = 8.7, 1.5 Hz),
0
0
0
7.30–7.34 (m, 2H, H5 ;6 ), 7.42 (s, 1H, H2 ), 7.63 (d, 1H,
0
4.4. 3-Amino-7-chloro-9(40-methylphenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6l)
H4 , J = 6.0 Hz), 8.07 (d, 1H, H5, J = 8.7 Hz). 13C NMR
88.9, 108.5, 115.6, 123.4, 123.6, 125.3, 127.6, 129.7,
131.5, 132.5, 136.9, 138.9, 139.5, 139.7, 140.3, 149.6. Anal.
Calcd for C16H10Cl2N2O2S2: C, 48.37; H, 2.54; N, 7.05.
Found: C, 48.61; H, 2.43; N, 6.89.
Yield 71%, mp 228–230 ꢁC; IR (KBr cmꢀ1): 3444 NH2,
1466 SO2; 1H NMR d CDCl3: 2.50 (s, 3H, CH3), 4.43 (br
s, 2H, NH2), 5.55 (s, 1H, H2), 6.53 (d, 1H, H8,
J = 2.0 Hz), 7.20 (dd, 1H, H6, J = 8.7, 2.0 Hz), 7.23 (d,
4.6.3. 3-Amino-7-chloro-9(40-chlorophenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6q). Yield 57%, mp 282–
284 ꢁC; IR (KBr cmꢀ1): 3344 NH2, 1462 SO2; 1H
NMR d CDCl3: 4.23 (br s, 2H, NH2), 5.58 (s, 1H,
H2), 6.50 (d, 1H, H8, J = 1.9 Hz), 7.23 (dd, 1H, H6,
0
0
0
0
2H, H2 ;6 , J = 8.4 Hz), 7.44 (d, 2H, H3 ;5 , J = 8.4 Hz),
8.06 (d, 1H, H5, J = 8.7 Hz). 13C NMR 21.5, 88.8,
107.9, 115.8, 122.9, 123.4, 125.1, 128.8, 132.1, 135.9,
138.6, 140.1, 140.2, 141.4, 150.5. Anal. Calcd for
C17H13ClN2O2S2: C, 54.18; H, 3.48; N, 7.43. Found:
C, 53.98; H, 3.67; N, 7.63.
0
J = 8.7, 1.9 Hz), 7.34 (d, 2H, H2 ;6 , J = 8.7 Hz), 7.64
0
0
0
(d, 2H, H3 ;5 ; J = 8.7 Hz), 8.07 (d, 1H, H5, J = 8.7 Hz).
13C NMR 88.4, 107.9, 115.76, 123.3, 124.1, 125.9,
131.9, 132.0, 132.1, 137.6, 137.6, 139.8, 141.6, 149.8.
Anal. Calcd for C16H10Cl2N2O2S2: C, 48.37; H, 2.54;
N, 7.05. Found: C, 48.57; H, 2.57; N, 7.17.
4.5. 3-Amino-7-chloro-9(20,50-dimethylphenyl)-thieno-
[3,2-b]benzothiazine 4,4-dioxide (6m)
Yield 47%, mp 190–192 ꢁC; IR (KBr cmꢀ1): 3408 NH2,
1462 SO2; 1H NMR d CDCl3: 2.00 (s, 3H, CH3), 2.50 (s,
3H, CH3), 4.52 (br s, 2H, NH2), 5.55 (s, 1H, H2), 6.45 (d,
4.6.4. 3-Amino-7-chloro-9(30,40-dichlorophenyl)-thieno-
[3,2-b]benzothiazine 4,4-dioxide (6r). Yield 49%, mp
1
0
1H, H8, J = 2.0 Hz), 7.07 (s, 1H, H6 ), 7.22 (dd, 1H, H6,
0
J = 8.7, 2.0 Hz), 7.32 (d, 1H, H4 , J = 8.4 Hz), 7.37 (d,
230–232 ꢁC; IR (KBr cmꢀ1): 3430 NH2, 1464 SO2; H
NMR d CDCl3: 4.24 (br s, 2H, NH2), 5.59 (s, 1H,
H2), 6.51 (d, 1H, H8, J = 1.9 Hz), 7.25 (dd, 1H, H6,
13
1H, H5 , J = 8.4 Hz), 8.08 (d, 1H, H5, J = 8.7 Hz). C
0
0
J = 8.7, 1.9 Hz), 7.28 (dd, 1H, H6 , J = 8.4, 2.2 Hz),
NMR 16.7, 21.0, 88.6, 107.9, 115.2, 123.0, 123.4,
125.3, 129.5, 132.0, 132.7, 133.9, 136.9, 138.9, 139.1,
140.2, 149.6. Anal. Calcd for C18H15ClN2O2S2: C,
53.31; H, 3.87; N, 7.16. Found: C, 53.12; H, 3.81; N,
7.18.
0
0
7.54 (d, 1H, H2 ; J = 2.2 Hz), 7.76 (d, 1H, H5 ,
J = 8.4 Hz), 8.06 (d, 1H, H5, J = 8.7 Hz). 13C NMR
88.9, 108.7, 115.4, 123.6, 123.7, 125.4, 128.8, 131.5,
133.3, 135.6, 135.9, 137.5, 138.9, 139.5, 140.4, 149.3.
Anal. Calcd for C16H9Cl3N2O2S2: C, 44.51; H, 2.10;
N, 6.49. Found: C, 44.37; H, 1.87; N, 6.37.
4.6. 3-Amino-7-chloro-9(30-methoxylphenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6n)
Yield 43%, mp 158–160 ꢁC; IR (KBr cmꢀ1): 3376 NH2,
5. X-ray crystallography
1
1462 SO2; H NMR d CDCl3: 3.85 (s, 3H, OCH3), 4.37
(br s, 2H, NH2), 5.56 (s, 1H, H2), 6.57 (d, 1H, H8,
Crystals suitable for X-ray diffraction for all compounds
were obtained by slow evaporation of solutions in etha-
nol. Crystal data, intensity data collection parameters
and final refinement results are summarized in Table 4.
0
J = 1.7 Hz), 6.88 (t, 1H, H2 J = 2.2 Hz), 6.95 (d, 1H,
0
H4 , J = 7.9 Hz), 7.16 (dd, 2H, H6 , J = 7.9, 2.2 Hz),
0
0
7.22 (dd, 1H, H6, J = 8.7, 1.7 Hz), 7.56 (t, 1H, H5 ,
J = 7.9 Hz), 8.06 (d, 1H, H5, J = 8.7 Hz). 13C NMR
55.8, 88.8, 107.0, 114.2, 115.8, 116.9, 120.8, 123.0,
123.4, 125.1, 132.1, 138.7, 139.5, 139.9, 140.1, 150.1,
161.9. Anal. Calcd for C17H13ClN2O3S2: C, 51.97; H,
3.34; N, 7.13. Found: C, 52.15; H, 3.37; N, 7.23.
Unit cell and intensity measurements were carried out
on a Bruker Smart CCD area-detector diffractometer,
using graphite-monochromated Mo-Ka radiation
˚
(k = 0.71073 A). The structures were solved by direct
methods and refined on F2 by full-matrix least-squares,
using all reflections, anisotropic displacement parame-
4.6.1.
3-Amino-7-chloro-9(40-methoxylphenyl)-thieno-
[3,2-b]benzothiazine 4,4-dioxide (6o). Yield 47%, mp
2
ters and weights w ¼ ½r2ðF o2Þ þ ðaPÞ þ bPꢂꢀ1, with
1
226–228 ꢁC; IR (KBr cmꢀ1): 3424 NH2, 1465 SO2; H
P ¼ ðF 2o þ 2F 2cÞ=3. The C- and N(imino)-bonded H
NMR d CDCl3: 3.91 (s, 3H, OCH3), 4.39 (br s, 2H,
NH2), 5.55 (s, 1H, H2), 6.54 (d, 1H, H8, J = 1.7 Hz),
atoms were placed in calculated positions, and refined
˚
using a riding atom model with fixed C–H [0.93 A for
2
3
˚
˚
0
7.12 (d, 2H, H2 ;6 , J = 8.9 HZ), 7.20 (dd, 1H, H6,
0
C(sp ), 0.96 A for C(sp )] and N–H [0.86 A] distances,
and Uiso = p Ueq(parent atom) [p = 1.2 for C(sp2) and
N; 1.5 for C(sp3)]. The N(amino)-bonded H atoms were
located in difference Fourier syntheses and refined iso-
tropically. The final difference Fourier syntheses were
featureless.
0
0
J = 8.7, 1.7 Hz), 7.27 (d, 2H, H3 ;5 ; J = 8.9 Hz), 8.05
(d, 1H, H5, J = 8.7 Hz). 13C NMR 55.7, 88.7, 108.3,
115.8, 116.5, 122.9, 123.7, 125.1, 130.4, 138.6, 138.7,
140.3, 140.4, 150.9, 161.2. Anal. Calcd for
C17H13ClN2O3S2: C, 51.97; H, 3.34; N, 7.13. Found:
C, 51.86; H, 3.47; N, 6.90.
The following computer programs were used: data col-
lection, SMART;21a data reduction and cell refinement,
SAINT;21b absorption correction, SADABS;21c struc-
4.6.2. 3-Amino-7-chloro-9(30-chlorophenyl)-thieno-[3,2-
b]benzothiazine 4,4-dioxide (6p). Yield 38%, mp 235–

3672
A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
Table 4. Crystal data, intensity data-collection parameters, and final
refinement results
6. Computational methods
All the calculations reported here were carried out
using the program Gaussian 03.26 In the first step, a
conformational search was carried out using explor-
atory RHF/3-21G calculations. Then more accurate
B3LYP/6-31G (d) calculations were carried out to
confirm the critical points obtained. Minima were
characterized through harmonic frequency analysis
using B3LYP/6-31G (d) calculations. The electronic
study of the molecules was carried out using molecu-
lar electrostatic potentials (MEPs).17,18 MEPs were
calculated from B3LYP/6-31G(d) wave functions.
MEP graphical presentations were created using the
Molekel program.27
Compound
4d
5d
CCDC deposit No.
CCDC 650290
CCDC 650291
Crystal data
Formula
MW
C
18H15ClN2O2S2 C21H19ClN2O4S2
390.89
Colorless
Prism
462.95
Colorless
Prism
Color
Morphology
Specimen size (mm)
T (K)
0.24 · 0.18 · 0.13 0.30 · 012 · 0.11
296(2)
296(2)
˚
a (A)
9.8708(8)
9.9252(8)
11.7038
91.235(2)
111.7120(10)
116.8240(10)
925.75(13)
Triclinic
P ꢀ1 (#2)
2
11.1580(8)
10.1225(7)
19.1247(14)
˚
b (A)
˚
c (A)
a (ꢁ)
b (ꢁ)
c (ꢁ)
91.851(2)
3
˚
V (A )
2158.9(3)
Monoclinic
P21/c (#14)
4
7. Biological assays
Crystal system
Space group (No.)
Z
7.1. Inhibition of b-hematin formation
Dc (g cmꢀ3
)
1.402
404
1.424
960
The b-hematin formation assay was performed with a
solution of hemin chloride (50 lL, 4 mM), dissolved in
DMSO (5.2 mg/mL), distributed in 96-well micro-
plates.28 Different concentrations (50–5 mM) of the
compounds dissolved in DMSO were added in triplicate
in test wells (50 lL). Controls contained either water
(50 lL) or DMSO (50 lL). b-Hematin formation was
initiated by the addition of acetate buffer (100 lL,
0.2 M, pH 4.4). Plates were incubated at 37 ꢁC for
48 h to allow for completion of the reaction and centri-
fuged (4000 RPM · 15 min, IEC-CENTRA, MP4R).
After discarding the supernatant, the pellet was washed
twice with DMSO (200 lL) and finally, dissolved in
NaOH (200 lL, 0.2 N). The solubilized aggregates were
further diluted 1:2 with NaOH (0.1 N) and absorbance
recorded at 405 nm (Microplate Reader, BIORAD-
550). The results were expressed as a percentage of inhi-
bition of b-hematin formation.
F(000)
l(Mo-Ka) (mmꢀ1
h range (ꢁ) for cell
No. refls. for cell
)
0.446
2.4–26.7
1897
0.401
2.3–21.5
2139
Data collection
h range (ꢁ)
h range
1.9–29.0
ꢀ13, 13
ꢀ13, 13
ꢀ10, 15
<1
1.8–29.0
ꢀ14, 8
ꢀ13, 13
ꢀ25, 25
<1
k range
l range
Mean DI for checks (%)
No. refls. measured
No. refls. unique
No. refls. I > 2r(I)
Rint
6438
4320
14649
5328
3341
3099
0.0147
0.0368
Refinement (last cycle)
Weighting scheme (a,b)
Trans. coeff. (Tmin, Tmax
No. params. refined
R1 [I > 2r(I)]
R1 (all data)
wR2 [I > 2r(I)]
wR2 (all data)
0.0586, 0.1562
0.919–0.944
229
0.0636, 0.5070
0.913–0.957
282
)
0.0464
0.0664
0.0567
0.0998
7.2. Parasite, experimental host, and strain maintenance
0.1153
0.1253
0.1286
0.1482
Male Balb-C mice, weighing 18–22 g were maintained
on a commercial pellet diet and housed under conditions
approved by the Ethics Committee. Plasmodium berghei
(ANKA strain chloroquine sensible), a rodent malaria
parasite, was used for infection. Mice were infected by
ip passage of 1 · 106 infected erythrocytes diluted in
phosphate buffered saline solution (PBS, 10 mM, pH
7.4, 0.1 mL). Parasitemia was monitored by microscopic
examination of Giemsa stained smears.29
S (g.o.f.) (all data)
D/r max.
D/r mean
1.043
0.014
1.029
0.001
<0.0005
ꢀ0.29, 0.42
<0.0005
ꢀ0.59, 0.65
ꢀ3
˚
Dqr (min, max) (e A
)
ture solution, SHELXS-97;22 structure refinement,
SHELXL-97;23 molecular graphics, ORTEP 3.24 The
structure solutions, the refinements and the drawings
were carried out with the aid of the WinGX25 suite of
programs.
7.3. Parasite extracts
Blood of infected animals with a high level of parasite-
mia (30–70%), was collected by cardiac puncture with
a heparinized syringe and the blood pool was centri-
fuged (500g · 10 min, 4 ꢁC). Plasma and buffy coat were
removed and the red blood cell (RBC) pellet was washed
twice with chilled PBS–Glucose (5.4%). The washed
RBC pellet was centrifuged on a discontinuous percoll
gradient (80–70% percoll in PBS–Glucose, 20,000g
· 30 min · 4 ꢁC).30 The upper band (mature forms)
Crystallographic data (excluding structure factors) for
the structures in this paper have been deposited with
the Cambridge Crystallographic Data Centre as supple-
mentary publication Nos. CCDC 650290 and 650291.
Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK, fax: +44 (0) 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk.

A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
3673
was removed by aspiration, collected in eppendorf tubes
Acknowledgments
and washed twice with chilled PBS–Glucose and the in-
fected erythrocytes were lysed with the non-ionic deter-
gent saponin (0.1% in PBS · 10 min). Cold PBS (1 mL)
was added and the samples were centrifuged (13,000g
· 5 min, 4 ꢁC) to remove erythrocyte cytoplasm content
(including erythrocyte hemoglobin). The free parasites
were mixed in PBS–Glucose (5.4%), and subjected to
three freeze–thaw cycles (ꢀ70 ꢁC + 37 ꢁC). The final
homogenate was used in the hemoglobin hydrolysis inhi-
bition assay.31
We thank the IIF-FF and CDCH-UCV (Grants IIF:
03.2006, PG. 06-00-6502-2006), CONICIT PCP and
CYTED-RIDIMEDCHAG programs for financial sup-
port. R.D. Enriz is a member of the Consejo Nacional
´
´
de Investigaciones Cientıficas y Tecnologicas (CONI-
CET—Argentina) staff. S.A. Andujar Thanks a CONI-
CET—fellowship.
References and notes
7.4. Mice native hemoglobin
1. WHO World Malaria Report 2005, http://rbm.who.int/
wmr 2005.
2. Miller, L. H.; Baruch, D. I.; Marsh, K.; Doumbo, O. K.
Nature (London) 2002, 415, 673.
3. (a) Joshi, A. A.; Viswanathan, C. L. Anti-Infec. Agents
Native hemoglobin from non-infected mice was ob-
tained by treating one volume of pellet erythrocytes with
two volumes of water. The resulting solution was used
as the substrate in the inhibition of the hemoglobin
hydrolysis assay.
´
Med. Chem. 2006, 5, 105; (b) Domınguez, J. N. Curr.
Trop. Med. Chem. 2002, 2, 1171.
4. Sullivan, D. J.; Gluzman, I. Y.; Russell, D. G.; Goldberg,
D. E. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11865.
5. Tilley, L.; Loria, P.; Foley, M. Chloroquine and other
quinoline antimalarials. In Antimalarial Chemotherapy;
Rosenthal, P. J., Ed.; Humana Press Inc.: Totowa, NJ,
2001; p 87.
6. Slater, A.; Swiggard, W.; Orton, B.; Flitter, W.; Goldberg,
D.; Cerami, A.; Henderson, G. Proc. Natl. Acad. Sci.
U.S.A. 1991, 88, 325.
7. (a) Pagola, S.; Stephens, P.; Bohle, D.; Kosar, A.;
Madesen, S. Nature (London) 2000, 404, 307; (b) Egan,
T. J. J. Inorg. Chem. 2006, 100, 916.
8. Anquetin, G.; Rouquayrol, M.; Mahmoudi, N.; Santill-
ana-Hayat, M.; Gonzalbes, R.; Greiner, J.; Farhati, K.;
Derouin, F.; Guedj, R.; Vierling, P. Bioorg. Med. Chem.
Lett. 2004, 14, 2773.
7.5. Inhibition of hemoglobin hydrolysis
The proteolytic effect of the parasite extract on the na-
tive mice hemoglobin was assayed using 96-well tissue
culture plate (Greiner Bio-One). The assay mixture con-
tained: mice native hemoglobin (10 lL), parasite extract
(50 lL), GSH (10 lL, 10 lM), and acetate buffer (0.2 M,
pH 5.4) to a final volume of 200 lL. The compounds,
chloroquine, leupeptin, and pepstatin (2.5 mM) were
incorporated into the incubation mixture dissolved in
DMSO. The incubations were carried out at 37 ꢁC for
18 h and the reactions were stopped by the addition of
reduced sample buffer. The degree of digestion was eval-
uated electrophoretically by SDS–PAGE by visual com-
parison of the globin bands (14.4 kDa). A DMSO
control was electrophoresed at the same time. Once
the bands were obtained, the densitometer registered
the band densities reported as intensity/mm2 SD, so
we proceeded to check the densities in order to have a
percentage of inhibition of hemoglobin hydrolysis.
´
9. (a) Charris, J. E.; Domınguez, J. N.; Gamboa, N.;
Rodrigues, J.; Angel, J. E. Heterocycl. Commun. 2005,
´
11, 419; (b) Charris, J.; Barazarte, A.; Domınguez, J.;
Lobo, G.; Camacho, J.; Ferrer, R.; Gamboa, N.; Rodri-
gues, J.; Capparelli, M. J. Heterocycl. Chem. 2007, 44, 639.
10. (a) Leysen, D. C.; Zhang, M. Q.; Haemers, A.; Bollaert,
W. Pharmazie 1991, 46, 485; (b) Brown, C.; Davinson, R.
M. Adv. Heterocycl. Chem. 1985, 38, 135; (c) Teller, J.
Methods Org. Chem. (Houben-Weyl) 1997, E9, 450.
11. (a) Karchgaudhuri, N.; De, A.; Mitra, A. K. J. Chem. Res.
(S) 2001, 180; (b) Kappe, C. O. Angew. Chem. Int. Ed.
2004, 43, 6250; (c) Bourgin, K.; Loupy, A.; Soufiaoui, M.
J. Photochem. Photobiol. C: Phochem. Rev. 2005, 6, 139;
(d) Kappe, C. O.; Stadler, A. Microwave in organic and
medicinal chemistry. In Mannhold, R., Kubinyi, H.,
Folkers, G., Eds.; Whiley-VCH Verlag Gmbh & Co.
KGaA: Weinhein, 2005; (e) Microwave-Assisted Synthesis
of Heterocycles Series: Topics in Heterocyclic Chemistry,
Gupta, R. R., Series Ed.; Van der Eycken, E., Kappe, C.
O., Volume Editors; Springer, 2006; Vol. 1.
7.6. Four-day suppressive test
NIH mice (18–22 g) were infected ip with 1 · 107 P.
berghei parasites. Two hours after infection, treatment
began with the best compounds tested in the globin
hydrolysis assay. These were dissolved in DMSO
(0.1 M), diluted with Saline–Tween 20 solution (2%).
Each compound (20 mg/kg) was administered once by
ip for 4 days. At day four, the parasitemia was counted
by examination of Giemsa stained smears. The chloro-
quine (25 mg/kg) was used as a positive control. The sur-
vival time after infection was recorded. The results were
expressed as a percentage of parasitemia at four days
post-infection and survival time after infection com-
pared to control mice.32
´
12. Gomez, L.; Charris, J.; Domınguez, J.; Lobo, G.; Basante,
W.; Riggione, F.; Sanchez, E.; Duerto, Z. Rev. Fac.
´
´
Farmacia 2001, 64, 56.
13. Unpublished results. Crystallographic data is deposited
with the Cambridge Crystallographic Data Centre (3c:
CCDC 650288, 3e: CCDC 650289).
´
14. (a) Charris, J.; Barazarte, A.; Domınguez, J.; Gamboa, N.
J. Chem. Res. 2005, 27; (b) Charris, J.; Barazarte, A.;
7.7. Data analysis
Ferrer, R.; Camacho, J.; Gamboa, N.; Rodrigues, J.;
´
Data were statistically analyzed using t-tests for specific
group comparisons, assuming 95% confidence according
GraphPad Prism 3.02.33
Atencio, R.; Gonzalez, T. J. Chem. Res. 2007, 16.
15. (a) Kirsch, G.; Cagniant Mme, D.; Cagniant, P. J.
Heterocycl. Chem. 1982, 19, 443; (b) Zambias, R.; Ham-

3674
A. Barazarte et al. / Bioorg. Med. Chem. 16 (2008) 3661–3674
mond, M. Synth. Commun. 1991, 21, 859; (c) Sthephens,
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;
Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene,
M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R.
E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth,
G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V.
G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas,
O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.;
Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R.
L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C.
Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.;
Pople, J. A. Gaussian 03 Revision B.05; Gaussian, Inc.:
Pittsburgh, PA, 2003.
C.; Price, M.; Sowell, W. J. Heterocycl. Chem. 1999, 36,
659; (d) Comel, A. J. Heterocycl. Chem. 2001, 38, 1167; (e)
Owton, W. J. Tetrahedron Lett. 2003, 44, 7147.
16. Allen, F. H.; Kennard, O.; Watson, D. G.; Brammer, L.;
Orpen, A. G.; Taylor, R. J. Chem. Soc. Perkin Trans. II
1987, 1–19.
17. Polilzer, P.; Truhlar, D. G. Chemical Applications of
Atomic and Molecular Electrostatic Potential; Plenum:
New York, 1991.
´
18. Carrupt, P. A.; El Tayar, N.; Karle, A.; Festa, B. Methods
Enzymol. 1991, 202, 638.
19. Politzer, P., Murray, J. S., Lipkowitz, K. B., Boyd, D. B.,
Eds.; Reviews in Computational Chemistry; Springer: New
York, 1991; Vol. 2, Chapter 7.
20. Naray-Szabo, G.; Ferenczy, G. G. Chem. Rev. 1995, 95,
829.
21. (a) Bruker. SMART, version 5.63. Bruker AXS Inc.,
Madison, WI, USA, 2002.; (b) Bruker. SAINT, version
6.22. Bruker AXS Inc., Madison, WI, USA, 2001.; (c)
Bruker. SADABS, version 2.05. Bruker AXS Inc., Mad-
ison, WI, USA, 2001.
27. Flukiger, P.; Luthi, H. P.; Portmann, S.; Weber, J.
¨
¨
MOLEKEL 4.0; Swiss Center for Scientific Computing:
Manno, Switzerland, 2000.
22. Sheldrick, G. M. SHELXS—A Program for Crystal
Structure Solution. Release 97-2. University of Go¨ttingen,
Germany, 1997.
23. Sheldrick, G.M. SHELXL—A Program for Crystal Struc-
ture Refinement. Release 97-2. University of Go¨ttingen,
Germany, 1997.
28. Baelmans, R.; Deharo, E.; Mun˜oz, V.; Sauvain, M.;
Ginsburg, H. Exp. Parasitol. 2000, 96, 243.
29. Dorn, A.; Stoffel, R.; Matile, H.; Bubendorf, A.; Ridley,
R. Nature (London) 1995, 374, 269.
30. Deharo, E.; Gautret, P.; Ginsburg, H.; Chabaud, A.;
Landau, I. Parasitol. Res. 1994, 80, 159.
24. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
25. Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837.
26. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria,
G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J.
A., ; Vreven, T., Jr.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
31. Rosenthal, P. Exp. Parasitol. 1995, 80, 272.
32. Peters, W.; Robinson, B. L. Parasitic infection models. In
Handbook of Antimalarial Models of Infection; Zak, O.,
Sande, M., Eds.; Academic Press: London, 1999; p 757.
33. Graph Pad Prism Software Inc. 4.02 for windows. May
17th, 1992–2004.