Synthesis and antimalarial activity of pyrazolo and pyrimido benzothiazine dioxide derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.08.005

A series of phenylsubstituted pyrazolo and pyrimido benzothiazine dioxide derivatives were synthesized and investigated for their abilities to inhibit β-hematin formation, hemoglobin hydrolysis and in vivo for their antimalarial efficacy in rodent Plasmod

Full text of DOI:10.1016/j.ejmech.2008.08.005

European Journal of Medicinal Chemistry 44 (2009) 1303–1310
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimalarial activity of pyrazolo and pyrimido benzothiazine
dioxide derivatives
Arthur Barazarte a, Gricela Lobo a, Neira Gamboa b, Juan R. Rodrigues b, Mario V. Capparelli c,
d
e
a,
*
´
a
´
´
´
Angel Alvarez-Larena , Simon E. Lopez , Jaime E. Charris
´
Laboratorio de S´ıntesis Organica, Universidad Central de Venezuela, Aptdo. 47206, Los Chaguaramos, 1041-A Caracas, Venezuela
b Unidad de Bioqu´ımica, Facultad de Farmacia, Universidad Central de Venezuela, Aptdo. 47206, Los Chaguaramos, 1041-A Caracas, Venezuela
cEscuela de Qu´ımica, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela
d Servicio de Difraccio´n de Rayos X, Universidad Auto´noma de Barcelona, Bellaterra, Spain
e
´
´
´
Departamento de Quımica, Universidad Simon Bolıvar, Caracas, Venezuela
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2008
Received in revised form 7 August 2008
Accepted 8 August 2008
Available online 26 August 2008
A series of phenylsubstituted pyrazolo and pyrimido benzothiazine dioxide derivatives were synthesized
and investigated for their abilities to inhibit b-hematin formation, hemoglobin hydrolysis and in vivo for
their antimalarial efficacy in rodent Plasmodium berghei. Compounds 3-amino-7-chloro-9-(20-methyl-
pheyl)-1,9-dihydro-pyrazolo-[4,3-b]benzothiazine 4,4-dioxide 2b and 2,4-diamino-8-chloro-10H-
phenyl-pyrimido-[5,4-b]benzothiazine 5,5-dioxide 3a were the most promising as inhibitors of hemo-
globin hydrolysis, however, their effect as inhibitors of b-hematin formation was marginal, except for
Keywords:
Benzothiazines
Pyrazol
Pyrimidine
Plasmodium berghei
compound 3-amino-7-chloro-9-(30-chlorophenyl)-1,9dihydro-pyrazolo-[4,3-b]benzothiazine 4,4-dioxide
2g. The most active compound to emerge from the in vitro and in vivo murine studies was 2b, suggesting
an antimalarial activity via inhibition of hemoglobin hydrolysis, however, not as efficient as chloroquine.
Ó 2008 Published by Elsevier Masson SAS.
b
-Hematin
Hemoglobin
1. Introduction
drugs that attack the same vital target of chloroquine but that are
not subject to the same resistance mechanism would be highly
Malaria, a major tropical infectious disease caused primarily by
the protozoan parasite Plasmodium falciparum, is one of the most
serious health problems worldwide and is responsible for the death
of over 1 million individuals every year with more than 40% of the
global population at risk [1]. Since resistance to currently used
antimalarials is spreading rapidly, there is a great need for new
drugs. Thus, there is a compelling and urgent necessity for new
antimalarials, with mechanisms of action different from the exist-
ing ones, and to identify new drug targets [2]. Chloroquine has
recently been shown to inhibit hemozoin formation within the
parasite food vacuole [3]. This process is also thought to be the
molecular target of other quinoline antimalarials [4]. Hemozoin
was originally considered to be formed by the polymerization of
heme, but it has now been demonstrated to be a crystalline cyclic
dimer of ferriprotoporphyrin IX [5–8]. Thus, hemozoin synthesis,
a process unique to the malaria parasite, offers a logical and valu-
able potential target for new antimalarial drug development. New
desirable. Fluoroquinolones are widely used clinically. Some of
these quinolones such as ciprofloxacin, gatifloxacin, moxifloxacin
and trovafloxacin, display a diverse array of biological activities
including antiplasmodial efficacy [9]. We have recently described
the preparation and antimalarial activities of several tricyclic qui-
nolone and benzothiazine analogs [10–12]. In continuation of our
studies directed toward synthesis of quinolones and benzothia-
zines annelated with various five- and six-member heterocycles,
we reported here the synthesis of 3-amino-6 or 7-chloro-9-
(phenylsubstituted)-1,9-dihydro-pyrazolo-[4,3-b]benzothiazine 4,
4-dioxide 2a–t and 2,4-diamino-7 or 8-chloro-10-(phenyl-
substituted)-10H-pyrimido-[5,4-b]benzothiazine 5,5-dioxide 3a–t,
their in vitro abilities to inhibit
b-hematin formation and hemo-
globin hydrolysis and their in vivo efficacy against rodent Plasmo-
dium berghei.
2. Chemistry
2,3-Substituted 6 or 7 chloro-N-phenylbenzothiazine 1a–t were
obtained following the method previously reported [10–12].
Products 2a–t and 3a–t were obtained when 1a–t were reacted
* Corresponding author. Tel.: þ58 212 6052722; fax: þ58 212 6052707.
E-mail address: charrisj@ucv.ve (J.E. Charris).
0223-5234/$ – see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.08.005

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A. Barazarte et al. / European Journal of Medicinal Chemistry 44 (2009) 1303–1310
with hydrazine hydrate or guanidine hydrochloride, dry pyridine or
DMF under an inert atmosphere of nitrogen, respectively, (Scheme
1).
It is important to mention that in the 1H NMR spectra of these
compounds, protons at positions 8 and 9 from derivatives 2a–t and
3a–t appeared as doublets around 6.0 ppm with coupling constants
ranging between 0.9–2.5 and 7.0–8.0 Hz, clearly indicating the
smaller chemical shift of the proton on these positions by the effect
of the phenyl group on position 9 or 10, respectively. Additional
supports for these structures were obtained from 13C NMR.
The molecular structure of 3p was confirmed by X-ray crystal-
lography (Fig.1). The X-ray crystal structure analysis showed that all
the bond distances are within expected values [13]. In the tricyclic
system, the central ring displays an approximate sofa conformation,
with S1 out of the plane [0.600(2) Å], and O1 and O2 in equatorial
and axial positions, respectively. The N-bonded phenyl ring is
approximately perpendicular to the tricyclic system [dihedral angle
between mean planes: 84.83(5)ꢀ]. The molecule forms an N–H/O
(sulfonyl) intramolecular hydrogen bond. In addition, in the crystal
structure there are a number of intermolecular hydrogen bonds of
the types N–H/O(sulfonyl), N–H/O(methoxy), N–H/Cl, N–H/S,
C–H/O(sulfonyl), C–H/O(methoxy), C–H/N(amino) and C–H/
N(pyrimidine, N4) which link the molecules to form a three
dimensional network (see CIF file for details).
3. Biological results and discussion
Fig. 1. Molecular structure of compound 3p showing the atomic numbering. The
displacement ellipsoids are drawn at 50% probability. A dashed line indicates an
intramolecular hydrogen bond.
Previous reports showed that tricyclic benzothiazines exhibited
antimalarial activities [10,12]. All analogs of those derivatives were
tested in vitro for their effects as inhibitors of b-hematin formation,
and inhibition of hemoglobin hydrolysis (Table 1). Only 2a, 2g, 3a
derivatives were tested in vivo for their efficacy in a murine model
(Table 3). The first mentioned in vitro assay was used to assess the
abilities of the N-phenylpyrazolo[4,3-b]benzothiazine and N-phe-
nylpyrimido[5,4-b]benzothiazine S,S-dioxide derivatives to inhibit
Compounds 2a–t and 3a–t were also tested for the inhibition of
globin proteolysis using in vitro assays which used rich extracts of
trophozoites to digest the native hemoglobin of mice. Electropho-
retic analyses indicated that compounds 2a–j and 3a–j were
effective as inhibitors of hemoglobin degradation; however,
compounds 2b and 3a were the most effectives (92.32 ꢁ 1.1 and
83.72 ꢁ 2.13%) compared to leupeptin and pepstatin (89.06 ꢁ 0.69
and 92.94 ꢁ 0.67), respectively (Table 1).
b-hematin formation, where hemin was allowed to form b-hematin
under acidic conditions. Among the 40 compounds tested, only one
2g showed a measurable activity (85.42 ꢁ 6.14%) compared to
chloroquine (86.6 ꢁ 2.75%).
O
O
4
NH2
3
5
S
R1
N
1
N
H
N
9
8
i
R2
O
O
8
1
CN
S
2a-t
R1
S
CH3
N
5
4
NH2
4
O
O
5
6
ii
S
R2
N
R1
N
N
1
NH2
10
9
1a-t
R2
3a-t
Scheme 1. Synthesis of pyrazolo and pyrimido benzothiazine dioxide derivatives 2a–t, 3a–t. (i) N2H4 hydrate, pyridine,
. R1: Cl; R2: H, CH3, OCH3, Cl, Br.
D. (ii) Guanidine hydrochloride, potassium carbonate, DMF,
D

A. Barazarte et al. / European Journal of Medicinal Chemistry 44 (2009) 1303–1310
1305
Table 1
Table 2
Inhibition of
b-hematin synthesis (I
b
HS) and globin proteolysis (IGP) (%) by ben-
Effect of benzothiazine derivatives (20 mg/kg) on parasitemia at fourth day post-
zothiazine derivatives
infection (%P) and survival days (SD) of P. berghei infected mice
No.
R2
I
bHS
IGP
Treatment
P (%)
SD
2a
2b
2c
2d
2e
2f
2g
2h
H
<5
<5
<5
<5
<5
<5
76.39 ꢁ 1.52
Saline solution
2b
2g
3a
21.8 ꢁ 2.31
11.66 ꢁ 1.66
13 ꢁ 1.26
12.5 ꢁ 2.13**
10.8 ꢁ 1.59
30
2-CH3
4-CH3
2,5-CH3
3-OCH3
4-OCH3
3-Cl
4-Cl
3,4-Cl
4-Br
92.32 ꢁ 1.1**
9.75 ꢁ 3.01*
10.33 ꢁ 2.43*
12.7 ꢁ 2.11
1.3 ꢁ 0.3
78.78 ꢁ 0.41
69.39 ꢁ 1.17
47.27 ꢁ 1.79
Chloroquine
54.77 ꢁ 1.58
The results are expressed by the mean ꢁ standard error of the mean.
*p < 0.05 and **p < 0.01 compared to untreated mice (saline). n ¼ 6.
85.42 ꢁ 6.14y
58.89 ꢁ 2.14
<5
<5
<5
<5
55.80 ꢁ 1.74
2i
2j
2k
67.63 ꢁ 1.28
26.66 ꢁ 1.31
activity. Thus, the poor inhibition of b-hematin formation appears
H
0
2l
2m
2n
2o
2p
2q
2r
2s
2t
3a
3b
3c
3d
3e
3f
3g
2-CH3
4-CH3
2,5-CH3
3-OCH3
4-OCH3
3-Cl
4-Cl
3,4-Cl
4-Br
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
0
0
0
0
0
0
0
0
to have close relation with this substitution pattern.
The presence of a methyl group as substituent in the aromatic
ring appeared to be favourable for the antimalarial activity, since
most of the compounds displaying this group showed measurable
levels of inhibition of hemoglobin degradation, regardless of the
nature of the substitutions in the aromatic ring.
Previously we observed that compounds with different
substituents on phenyl ring showed different activities [12]. Thus,
compounds without substituents or with methyl on phenyl ring
0
H
83.72 ꢁ 2.13 **
2-CH3
4-CH3
2,5-CH3
3-OCH3
4-OCH3
3-Cl
72.64 ꢁ 2.01
50.09 ꢁ 0.79
64.60 ꢁ 0.71
56.34 ꢁ 1.20
Table 3
40.89 ꢁ 1.73
Crystal data, intensity data collection parameters and final refinement results for
compound 3p
0
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
4-Cl
3,4-Cl
4-Br
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
–
–
67.19 ꢁ 2.03
CCDC deposit No.
Crystal data
Formula
MW
CCDC 685720
76 ꢁ 1.22
49.04 ꢁ 1.41
C17H14ClN5O3S2
435.91
Colourless
Prism
H
0
0
0
0
0
0
0
0
2-CH3
4-CH3
2,5-CH3
3-OCH3
4-OCH3
3-Cl
4-Cl
3,4-Cl
4-Br
Colour
Morphology
Specimen size (mm)
T (K)
a (Å)
b (Å)
0.50 ꢂ 0.12 ꢂ 0.09
296(2)
8.2872(5)
7.6900(5)
28.0940(19)
90.000
92.396(1)
90.000
1788.8(2)
Monoclinic
P21/n (No. 14)
4
1.500
832
0.360
2.5–26.0
3080
c (Å)
0
0
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
3t
Leupeptin
Pepstatin
Chloroquine
–
–
–
89.06 ꢁ 0.69
92.94 ꢁ 0.67
24.12 ꢁ 1.16
V (Å3)
86.6 ꢁ 2.75
Crystal system
Space group (No.)
Z
R1 (2a–j) ¼ 7-Cl; R1 (2k–t) ¼ 6-Cl; R1 (3a–j) ¼ 8-Cl; R1 (3k–t) ¼ 7-Cl.
The results are expressed by the mean ꢁ standard error of the mean.
yp > 0.05 compared to chloroquine, **p > 0.05 compared to leupeptin (LEP) and
Dc (g cmꢃ3
F(000)
)
pepstatin (PEP).
m
q
(Mo K
a
) (mmꢃ1
)
range (ꢀ) for cell
No. of reflections for cell
Compounds 2b, 2g, and 3a were tested in infected mice with P.
berghei ANKA, a chloroquine-susceptible strain of murine malaria.
Compounds were given to mice (chloroquine or 2b, 2g and 3a, in
20 mg/kg, i.p. once daily) for four consecutive days (days 1–4 post-
infection). At day fourth post-infection, the parasitemia was
determined; the survival days were monitored and compared with
control mice receiving a saline solution (untreated mice). Control
mice died within 12 days post-infection, compound 2b increased
the survival time for 13 days, while chloroquine prolonged the
survival time of the infected mice to 30 days. Compound 2b was
able to reduce and delay the progression of malaria (9.75 ꢁ 3.01%)
but did not eradicate the infection (Table 2).
Data Collection
q
range (ꢀ)
1.5–29.0
ꢃ9, 11
h range
k range
l range
Mean DI for checks (%)
No. of reflections measured
No. of reflections unique
No. of reflections I > 2
Abs. correction
Trans. coeff. (Tmin, Tmax
Rint
ꢃ9, 10
ꢃ38, 30
0.4
11963
4378
3330
Multi-scan
0.759–0.968
0.0229
s
(I)
)
Refinement (last cycle)
It should be emphasized that compounds 2a–j and 3a–j, which
bear methyl groups on the N-bonded phenyl ring and Cl atoms on
positions 7 or 8 of the benzothiazine nucleus, showed the highest
activity. It is interesting to note that the presence of Cl at positions 7
or 8 appears to be necessary, but not sufficient by itself to produce
the biological response. The lack of inhibitory effect of compounds
2k–t and 3k–t illustrates this situation. Compounds having a Cl on
positions 6 or 7 of the benzothiazine nucleus, and the same
substitution pattern on the phenyl group markedly decrease the
Weighting scheme (a, b)
No. of parameters refined
0.0614, 0.3861
261
0.0463
0.0636
0.1139
0.1230
1.045
<0.0005
<0.0005
ꢃ0.22, 0.41
R1 [I > 2
R1 (all data)
wR2 [I > 2 (I)]
s(I)]
s
wR2 (all data)
S (g.o.f.) (all data)
D/s
max
D
/
s
mean
Drr (min., max.) (e Åꢃ3
)

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A. Barazarte et al. / European Journal of Medicinal Chemistry 44 (2009) 1303–1310
having a large-size positive zone near to the phenyl group, were the
most active molecules in these series. Compounds with methoxy
groups have a reduced zone of positive charge due to the presence
of the oxygen atom. These compounds display only mediocre
activity. In contrast, compounds having electron withdrawing
substituents such as Cl or Br were devoid of any inhibitory activity.
These results clearly indicate that an increase of the lipophilic
property, with appropriate groups on the phenyl substituent,
produces good inhibitory activity for hemoglobin degradation.
The fact that the activity is markedly affected by altering the
substituents on phenyl ring suggests that this aromatic ring makes
a specific contribution to the binding via an aromatic ring orien-
tation. 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 (van der Waals) forces. Our
results indicate that a characteristic electronic distribution on the
phenyl ring might be important to produce the adequate interac-
tion. Whereas methyl substituents produce major inhibitions, only
mediocre activity was obtained with methoxy derivatives [12].
Compounds 2b, 2g and 3a were tested in mice infected with P.
berghei ANKA, a chloroquine-susceptible strain of murine malaria.
Mice were given the compound (chloroquine or 2b, 2g and 3a in
20 mg/kg, ip once daily) for 4 consecutive days (days 1–4 post-
infection). The parasitemia at fourth day post-infection and their
survival times were monitored and compared with control mice
receiving a saline solution (untreated mice). Control mice died at
day 11.66 ꢁ 1.66 post-infection, compound 2b only slightly
increased the survival time 13 ꢁ1.26 days, while 2g and 3a
increased that time for 12.5 ꢁ 2.13 and 10.8 ꢁ 1.5 days, respectively
(Table 2). Particular attention was paid to compound 2b which
was able to reduce and delay the progression of malaria but did
not eradicate the infection 9.75 ꢁ 3.01 days (Table 2). Although
compounds 2b, 2g and 3a 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, cysteine or metalloproteases due to the presence of
a globin band. The 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 may be partially responsible for the poor in
vivo activity observed.
5 h. The solvent was evaporated to dryness under reduced pressure,
water was added (10 mL) and the solid thus obtained was collected
by filtration. Further purification was accomplished by recrystalli-
zation from ethanol–water (4/1).
4.1.1.1. 3-Amino-7-chloro-9-phenyl-1,9-dihydro-pyrazolo-[4,3-b]bꢃen1
zothiazine 4,4-dioxide 2a. Yield 65%; mp 316–318 ꢀC; IR (KBr) cm
:
3317, 2935 (NH), 1497, 1115 (SO2). 1H NMR DMSO-d6:
d 6.35(s, 1H,
H8), 6.39 (br s, 2H, NH2), 7.23 (dd, 1H, H6, J: 8.5, 2.1 Hz), 7.43 (d, 2H,
0
0
0
0
0
H2 ,6 , J: 7.4 Hz), 7.62 (d, 2H, H3 ,5 , J: 7.4 Hz), 7.65 (m, 1H, H4 ), 7.94 (d,
1H, H5, J: 8.5 Hz), 11.64 (br s, 1H, NH); 13C NMR: 87.8, 114.9, 121.1,
125.1, 125.9, 129.8, 130.2, 131.1, 137.7, 137.7, 140.8, 146.9, 147.8. Anal.
C15H11ClN4O2S: C, 51.95; H, 3.20; N, 16.16. Found: C, 52.03; H, 2.97;
N, 16.34%.
4.1.1.2. 3-Amino-7-chloro-9-(20-methylphenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2b. Yield 53; mp 280 ꢀC dec.; IR
(KBr) cmꢃ1: 3450, 3360 (NH), 1440, 1110 (SO2). 1H NMR DMSO-d6:
d
1.95 (s, 3H, CH3), 6.24 (d, 1H, H8, J: 1.5 Hz), 6.41 (br s, 2H, NH2), 7.21
(dd, 1H, H6, J: 8.4, 1.5 Hz), 7.48 (m, 4H, Ar), 7.96 (d, 1H, H5, J: 8.4 Hz),
11.60 (br s, 1H, NH); 13C NMR: 22.1, 92.4, 118.8, 125.8, 129.8, 130.9,
133.5, 134.9, 135.2, 137.1, 141.2, 142.0, 142.5, 144.7, 151.6. Anal.
C16H13ClN4O2S: C, 53.26; H, 3.63; N, 15.53. Found: C, 53.37; H, 3.87;
N, 15.71%.
4.1.1.3. 3-Amino-7-chloro-9-(40-methylphenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2c. Yield 62%; mp 274–276 ꢀC; IR
(KBr) cmꢃ1: 3460, 3350 (NH), 1445, 1128 (SO2). 1H NMR DMSO-d6:
d
2.42 (s, 3H, CH3), 6.38 (br s, 3H, H8, NH2), 7.19 (d, 1H, H6, J: 8.5 Hz),
0
0
0
0
7.28 (d, 2H, H2 ,6 , J: 7.6 Hz), 7.42 (d, 2H, H3 ,5 , J: 7.6 Hz), 7.93 (d, 1H,
H5, J: 8.5 Hz), 11.60 (br s, 1H, NH); 13C NMR: 21.4, 87.7, 115.0, 120.9,
125.1, 125.4, 129.9, 131.6, 135.2, 137.5, 139.3, 141.1, 146.9, 150.2. Anal.
C16H13ClN4O2S: C, 53.26; H, 3.63; N, 15.53. Found: C, 53.40; H, 3.45;
N, 15.61%.
4.1.1.4. 3-Amino-7-chloro-9-(20,50-dimethylphenyl)-1,9-dihydro-pyr-
azolo-[4,3-b]benzothiazine 4,4-dioxide 2d. Yield 50%; mp > 320 ꢀC;
IR (KBr) cmꢃ1: 3435, 3180 (NH), 1459, 1130 (SO2). 1H NMR DMSO-
d6:
d
1.90 (s, 3H, CH3), 2.33 (s, 3H, CH3), 6.26 (d, 1H, H8, J: 1.5 Hz),
0
6.39 (br s, 2H, NH2), 7.14 (s, 1H, H6 ), 7.22 (dd, 1H, H6, J: 8.7, 1.5 Hz),
0
0
7.28 (d, 1H, H4 , J: 7.8 Hz), 7.38 (d, 1H, H3 , J: 7.8 Hz), 7.95 (d, 1H, H5, J:
8.7 Hz), 11.58 (s, 1H, NH); 13C NMR: 16.8, 20.9, 87.6, 114.2, 121.3,
124.7, 126.0, 130.4, 130.9, 132.2, 133.8, 135.9, 138.0, 138.4, 139.9,
146.9, 147.9. Anal. C17H15ClN4O2S: C, 54.47; H, 4.03; N, 14.95. Found:
C, 54.52; H, 4.19; N, 15.01%.
4. Experimental
4.1. Chemistry
4.1.1.5. 3-Amino-7-chloro-9-(30-methoxyphenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2e. Yield 48%; mp 312–314 ꢀC; IR
(KBr) cmꢃ1: 3312, 3128 (NH), 1499, 1115 (SO2). 1H NMR DMSO-d6:
Melting points were determined on a Thomas micro-hot stage
apparatus and are uncorrected. Infrared spectra were determined
as KBr pellets on a Shimadzu model 470 spectrophotometer. The 1H
NMR and 13C NMR spectra were recorded using a Jeol Eclipse 270
(270 MHz/67.9 MHz) spectrometer using DMSO-d6, and are repor-
ted in parts per million downfield from the residual DMSO.
Elemental analyses were performed on a Perkin Elmer 2400 CHN
analyser, results were within ꢁ0.4% of the predicted values for all
compounds. Chemical reagents were obtained from Aldrich
Chemical Co., USA. All solvents were distilled and dried in the usual
manner. 2,3-Substituted 6 or 7 chloro-N-phenylbenzothiazines 3a–
k and 4a–k were obtained following the method previously
reported [12,14].
0
0
0
d
3.78 (s, 3H, OCH3), 6.41 (br s, 3H, H8, NH2), 7.13 (m, 4H, H6,2 ,4 ,6 ), 7.53
(t, 1H, H5 , J: 8.1 Hz), 7.95 (d, 1H, H5, J: 8.5 Hz), 11.61 (br s, 1H, NH); 13C
NMR: 56.1, 87.7, 114.9, 115.6,115.7, 121.1, 122.0, 125.1, 125.9, 131.8, 137.5,
138.9, 140.7, 147.0, 147.9. Anal. C16H13ClN4O3S: C, 51.00; H, 3.48; N,
14.87. Found: C, 50.89; H, 3.63; N, 15.19%.
0
4.1.1.6. 3-Amino-7-chloro-9-(40-methoxyphenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2f. Yield 67%; mp > 320 ꢀC; IR (KBr)
cmꢃ1: 3344, 3184 (NH), 1469, 1130 (SO2). 1H NMR DMSO-d6:
d
3.84 (s,
3H, OCH3), 6.38 (br s, 2H, NH2), 6.41 (d, 1H, H8, J: 1.8 Hz), 7.15 (m, 3H,
0
0
0
0
H6, 2 ,6 ), 7.33 (d, 2H, H3 ,5 , J: 7.9 Hz), 7.93 (d, 1H, H5, J: 8.2 Hz), 11.60 (br
s, 1H, NH); 13C NMR: 56.0, 87.5, 115.0, 116.2, 120.6, 125.1, 125.9, 130.1,
131.3, 137.4, 141.2, 146.9, 147.9, 160.0. C16H13ClN4O3S: C, 51.00; H, 3.48;
N, 14.87. Found: C, 51.13; H, 3.48; N, 14.83%.
4.1.1. General procedure for the synthesis of 3-amino-6 or 7-chloro-
9-(phenylsubstituted)-1,9-dihydro-pyrazolo-[4,3-b]benzothiazine
4,4-dioxide 2a–t
A mixture of the appropriate benzothiazine (1.3 mmol), hydra-
zine hydrate (1.3 mmol), in dry pyridine (10 mL) was refluxed for
4.1.1.7. 3-Amino-7-chloro-9-(30-chlorophenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2g. Yield 64%; mp 306–308 ꢀC; IR

A. Barazarte et al. / European Journal of Medicinal Chemistry 44 (2009) 1303–1310
1307
(KBr) cmꢃ1: 3390, 3370 (NH), 1489, 1150 (SO2). 1H NMR DMSO-d6:
6.42 (d, 1H, H8, J: 1.5 Hz), 6.43 (br s, 2H, NH2), 7.23 (dd, 1H, H6, J:
dec.; IR (KBr) cmꢃ1: 3345, 3180 (NH), 1459, 1130 (SO2). 1H NMR
DMSO-d6: 1.88 (s, 3H, CH3), 2.32 (s, 3H, CH3), 6.32 (br s, 2H, NH2),
d
d
0
0
8.8,1.5 Hz), 7.65 (m, 4H, Ar), 7.95 (d,1H, H5, J: 8.9 Hz),11.63 (br s,1H,
NH); 13C NMR: 87.6, 114.9, 121.4, 125.4, 126.0, 129.2, 130.7, 132.7,
134.9, 137.5, 140.4, 146.8, 147.9. C15H10Cl2N4O2S: C, 47.26; H, 2.64; N,
14.70. Found: C, 47.17; H, 2.80; N, 14.77%.
6.39 (d, 1H, H8, J: 9.1 Hz), 7.12 (s, 1H, H6 ), 7.26 (d, 1H, H4 , J: 7.8 Hz),
0
7.36 (d, 1H, H3 , J: 7.8 Hz), 7.48 (dd, 1H, H7, J: 9.1, 2.1 Hz), 7.85 (d, 1, H5,
J: 2.1 Hz), 11.73 (s, 1H, NH); 13C NMR: 17.0, 21.0, 87.5, 117.5, 122.8,
125.1, 127.1, 130.0, 130.3, 130.6, 132.2, 132.9, 133.3, 134.1, 140.0, 147.1,
147.5. Anal. C17H15ClN4O2S: C, 54.47; H, 4.03; N, 14.95. Found: C,
54.31; H, 3.91; N, 15.17%.
4.1.1.8. 3-Amino-7-chloro-9-(40-chlorophenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2h. Yield 60%; mp 248–250 ꢀC; IR
(KBr) cmꢃ1: 3444, 3317 (NH), 1499, 1125 (SO2). 1H NMR DMSO-d6:
4.1.1.15. 3-Amino-6-chloro-9-(30-methoxyphenyl)-1,9-dihydro-pyraz
olo-[4,3-b]benzothiazine 4,4-dioxide 2o. Yield 42%; mp 294–296 ꢀC;
IR (KBr) cmꢃ1: 3343, 3175 (NH), 1461, 1113 (SO2). 1H NMR DMSO-d6:
d
6.42 (m, 3H, H8, NH2), 7.19 (dd, 1H, H6, J: 8.4, 2.0 Hz), 7.49 (d, 2H,
0
0
0
0
H2 ,6 , J: 8.6 Hz), 7.69 (d, 2H, H3 ,5 , J: 8.6 Hz), 7.93 (d, 1H, H5, J:
8.4 Hz), 11.65 (br s, 1H, NH); 13C NMR: 87.9, 114.9, 121.5, 125.1, 126.1,
131.3, 132.3, 134.4, 136.4, 137.8, 140.9, 146.8, 147.8. C15H10Cl2N4O2S:
C, 47.26; H, 2.64; N, 14.70. Found: C, 46.95; H, 2.53; N, 14.83%.
d
3.74 (s, 3H, OCH3), 6.47 (br s, 2H, NH2), 6.41 (d, 1H, H8, J: 9.0 Hz),
0
0
0
0
7.18 (m, 3H, H2 ,4 ,6 ), 7.36 (t, 1H, H5 , J: 8.1 Hz), 7.48 (dd, 1H, H7, J: 9.0,
1.8 Hz), 7.91 (d, 1H, H5, J: 1.8 Hz), 11.78 (br s, 1H, NH); 13C NMR: 56.9,
87.7, 118.9, 124.6, 125.7, 128.0, 130.9, 131.3, 131.8, 132.2, 133.1, 133.8,
138.9, 140.7, 147.0, 160.4. Anal. C16H13ClN4O3S: C, 51.00; H, 3.48; N,
14.87. Found: C, 51.12; H, 3.45; N, 14.91%.
4.1.1.9. 3-Amino-7-chloro-9-(30,40-dichlorophenyl)-1,9-dihydro-pyr-
azolo-[4,3-b]benzothiazine 4,4-dioxide 2i. Yield 60%; mp > 320 ꢀC;
IR (KBr) cmꢃ1: 3395, 3399 (NH), 1455, 1124 (SO2). 1H NMR DMSO-
d6:
d
6.43 (br s, 2H, NH2), 6.55 (s, 1H, H8), 7.23 (dd, 1H, H6, J: 8.4,
4.1.1.16. 3-Amino-6-chloro-9-(40-methoxyphenyl)-1,9-dihydro-pyraz
olo-[4,3-b]benzothiazine 4,4-dioxide 2p. Yield 63%; mp 300 ꢀC dec.;
IR (KBr) cmꢃ1: 3344, 3184 (NH), 1469, 1130 (SO2). 1H NMR DMSO-
0
0
0
2.0 Hz), 7.50 (d, 1H, H6 , J: 8.4 Hz), 7.89 (m, 2H, H2 ,5 ), 7.95 (d, 1H, H5,
J: 8.4 Hz), 11.65 (br s, 1H, NH); 13C NMR: 87.6, 115.1, 121.6, 125.5,
126.0, 130.9, 132.8, 133.9, 133.1, 137.6, 137.6, 140.4, 147.0, 147.9.
C15H9Cl3N4O2S: C, 43.34; H, 2.18; N, 13.48. Found: C, 43.51; H, 2.33;
N, 13.61%.
d6:
d 3.83 (s, 3H, OCH3), 6.37 (br s, 2H, NH2), 6.54 (d, 1H, H8, J:
0
0
0
0
9.4 Hz), 7.13 (d, 2H, H 2 ,6 , J: 8.7 Hz), 7.31 (d, 2H, H3 ,5 , J: 8.7 Hz), 7.47
(dd, 1H, H7, J: 9.4, 2.0 Hz), 7.83 (d, 1H, H5, J: 2.0 Hz), 11.59 (br s, 1H,
NH); 13C NMR: 56.1, 87.5, 115.0, 116.11, 18.2, 122.6, 124.6, 127.2, 131.2,
133.0,135.1,138.9, 146.9, 148.3,159.9. Anal. C16H13ClN4O3S: C, 51.00;
H, 3.48; N, 14.87. Found: C, 51.07; H, 3.41; N, 14.73%.
4.1.1.10. 3-Amino-7-chloro-9-(40-bromophenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2j. Yield 58%; mp > 320 ꢀC; IR (KBr)
cmꢃ1: 3367, 2922 (NH), 1499, 1122 (SO2). 1H NMR DMSO-d6:
d
6.48
4.1.1.17. 3-Amino-6-chloro-9-(30-chlorophenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2q. Yield 54%; mp 188–190 ꢀC; IR
(KBr) cmꢃ1: 3390, 3370 (NH), 1489, 1150 (SO2). 1H NMR DMSO-d6:
0
0
0
(m, 3H, H8, NH2), 7.22 (dd, 1H, H6, J: 8.7, 1.9 Hz), 7.49 (d, 2H, H2 ,6 , J:
0
0
8.4 Hz), 7.81 (d, 2H, H3 ,5 , J: 8.4 Hz), 7.94 (d,1H, H5, J: 8.7 Hz),11.63 (br
s,1H, NH); 13C NMR: 87.7, 114.9,121.5,123.0, 125.1, 125.9,132.6, 134.2,
136.9, 137.7, 140.4, 146.9, 147.9. C15H10BrClN4O2S: C, 42.32; H, 2.37; N,
13.16. Found: C, 42.35; H, 2.41; N, 13.23%.
d
6.19 (br s, 2H, NH2), 6.79 (d, 1H, H8, J: 9.1 Hz), 7.18 (s, 1H, H6 ), 7.66
0
0
0
(dd, 1H, H7, J: 7.1, 2.0 Hz), 7.68 (m, 3H, H2 ,4 ,5 ), 8.25 (d, 1H, H5, J:
2.0 Hz), 11.10 (br s, 1H, NH); 13C NMR: 87.5, 119.8, 122.0, 124.0,
127.2, 130.3, 130.8, 133.8, 134.0, 133.8, 134.5, 141.6, 143.7.
C15H10Cl2N4O2S: C, 47.26; H, 2.64; N, 14.70. Found: C, 47.23; H, 2.68;
N, 14.77%.
4.1.1.11. 3-Amino-6-chloro-9-phenyl-1,9-dihydro-pyrazolo-[4,3-
b]benzothiazine 4,4-dioxide 2k. Yield 60%; mp > 320 ꢀC; IR (KBr)
cmꢃ1: 3317, 2935 (NH), 1497, 1115 (SO2). 1H NMR DMSO-d6:
d
6.42
0
0
(br s, 2H, NH2), 6.49 (d, 1H, H8, J: 9.1 Hz), 7.41 (d, 2H, H3 ,6 , J: 7.4 Hz),
7.65 (m, 4H, H7,2 ,4 ,6 ), 7.85 (d, 1H, H5, J: 2.1 Hz), 11.61 (br s, 1H, NH);
13C NMR: 87.6, 118.2, 122.7, 124.9, 127.3, 129.6, 130.3, 131.0, 133.0,
135.3, 138.5, 145.9, 147.1. Anal. C15H11ClN4O2S: C, 51.95; H, 3.20; N,
16.16. Found: C, 51.73; H, 3.19; N, 16.25%.
4.1.1.18. 3-Amino-6-chloro-9-(40-chlorophenyl)-1,9-dihydro-pyrazolo-
0
0
0
[4,3-b]benzothiazine 4,4-dioxide 2r. Yield 60%; mp > 320 ꢀC; IR (KBr)
cmꢃ1: 3444, 3317 (NH), 1499, 1125 (SO2). 1H NMR DMSO-d6:
d
6.41
0
0
(br s, 2H, NH2), 6.55 (d,1H, H8, J: 9.2, Hz), 7.48 (m, 3H, H7,2 ,6 ), 7.67 (d,
2H, H3 ,5 , J: 8.2 Hz), 7.85 (d, 1H, H5, J: 2.0 Hz), 11.64 (br s, 1H, NH); 13
C
0
0
4.1.1.12. 3-Amino-6-chloro-9-(20-methylphenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2l. Yield 56%; mp 294 ꢀC dec; IR
(KBr) cmꢃ1: 3450, 3360 (NH), 1440, 1110 (SO2). 1H NMR DMSO-d6:
NMR: 87.5, 118.2, 122.7, 125.3, 127.4, 131.0, 132.4, 133.1, 134.1, 136.9,
138.3, 146.9, 147.9. C15H10Cl2N4O2S: C, 47.26; H, 2.64; N,14.70. Found:
C, 47.43; H, 2.82; N, 14.73%.
d
1.94 (s, 3H, CH3), 6.37 (d, 1H, H8, J: 9.4 Hz), 6.40 (br s, 2H, NH2), 7.31
(d, 1H, H6 , J: 6.9 Hz), 7.47 (m, 4H, H7,3 ,4 ,5 ), 7.86 (d, 1H, H5, J: 1.9 Hz),
11.59 (br s, 1H, NH); 13C NMR: 16.8, 87.3, 117.4, 123,0, 125.0, 128.6,
130.8, 132.4, 133.4, 136.6, 136.9, 137.5, 137.6, 138.0, 142.6, 147.0. Anal.
C16H13ClN4O2S: C, 53.26; H, 3.63; N,15.53. Found: C, 52.96; H, 3.62; N,
15.49%.
4.1.1.19. 3-Amino-6-chloro-9-(30,40-dichlorophenyl)-1,9-dihydro-pyr-
azolo-[4,3-b]benzothiazine 4,4-dioxide 2s. Yield 58%; mp 218–
220 ꢀC; IR (KBr) cmꢃ1: 3367, 2922 (NH), 1499, 1122 (SO2). 1H NMR
0
0
0
0
DMSO-d6:
d 6.28 (br s, 2H, NH2), 7.25 (d, 1H, H8, J: 9.2 Hz), 7.35 (d,
0
0
0
1H, H6 , J: 8.9 Hz), 7.72 (m, 3H, H7,2 ,5 ), 8.24 (d, 1H, H5, J: 2.3 Hz),
11.67 (br s, 1H, NH); 13C NMR: 86.3, 117.5, 117.0, 121.1, 130.4, 131.0,
131.5, 132.5, 132.7, 134.0, 134.1, 134.3, 134.9, 135.2, 141.6, 147.6.
C15H9Cl3N4O2S: C, 43.34; H, 2.18; N, 13.48. Found: C, 43.29; H, 2.27;
N, 13.60%.
4.1.1.13. 3-Amino-6-chloro-9-(40-methylphenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2m. Yield 67%; mp 300 ꢀC dec.; IR
(KBr) cmꢃ1: 3460, 3350 (NH), 1445, 1128 (SO2). 1H NMR DMSO-d6:
d
2.40 (s, 3H, CH3), 6.35 (br s, 2H, NH2), 6.51 (d, 1H, H8, J: 9.4 Hz), 7.27
(d, 2H, H2 ,6 , J: 7.9 Hz), 7.42 (d, 2H, H3 ,5 , J: 7.9 Hz), 7.47 (dd, 1H, H7, J:
9.4, 1.7 Hz), 7.84 (d, 1H, H5, J: 1.7 Hz), 11.62 (br s, 1H, NH); 13C NMR:
21.4, 88.0, 118.2, 122.6, 124.7, 127.2, 130.0, 131.5, 133.0, 135.4, 138.6,
139.1, 145.4, 149.0. Anal. C16H13ClN4O2S: C, 53.26; H, 3.63; N, 15.53.
Found: C, 53.18; H, 3.65; N, 15.68%.
4.1.1.20. 3-Amino-6-chloro-9-(40-bromophenyl)-1,9-dihydro-pyrazolo-
[4,3-b]benzothiazine 4,4-dioxide 2t. Yield 53%; mp 304–306 ꢀC; IR
(KBr) cmꢃ1: 3383, 3117 (NH), 1463, 1117 (SO2). 1H NMR DMSO-d6:
0
0
0
0
0 0
d
6.35 (br s, 2H, NH2), 7.23 (d, 1H, H8, J: 9.1 Hz), 7.40 (m, 3H, H7,2 ,6 ),
0
0
7.93 (d, 2H, H3 ,5 , J: 8.4 Hz), 8.19 (d, 1H, H5, J: 2.0 Hz), 11.69 (br s, 1H,
NH); 13C NMR: 87.3, 118.5,121.7, 129.0, 131.3, 131.9, 132.8, 135.6, 136.9,
137.9, 142.3, 145.8, 148.9. C15H10BrClN4O2S: C, 42.32; H, 2.37; N,13.16.
Found: C, 42.60; H, 2.35; N, 12.97%.
4.1.1.14. 3-Amino-6-chloro-9-(20,50-dimethylphenyl)-1,9-dihydro-pyr-
azolo-[4,3-b]benzothiazine 4,4-dioxide 2n. Yield 50%; mp 302 ꢀC

1308
A. Barazarte et al. / European Journal of Medicinal Chemistry 44 (2009) 1303–1310
4.1.2. General procedure for the synthesis of 2,4-diamino-7 or 8-
chloro-10-(phenylsubstituted)-pyrimido-[5,4-b]benzothiazine 5,5-
dioxide 3a–t
124.5, 124.7, 130.9, 131.5, 137.9, 140.6, 157.3, 159.7, 160.7. Anal.
17H14ClN5O3S: C, 50.56; H, 3.49; N, 17.34. Found: C, 50.72; H, 3.40; N,
17.62%.
C
A mixture of the appropriate benzothiazine (1.3 mmol), guani-
dine hydrochloride (1.3 mmol), and potassium carbonate
(1.6 mmol) in dry DMF 10 mL was refluxed for 5 h. The solvent was
evaporated to dryness under reduced pressure, water-ice was
added (10 mL) and the solid thus obtained was collected by filtra-
tion. Further purification was accomplished by recrystallization
from ethanol–water (4/1).
4.1.2.7. 2,4-Diamino-8-chloro-10H-(30-chlorophenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3g. Yield 43%; mp > 320 ꢀC; IR (KBr)
cmꢃ1: 3473 (NH2), 1488, 1126 (SO2). 1H NMR DMSO-d6:
d 6.65 (d,
1H, H9, J: 1.7 Hz), 6.75 (br s, 2H, NH2), 7.43–7.55 (m, 3H, Ar), 7.66–
7.70 (m, 1H, H4 ), 7.80 (d, 1H, H6, J: 8.7 Hz); 13C NMR: 88.4, 117.5,
0
123.7, 124.5, 125.6, 129.4, 129.9, 130.9, 132.4, 134.7, 137.9, 140.0,
157.1, 160.7, 162.8. Anal. C16H11Cl2N5O2S: C, 47.07; H, 2.72; N, 17.15.
Found: C, 46.93; H, 2.77; N, 17.31%.
4.1.2.1. 2,4-Diamino-8-chloro-10H-phenyl-pyrimido-[5,4-b]benzothi
azine 5,5-dioxide 3a. Yield 59%; mp > 320 ꢀC; IR (KBr) cmꢃ1: 3480
(NH2), 1450, 1139 (SO2). 1H NMR DMSO-d6:
d
6.30 (d, 1H, H9, J:
4.1.2.8. 2,4-Diamino-8-chloro-10H-(40-chlorophenyl)-pyrimido-[5,4-
2.0 Hz), 6.64 (br s, 2H, NH2), 7.41 (dd, 1H, H7, J: 8.5, 2.1 Hz), 7.66 (m,
5H, Ar), 7.99 (d, 1H, H6, J: 8.4 Hz); 13C NMR: 88.3, 117.6, 123.6, 124.7,
124.9, 129.7, 130.5, 130.9, 138.4, 157.2, 160.8, 162.8. Anal.
C16H12ClN5O2S: C, 51.41; H, 3.24; N, 18.73. Found: C, 51.37; H, 3.19;
N, 18.96%.
b]benzothiazine 5,5-dioxide 3h. Yield 51%; mp 236–238 ꢀC; IR (KBr)
cmꢃ1: 3475 (NH2),1415,1129 (SO2). 1H NMR DMSO-d6:
d
6.39 (d,1H,
H9, J: 1.5 Hz), 6.70 (br s, 2H, NH2), 7.41 (dd, 1H, H7, J: 8.7, 1.5 Hz), 7.43
0
0
0
0
(d, 2H, H2 ,6 , J: 8.4 Hz), 7.68 (d, 2H, H3 ,5 , J: 8.4 Hz), 7.80 (d, 1H, H6, J:
8.7 Hz); 13C NMR: 88.4, 117.5, 123.7, 124.7, 124.9, 130.9, 132.6, 134.2,
137.3, 138.0, 140.2, 157.2, 160.8, 162.8. Anal. C16H11Cl2N5O2S: C,
47.07; H, 2.72; N, 17.15. Found: C, 47.03; H, 2.75; N, 17.28%.
4.1.2.2. 2,4-Diamino-8-chloro-10H-(20-methylphenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3b. Yield 63%; mp 256–258 ꢀC; IR (KBr)
cmꢃ1: 3472 (NH2), 1450, 1139 (SO2). 1H NMR DMSO-d6:
d
1.95 (s, 3H,
4.1.2.9. 2,4-Diamino-8-chloro-10H-(30,40-dichlorophenyl)-pyrimido[5,
4-b]benzothiazine 5,5-dioxide 3i. Yield 57%; mp 242–244 ꢀC; IR
CH3), 6.26 (d,1H, H9, J: 1.9 Hz), 6.66 (br s, 2H, NH2), 7.48–7.24 (m, 5H,
H7,3 ,4 ,5 ,6 ), 8.01 (d, 1H, H6, J: 8.2 Hz); 13C NMR: 17.4, 88.3, 116.8,
123.8,124.7,124.9,128.4,129.9,130.6,132.2,137.0,137.2,138.3,139.5,
156.7, 160.8, 163.1. Anal. C17H14ClN5O2S: C, 52.65; H, 3.64; N, 18.06.
Found: C, 52.83; H, 3.49; N, 18.20%.
(KBr) cmꢃ1: 3460 (NH2), 1469, 1126 (SO2). 1H NMR DMSO-d6:
d 6.50
0
0
0
0
0
(d,1H, H9, J: 2.0 Hz), 6.74 (br s, 2H, NH2), 7.41–7.44 (m, 2H, H7,6 ), 7.85
0
0
(d, 1H, H2 , J: 1.7 Hz), 7.88 (d, 1H, H5 , J: 8.4 Hz), 8.00 (d, 1H, H6, J:
8.7 Hz); 13C NMR: 88.4, 117.5, 123.9, 124.7, 124.8, 131.2, 132.6, 132.9,
133.1, 138.1, 138.3, 139.8, 147.5, 157.0, 160.7, 162.7. Anal.
C16H10Cl3N5O2S: C, 43.41; H, 2.28; N, 15.82. Found: C, 43.40; H, 2.32;
N, 15.91%.
4.1.2.3. 2,4-Diamino-8-chloro-10H-(40-methylphenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3c. Yield 69%; mp > 320 ꢀC; IR (KBr)
cmꢃ1: 3480 (NH2), 1455, 1130 (SO2). 1H NMR DMSO-d6:
d 2.42 (s, 3H,
CH3), 6.35 (d, 1H, H9, J: 1.5 Hz), 6.66 (br s, 2H, NH2), 7.24 (d, 2H, H2 ,6
,
4.1.2.10. 2,4-Diamino-8-chloro-10H-(40-bromophenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3j. Yield 65%; mp 238–240 ꢀC; IR (KBr)
0
0
0
0
J: 8.0 Hz), 7.37 (dd, 1H, H7, J: 8.4, 1.5 Hz), 7.42 (d, 2H, H3 ,5 , J: 8.0 Hz),
7.98 (d, 1H, H6, J: 8.4 Hz); 13C NMR: 21.4, 88.3, 117.7, 123.5, 124.6,
124.7, 130.2, 131.4, 135.8, 137.9, 139.1, 140.6, 157.3, 160.8, 162.8. Anal.
C17H14ClN5O2S: C, 52.65; H, 3.64; N, 18.06. Found: C, 52.71; H, 3.84;
N, 18.17%.
cmꢃ1: 3472 (NH2), 1459, 1120 (SO2). 1H NMR DMSO-d6:
d
6.39 (d, 1H,
0
0
H9, J: 1.8 Hz), 6.70 (br s, 2H, NH2), 7.37 (d, 2H, H2 ,6 , J: 8.4 Hz), 7.41 (d,
0
0
1H, H7, J: 8.2 Hz), 7.82 (d, 2H, H3 ,5 , J: 8.4 Hz), 7.99 (d, 1H, H6, J:
8.2 Hz); 13C NMR: 88.4, 117.5, 122.9, 123.7, 124.7, 124.9, 132.9, 133.8,
137.8, 138.0, 140.2, 157.1, 160.7, 162.7. Anal. C16H11BrClN5O2S: C, 42.45;
H, 2.45; N, 15.47. Found: C, 42.63; H, 2.39; N, 15.60%.
4.1.2.4. 2,4-Diamino-8-chloro-10H-(20,50-dimethylphenyl)-pyrimido-
[5,4-b]benzothiazine 5,5-dioxide 3d. Yield 49%; mp 222–224 ꢀC; IR
(KBr) cmꢃ1: 3470 (NH2), 1472, 1133 (SO2). 1H NMR DMSO-d6:
d
1.90
4.1.2.11. 2,4-Diamino-7-chloro-10H-phenyl-pyrimido-[5,4-b]benzo-
(s, 3H, CH3), 2.35 (s, 3H, CH3), 6.28 (d, 1H, H9, J: 1.8 Hz), 6.70 (br s,
thiazine 5,5-dioxide 3k. Yield 49%; mp 268–270 ꢀC; IR (KBr) cmꢃ1
:
2H, NH2), 7.11 (s,1H, H6 ), 7.27 (d,1H, H4 , J: 7.3 Hz), 7.37 (d,1H, H3 , J:
3480 (NH2), 1450, 1139 (SO2). 1H NMR DMSO-d6:
d 6.44 (d, 1H, H9, J:
0
0
0
7.3 Hz), 7.41 (dd, 1H, H7, J: 8.4, 1.9 Hz), 8.01 (d, 1H, H6, J: 8.4 Hz); 13
C
8.9 Hz), 6.67 (br s, 2H, NH2), 7.36 (d, 2H, H2 ,6 , J: 7.4 Hz), 7.36 (dd,
1H, H8, J: 8.8,1.9 Hz), 7.56–7.62 (m, 3H, Ar), 7.94 (d,1H, H6, J: 1.9 Hz);
13C NMR: 87.9, 120.7, 121.4, 127.5, 129.5, 130.5, 130.7, 133.5, 138.2,
138.6, 157.1, 160.8, 162.8. Anal. C16H12ClN5O2S: C, 51.41; H, 3.24; N,
18.73. Found: C, 51.23; H, 3.29; N, 18.80%.
0
0
NMR: 17.1, 21.0, 88.3, 116.9, 123.7, 124.7, 124.9, 130.5, 130.7, 132.0,
135.5, 137.0, 137.8, 138.2, 139.5, 160.0, 160.9, 163.2. Anal.
C18H16ClN5O2S: C, 53.80; H, 4.01; N, 17.43. Found: C, 54.05; H, 4.13;
N, 17.52%.
4.1.2.5. 2,4-Diamino-8-chloro-10H-(30-methoxyphenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3e. Yield 63%; mp > 320 ꢀC; IR (KBr)
4.1.2.12. 2,4-Diamino-7-chloro-10H-(20-methylphenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3l. Yield 57%; mp 244–246 ꢀC; IR (KBr)
cmꢃ1: 3472 (NH2), 1488, 1126 (SO2). 1H NMR DMSO-d6:
d
3.80 (s, 3H,
cmꢃ1: 3472 (NH2), 1450, 1130 (SO2). 1H NMR DMSO-d6:
d 1.95 (s, 3H,
0
OCH3), 6.38 (d,1H, H9, J: 1.9 Hz), 6.70 (br s, 2H, NH2), 6.92 (d,1H, H4 , J:
CH3), 6.38 (d, 1H, H9, J: 9.2 Hz), 6.65 (br s, 2H, NH2), 7.24–7.48 (m, 4H,
Ar), 7.56 (dd,1H, H8, J: 9.2, 2.0 Hz), 7.94 (d,1H, H6, J: 1.9 Hz); 13C NMR:
19.1, 87.9, 119.9, 121.5, 127.1, 127.7, 127.9, 128.3, 129.8, 130.6, 132.1,
133.8, 137.1, 137.4, 156.6, 160.8, 163.1. Anal. C17H14ClN5O2S: C, 52.65;
H, 3.64; N, 18.06. Found: C, 52.67; H, 3.71; N, 18.15%.
0
0
8.2 Hz), 6.97 (s, 1H, H2 ), 7.12 (d, 1H, H6 , J: 8.2 Hz), 7.39 (d, 1H, H7, J:
8.1 Hz), 7.53 (t, 1H, H5 , J: 8.2 Hz); 13C NMR: 56.0, 88.3, 115.4, 116.1,
0
117.7, 122.4, 123.5, 124.5, 124.7, 131.5, 137.9, 139.6, 140.3, 157.1, 160.8,
161.2, 162.9. Anal. C17H14ClN5O3S: C, 50.56; H, 3.49; N,17.34. Found: C,
50.61; H, 3.59; N, 17.46%.
4.1.2.13. 2,4-Diamino-7-chloro-10H-(40-methylphenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3m. Yield 59%; mp 288–290 ꢀC; IR (KBr)
4.1.2.6. 2,4-Diamino-8-chloro-10H-(40-methoxyphenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3f. Yield 68%; mp 262–264 ꢀC; IR (KBr)
cmꢃ1: 3480 (NH2), 1455, 1130 (SO2). 1H NMR DMSO-d6:
d 2.41 (s, 3H,
cmꢃ1: 3490 (NH2), 1456, 1125 (SO2). 1H NMR DMSO-d6:
d
3.85 (s, 3H,
CH3), 6.47 (d, 1H, H9, J: 8.9 Hz), 6.66 (br s, 2H, NH2), 7.21 (d, 2H, H2 ,6
,
0
0
0
0
0
0
OCH3), 6.40 (d, 1H, H9, J: 2.0 Hz), 6.65 (br s, 2H, NH2), 7.14 (d, 2H, H2 ,6
,
J: 7.9 Hz), 7.40 (d, 2H, H3 ,5 , J: 7.9 Hz), 7.56 (dd, 1H, H8, J: 8.9, 2.0 Hz),
7.94 (d, 1H, H6, J: 1.9 Hz); 13C NMR: 21.2, 87.8, 120.8, 121.4, 126.9,
128.9, 127.4, 130.3, 131.2, 133.5, 136.1, 138.3, 157.3, 160.8, 162.8. Anal.
0
0
J: 8.6 Hz), 7.27 (d, 2H, H3 ,5 , J: 8.7 Hz), 7.38 (dd, 1H, H7, J: 8.4, 2.0 Hz),
7.97 (d, 1H, H6, J: 8.4 Hz); 13C NMR: 55.9, 88.3, 115.9, 116.7, 117.7, 123.5,

A. Barazarte et al. / European Journal of Medicinal Chemistry 44 (2009) 1303–1310
1309
C17H14ClN5O2S: C, 52.65; H, 3.64; N, 18.06. Found: C, 52.73; H, 3.72;
N, 18.37%.
2.0 Hz); 13C NMR: 88.3, 120.5, 121.7, 127.8, 128.4, 130.9, 131.4, 131.9,
134.7, 136.9, 137.9, 138.5, 142.3, 155.9, 161.2, 163.0. Anal.
C16H11BrClN5O2S: C, 42.45; H, 2.45; N,15.47. Found: C, 42.51; H, 2.63;
N, 15.75%.
4.1.2.14. 2,4-Diamino-7-chloro-10H-(20,50-dimethylphenyl)-pyrimido-
[5,4-b]benzothiazine 5,5-dioxide 3n. Yield 47%; mp 242–244 ꢀC; IR
(KBr) cmꢃ1: 3470 (NH2), 1472, 1133 (SO2). 1H NMR DMSO-d6:
d
1.89
4.2. X-ray crystallography
(s, 3H, CH3), 2.34 (s, 3H, CH3), 6.40 (d,1H, H9, J: 9.2 Hz), 6.68 (br s, 2H,
0
0
NH2), 7.08 (d, 1H, H6 , J: 2.1 Hz), 7.25 (d, 1H, H4 , J: 7.7 Hz), 7.36 (d, 1H,
0
Crystals of 3p suitable for X-ray diffraction were obtained by
slow evaporation of a solution in ethanol. Crystal data, intensity
data collection parameters and final refinement results are sum-
marised in Table 3.
Unit cell and intensity measurements were carried out on
a Bruker Smart CCD area-detector diffractometer, using graphite-
H3 , J: 7.7 Hz), 7.59 (dd, 1H, H8, J: 9.2, 2.0 Hz), 7.94 (d, 1H, H6, J:
2.0 Hz); 13C NMR: 17.2, 20.9, 87.8, 120.1, 121.5, 127.1, 127.5, 130.5,
130.6, 131.9, 132.0, 133.9, 134.0, 137.7, 156.9, 160.8, 163.1. Anal.
C18H16ClN5O2S: C, 53.80; H, 4.01; N, 17.43. Found: C, 53.77; H, 4.23;
N, 17.66%.
monochromated Mo K
a
radiation (
l
¼ 0.71073 Å). The structure
4.1.2.15. 2,4-Diamino-7-chloro-10H-(30-methoxyphenyl)-pyrimido-[5,
4-b]benzothiazine5,5-dioxide 3o. Yield 66%; mp 287–288 ꢀC; IR (KBr)
was solved by direct methods and refined on F2 by full-matrix least-
squares, using all reflections, anisotropic displacement parameters
cmꢃ1: 3490 (NH2), 1456, 1125 (SO2). 1H NMR DMSO-d6:
d 3.87 (s, 3H,
and weights w ¼ [
s
2(Fo2) þ (aP)2 þ bP]ꢃ1, with P ¼ (Fo2 þ 2F2c)/3. The
OCH3), 6.42 (d,1H, H9, J: 9.2 Hz), 6.66 (br s, 2H, NH2), 7.23 (m, 3H, Ar),
C-bonded H atoms were placed in calculated positions, and refined
using a riding atom model with fixed C–H [0.93 Å for C(sp2) and
0.96 Å for C(sp3)] distances, and Uiso ¼ pUeq(parent atom) [p ¼ 1.2
for C(sp2) and 1.5 for C(sp3)]. The N-bonded H atoms were located
in difference Fourier syntheses and refined isotropically.
0
7.42 (t,1H, H5 , J: 8.1 Hz), 7.58 (dd,1H, H8, J: 9.1,1.9 Hz), 7.92 (d,1H, H6,
J: 1.9 Hz); 13C NMR: 55.9, 88.5, 118.9, 120.6, 123.4, 128.8, 131.1, 131.3,
131.7, 133.5, 137.8, 156.7, 159.7, 161.0, 162.8. Anal. C17H14ClN5O3S: C,
50.56; H, 3.49; N, 17.34. Found: C, 50.62; H, 3.71; N, 17.53%.
The following computer programs were used: data collection,
SMART [15]; data reduction and cell refinement, SAINT [16];
absorption correction, SADABS [17]; structure solution, SHELXS-97
[18]; structure refinement, SHELXL-97 [19]; geometrical calcula-
tions, PLATON [20]; molecular graphics, ORTEP-3 [21]. The struc-
ture solution, the refinement and the drawings were carried out
with the aid of the WinGX [22] suite of programs.
Comprehensive crystallographic data (excluding structure
factors) for the structural analysis of 3p have been deposited with
the Cambridge Crystallographic Data Centre. Copies of the data (CIF
file) can be obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK, fax: þ44-(0)1223-336033, or
from http://www.ccdc.cam.ac.uk/products/csd/request/, quoting
deposition No. CCDC 685720.
4.1.2.16. 2,4-Diamino-7-chloro-10H-(40-methoxyphenyl)-pyrimido-[5,
4-b]benzothiazine5,5-dioxide 3p. Yield 62%; mp 276–378 ꢀC; IR (KBr)
cmꢃ1: 3496 (NH2), 1463, 1137 (SO2). 1H NMR DMSO-d6:
d
3.67 (s, 3H,
OCH3), 6.41 (d, 1H, H9, J: 9.2 Hz), 6.53 (br s, 2H, NH2), 7.17 (d, 2H,
0
0
0
0
H2 ,6 , J: 8.7 Hz), 7.25 (d, 2H, H3 ,5 , J: 8.7 Hz), 7.56 (dd, 1H, H8, J: 9.2,
1.7 Hz), 7.91 (d, 1H, H6, J: 1.7 Hz); 13C NMR: 55.9, 87.9, 115.8, 120.3,
121.3, 126.9, 127.3, 131.1, 131.5, 133.5, 137.1, 157.4, 159.7, 160.8, 162.9.
Anal. C17H14ClN5O3S: C, 50.56; H, 3.49; N, 17.34. Found: C, 50.45; H,
3.62; N, 17.41%.
4.1.2.17. 2,4-Diamino-7-chloro-10H-(30-chlorophenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3q. Yield 43%; mp 200 ꢀC dec.; IR (KBr)
cmꢃ1: 3472 (NH2), 1488,1126 (SO2). 1H NMR DMSO-d6:
d 6.45 (d,1H,
H9, J: 8.9 Hz), 6.69 (br s, 2H, NH2), 7.35–7.43 (m, 3H, H8, Ar), 7.60–
7.66 (m, 2H, Ar), 7.90 (d, 1H, H6, J: 1.7 Hz); 13C NMR: 87.6,119.8,121.1,
127.5, 128.5, 128.6, 129.4, 132.9, 133.1, 135.6, 137.1, 137.6, 157.1, 161.0,
162.4. Anal. C16H11Cl2N5O2S: C, 47.07; H, 2.72; N, 17.15. Found: C,
47.26; H, 2.73; N, 17.29%.
4.3. Biological assays
4.3.1. Inhibition of heme polymerization
The heme polymerization assay was performed according to
Ref. [23], briefly, a solution of hemin chloride (50 mL, 4 mM), dis-
solved in DMSO (5.2 mg/mL), was distributed in 96-well micro
4.1.2.18. 2,4-Diamino-7-chloro-10H-(40-chlorophenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3r. Yield 48%; mp 262–264 ꢀC; IR (KBr)
cmꢃ1: 3475 (NH2),1468,1129 (SO2). 1H NMR DMSO-d6:
d
6.52 (d, 1H,
plates. Different concentrations (100ꢃ5 mM) of the compounds
0
0
H9, J: 9.1 Hz), 6.68 (br s, 2H, NH2), 7.40 (d, 2H, H2 ,6 , J: 8.4 Hz), 7.56
dissolved in DMSO, were added in triplicate in test wells (50 mL).
0
0
(dd, 1H, H8, J: 9.1, 1.7 Hz), 7.67 (d, 2H, H3 ,5 , J: 8.4 Hz), 7.94 (d, 1H, H6,
J: 1.7 Hz); 13C NMR: 88.2, 120.7, 121.5, 127.1, 127.5, 127.6, 130.8, 132.6,
133.6,134.0,137.8,157.2, 160.8,162.8. Anal. C16H11Cl2N5O2S: C, 47.07;
H, 2.72; N, 17.15. Found: C, 47.22; H, 3.01; N, 17.43%.
Controls contained either water (50
Hematin formation was initiated by the addition of acetate buffer
(100
L 0.2 M, pH 4.4). The plates were incubated at 37 ꢀC for 48 h
mL) or DMSO (50 mL). b-
m
to allow for completion of the reaction and centrifuged
(4000 rpm ꢂ 15 min, IEC-CENTRA, MP4R). After discarding the
4.1.2.19. 2,4-Diamino-7-chloro-10H-(30,40-dichlorophenyl)-pyrimido-
[5,4-b]benzothiazine 5,5-dioxide 3s. Yield 45%; mp 316 ꢀC dec.; IR
supernatant, the pellet was washed twice with DMSO (200 mL) and
finally, dissolved in NaOH (200 mL, 0.2 N). The solubilized aggre-
(KBr) cmꢃ1: 3472 (NH2), 1459, 1120 (SO2). 1H NMR DMSO-d6:
d
6.60
gates were further diluted 1:2 with NaOH (0.1 N) and absorbances
recorded at 405 nm (Microplate Reader, BIORAD-550). The results
were expressed as a percentage of inhibition of flavoprotein (FP)
polymerization.
0
(d, 1H, H9, J: 9.2 Hz), 6.72 (br s, 2H, NH2), 7.41 (dd, 1H, H6 , J: 8.5,
0
2.3 Hz), 7.56 (dd,1H, H8, J: 9.2, 2.0 Hz), 7.82 (d,1H, H2 , J: 2.3 Hz), 7.88
(d, 1H, H5 , J: 8.5 Hz), 7.95 (d, 1H, H6, J: 2.0 Hz); 13C NMR: 88.2, 120.8,
0
121.6, 127.0, 127.7, 128.6, 131.2, 132.4, 132.5, 132.9, 133.1, 133.7, 137.5,
156.9, 160.8, 162.7. Anal. C16H10Cl3N5O2S: C, 43.41; H, 2.28; N, 15.82.
Found: C, 43.55; H, 2.37; N, 16.03%.
4.3.2. Parasite, experimental host and strain maintenance
Male Balb-C mice, weighing 18–22 g were maintained on
a commercial pellet diet and housed under conditions approved by
Ethics Committee. P. berghei (ANKA strain), a rodent malaria para-
site, was used for infection. Mice were infected by ip injection with
1 ꢂ106 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 [24].
4.1.2.20. 2,4-Diamino-7-chloro-10H-(40-bromophenyl)-pyrimido-[5,4-
b]benzothiazine 5,5-dioxide 3t. Yield 52%; mp 267–269 ꢀC; IR (KBr)
cmꢃ1: 3417 (NH2), 1451, 1135 (SO2). 1H NMR DMSO-d6:
d
6.67 (d, 1H,
0
0
H9, J: 9.1 Hz), 6.75 (br s, 2H, NH2), 7.43 (d, 2H, H2 ,6 , J: 8.4 Hz), 7.60
0
0
(dd,1H, H8, J: 9.0,1.9 Hz), 7.67 (d, 2H, H3 ,5 , J: 8.4 Hz), 8.16 (d,1H, H6, J:

1310
A. Barazarte et al. / European Journal of Medicinal Chemistry 44 (2009) 1303–1310
4.3.3. Parasite extracts
5. Data analysis
Blood of infected animals, at a high level of parasitemia (30–
50%), was collected by cardiac puncture with an heparinized
syringe and the blood pool was centrifuged (500 g ꢂ 10 min, 4 ꢀC).
Plasma and buffy coat were removed and the red blood cell (RBC)
pellets were 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, 20,000 g ꢂ 30 min ꢂ 4 ꢀC)
[25]. The upper band (mature forms) was removed by aspiration,
collected in Eppendorf tubes and washed twice with chilled PBS–
glucose and the infected erythrocytes were lysed with the nonionic
detergent saponin (0.1% in PBS ꢂ 10 min). One milliliter of cold PBS
was added and the samples were centrifuged (13,000 g ꢂ 5 min,
4 ꢀC) to remove erythrocyte cytoplasm content (including eryth-
rocyte hemoglobin). The free parasites were mixed PBS–glucose
(5.4%), and subjected to three freeze-thaw cycles (ꢃ70 ꢀC/þ37 ꢀC).
The final homogenate was used in the hemoglobin hydrolysis
inhibition assay [26].
Data were statistically analyzed using one-way ANOVA and t-
tests for specific group comparisons; assuming 95% of confidence
according GraphPad Prism 3.02 [28].
Acknowledgments
We thank the IIF-FF and CDCH-UCV (grants IIF: 03.2006, PG. 06-
00-6502-2006), FONACIT-PCP and CYTED-RIDIMEDCHAG pro-
grammes for financial support.
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4.3.6. 4-Day suppressive test
Balb-C mice (18–23 g) were infected i.v. (using caudal vein) with
106 infected red blood cells with P. berghei (n ¼ 6). Two hours after
infection, treatment began with the best compounds tested in the
in vitro assays. 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 4, the parasitemia was
counted by examination of Giemsa stained smears. Chloroquine
(25 mg/kg) was used as a positive control. The survival time beyond
the control group (without drug treatment) was recorded. The
results were expressed as percentage of parasitemia (% of para-
sitemia) and survival days of each compound-treated group over
the control (non-treated group) [27].
[27] W. Peters, B.L. Robinson, Parasitic Infection Models, in: O. Zak, M. Sande (Eds.),
Handbook of Antimalarial Models of Infection, Academic Press, London, 1999,
p. 757.
[28] Graph Pad Prism Software Inc. 4.02 for Windows. May 17th 1992–2004.