Design and synthesis of pyrimido[4,5-b][1,4]benzothiazine derivatives, as potent 15-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.12.022

A group of 2-substituted pyrimido[4,5-b][1,4]benzothiazines were designed, synthesized, and evaluated as potential inhibitors of 15-lipoxygenase (15-LO). Compounds 4d and 4e showed the best IC₅₀ of 15-LO inhibition (IC₅₀ = 18 and 34

Full text of DOI:10.1016/j.bmc.2006.12.022

Bioorganic & Medicinal Chemistry 15 (2007) 2120–2126
Design and synthesis of pyrimido[4,5-b][1,4]benzothiazine
derivatives, as potent 15-lipoxygenase inhibitors
M. Bakavoli,a,* M. Nikpour,a M. Rahimizadeh,a M. R. Saberib and H. Sadeghiana
aDepartment of Chemistry, School of Sciences, Ferdowsi University, Mashhad 91775-1436, Iran
bDepartment of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad 91775-1365, Iran
Received 13 November 2006; revised 11 December 2006; accepted 12 December 2006
Available online 14 December 2006
Abstract—A group of 2-substituted pyrimido[4,5-b][1,4]benzothiazines were designed, synthesized, and evaluated as potential
inhibitors of 15-lipoxygenase (15-LO). Compounds 4d and 4e showed the best IC50 of 15-LO inhibition (IC50 = 18 and 34 lM,
respectively). All compounds were docked into 15-LO. As a result the sulfur atom was oriented toward the iron atom of the
active site of 15-LO. We suggest the interaction of the iron atom is essential for the activity of the inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
sclerotic lesion.5 In addition, the immediate products of
15-LO oxidation of AA and linoleic acid (LA) have been
shown to be pro-inflammatory10 and pro-thrombotic.11
Our interest in pyrimido[4,5-b][1,4]benzothiazines as
lipoxygenase inhibitors emerges from the work of Hayuk-
awa and co-workers, as they reported lipoxygenase inhi-
bition by this class of heterocyclic compounds.1 It is
well documented that mammalian lipoxygenases (LOs)
are non-heme iron-containing enzymes responsible for
the oxidation of polyunsaturated fatty acids and esters
to hydroperoxy derivatives.2 There are heterogeneous
family of enzymes distributed widely throughout the
plant and animal kingdoms,3 and named according to
the position at which a key substrate, arachidonic acid
(AA), is oxidized. Among the mammalian lipoxygenases
involved in the etiology of human disease, 5-lipoxygenase
(5-LO) is now well established as a target for reducing the
production of leukotrienes (important in particular asth-
ma).4 More recently, 15-lipoxygenase (15-LO) has
emerged as an attractive target for therapeutic interven-
tion.5 15-LO has been implicated in the progression of cer-
tain cancers6 and chronic obstructive pulmonary disease
(COPD).7 Evidence for the inhibition of 15-LO in the
treatment of vascular disease is, however, most compel-
ling.8 Both transgenic and knockout studies implicate a
role for 15-LO in atherogenesis.9 The enzyme is abundant-
ly expressed in macrophages residing within the athero-
It is also found that 15-LO is linked to cardiovascular
complications since it is known to participate in oxida-
tive modification of low-density lipoproteins (LDL)
leading to the development of atherosclerosis.12
Three different strategies have been developed to inhibit
the LO’s pathway.13 They involve (i) redox inhibitors or
antioxidants, which interfere with the redox cycle of
15-LO, (ii) iron-chelator agents, and (iii) non-redox
competitive inhibitors, which compete with AA to bind
the enzyme active site.
In this study, eight novel 2-substituted pyrimido[4,5-
b][1,4]benzothiazines 4a–h were designed and synthe-
sized, and their activity was identified as the mean of
IC50 on soybean 15-LO. We report here (i) common
binding model of pyrimido[4,5-b][1,4]benzothiazine ana-
logues in 15-LO active site, (ii) QSAR study of inhibi-
tors to propose key features of this class of inhibitors,
(iii) synthesis of 2-substituted pyrimido[4,5-b][1,4]benzo-
thiazine analogues, and (iv) 15-LO inhibition study on
soybean enzyme.
Keywords: Pyrimido[4,5-b][1,4]benzothiazine; 5-Bromo-2,4-dichloro-6-
methylpyrimidine; 15-Lipoxygenase; Docking; Molecular modeling;
QSAR.
2. Chemistry
*
The synthesis of pyrimido[4,5-b][1,4]benzothiazines 4a–h
(Scheme 1) started from 5-bromo-2,4-dichloro-6-meth-
Corresponding author. Tel.: +98 511 8797022; fax: +98 511
8796416; e-mail: mbakavoli@yahoo.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.022

M. Bakavoli et al. / Bioorg. Med. Chem. 15 (2007) 2120–2126
2121
N
Cl
Cl
1,2-Aminothiophenol, CHCl
3
NH
2
NH2
EtOH, R2NH
N
N
Cl
S
R2N
S
N
Br
N
N
CH
3
Br
Br
CH
CH
3
3
2
3a-h
1
N
S
R2N
N
N
H
CH3
4a-h
Scheme 1.
ylpyrimidine 1 which was recently prepared by our
research group.14 This compound was converted to
output files were minimized under Semi-empirical
AM1 method (Convergence limit = 0.01; Iteration lim-
it = 50; RMS gradient = 0.05 kcal/mol; Fletcher-Reeves
optimizer algorithm) in HyperChem7.5.16
2-(5-bromo-2-chloro-6-methylpyrimidin-4-ylthio)benzen-
amine 2 by selective displacement of 4-chlorine atom
with 2-aminothiophenol in chloroform at room temper-
ature. The new key intermediates, 2-(5-bromo-6-methyl-
2-substituted-aminopyrimidin-4-ylthio)benzenamines
3a–h, were easily obtained by the reaction of compound
2 with secondary amines in boiling ethanol. The struc-
tures of products 2 and 3a–h were adequately supported
by spectral and microanalytical data. Treatment of these
compounds with sodamide in acetonitrile furnished a
host of pyrimido[4,5-b][1,4]benzothiazines 4a–h in good
yields.
Crystal structure of soybean lipoxygenase-3 (arachidon-
ic acid 15-lipoxygenase) complex with 13(S)-hydropr-
oxy-9(Z)-2,11(E)-octadecadienoic acid was retrieved
from RCSB Protein Data Bank (PDB entry: 1IK3).
3.2. Molecular docking
In ADT (Auto Dock Tools software)17 the torsion an-
gles of the ligands were identified, hydrogens were added
to the macromolecule, bond distances were edited, and
solvent parameters were added to the 15-LO 3D struc-
ture. Partial atomic charges were then assigned to the
macromolecule as well as ligands (Gasteiger for the
ligands and Kollman for the protein).
The structural assignments of compounds 4a–h were
based upon the spectral and microanalytical data. The
IR spectra did not exhibit the stretching vibration bands
at 3360 and 3440 cmÀ1 (br, NH2) due to precursors but
showed a sharp band at 3400 cmÀ1 for NH vibration.
The regions of interest of the enzyme were defined by
considering Fe as the central residue of a grid size of
50, 50, and 50 points in X, Y and Z axes. The docking
parameter files were generated using Genetic Algorithm
and Local Search Parameters (GALS) and number of
generations was set to 10. After docking procedure in
AD3 (Auto Dock 3),17 docking results were submitted
to Weblab Viwerlite 4.018 and Swiss-PdbViewer 3.7
(SP4)19 for further evaluations.
1
Further proof came from the H NMR spectra, which
showed the disappearance of a broad 2H signal belong-
ing to NH2 moiety of compounds 3a–h and the appear-
ance of a sharp 1H (NH) signal. The mass spectra of
compounds 4a–h confirm the elimination of HBr at
the final step.
In conclusion, the sequential treatment of the recently
prepared 5-bromo-2,4-dichloro-6-methylpyrimidine with
2-aminothiophenol and secondary amines which was
followed by interaction with sodamide in acetonitrile
and subsequent heterocyclization is a new, efficient,
and general access to pyrimido[4,5-b][1,4]benzothiazine
derivatives as potential lipoxygenase inhibitors.
The results of docking process (DGb, estimated free ener-
gy of binding; DGd, final docked energy; Ki, estimated
inhibition constant) are outlined in Table 1.
3.3. QSAR studies
QSAR studies were performed for optimized com-
pounds 4a–h in DRAGON 2.1.20 In this study, Morigu-
chi octanol–water partition coefficient (logP),21 polar
surface area (PSA),22 and hydrophilic factor (Hy)23 were
determined. The distance between sulfur atom of com-
pounds 4a–h and iron ion of 15-LO was also compared
in WebLab Viwerlite 4.0 for the best docked models
(Table 2).
3. Molecular modeling, docking, and QSAR study
3.1. Structure optimization
Structures (4a–h) were simulated in chem3D profession-
al; Cambridge software; using MM2 method (RMS gra-
dient = 0.05 kcal/mol).15 In the second optimization,

2122
M. Bakavoli et al. / Bioorg. Med. Chem. 15 (2007) 2120–2126
Table 1. Data obtained from docking studies
Six or more parallels of controls and three or more par-
allels for each test substance solution were measured. To
ensure constant enzyme activity throughout the experi-
ment, the enzyme solution was kept on ice, and controls
were measured at regular intervals. Calculation of en-
zyme activity was carried out as previously described24
and IC50 values were determined by linear interpolation
between the measuring points around to 50% activity
(Table 2).
R
N
S
N
N
H
Compound
R
(D)Gb
(kcal/mol) (kcal/mol)
(D)Gd
Ki
4a
À5.59
À7.04
À7.75
À7.91
À7.67
À6.07
À7.57
À8.09
À8.27
À8.42
7.94e-05
NH
NH
4b
4c
4d
4e
6.90e-06
2.10e-06
1.58e-06
2.38e-06
5. Results and discussion
2-Substituted pyrimido[4,5-b][1,4]benzothiazines 4a–h
were evaluated in vitro to determine their activity on
15-LO inhibition. The qualitative structure–activity rela-
tionship (QSAR) data acquired for these compounds
showed broad range (active to inactive) of 15-LO inhib-
itory activity (IC50 = 18 to >200 lM range; Table 2).
Compound 4d having a methylpiperazine substituent
was the most potent inhibitor of 15-LO at a concentra-
tion of 18 lM. The ethylpiperazine analogue was the
second inhibitor of 15-LO (IC50 = 34 lM). It was inter-
esting to see that neither phenylpiperazine nor 4-hydrox-
ypiperidine analogues 4g–h inhibited 15-LO activity
(IC50 > 200 lM). The pyrrolidine, piperidine, and mor-
pholine analogues exhibited moderate inhibition of
15-LO (Table 2), while compound 4c with a methylpi-
peridine substituent showed less 15-LO inhibitory
potency than its piperazine analogue 4b.
NH
N
NH
N
NH
O
4f
À8.04
À6.10
À7.25
À7.83
À6.49
À8.02
1.27e-06
3.39e-05
4.82e-06
NH
HO
4g
4h
NH
N
NH
DGb, estimated free energy of binding; DGd, final docked energy; Ki,
estimated inhibition constant.
The in vitro data acquired for this class of heterocycles
4a–h showed that 15-LO inhibition can be manipulated
by varying the substitution of pyrimido[4,5-b][1,4]benzo-
thiazine at C-2.
Table 2. Data obtained from 3D-QSAR analyses
Docking studies were carried out to investigate the opti-
mal binding conformations of the pyrimidobenzothia-
zines 4a–h within the 15-LO active site as shown in
Figure 3 (Selected conformations of 4a–h are based on
the best values of Ki and docking energy). The binding
conformation of 4-methyl-2-(4-methylpiperazinyl)pyrimi-
do[4,5-b][1,4]benzothiazine (4d, IC50 = 18 lM) within the
15-LO active site is illustrated in Figure 2. As shown in
the figures the sulfur atom of the pyrimidobenzothiazine
is directed toward the catalytic iron site of the 15-LO
Compound
logP
PSA
Hy
S–Fe
˚
(A)
IC50
(lM)
4a
4b
4c
4d
4e
4f
2.49
2.74
2.98
1.95
2.19
1.70
1.95
3.15
65.3
65.3
65.3
68.5
68.5
74.5
85.5
68.5
À0.266
À0.287
À0.305
À0.260
À0.279
À0.240
0.355
5.65
6.39
6.82
5.19
5.67
6.32
5.78
6.74
48
58
76
18
34
53
4g
4h
>200
>200
À0.342
˚
within 5.19–6.82 A (Table 2). Docking studies showed
(logP, Moriguchi octanol–water partition coefficient; PSA, polar sur-
face area; Hy, hydrophilic factor; S–Fe, the distance between sulfur
atom of compounds 4a–h and iron ion of 15-LO protein after docking
process).
no linear relationship between IC50 and other three Ki,
DGb, and DGd values (Table 1), while the data from
3D-QSAR studies (logP, Hy, and S–Fe distance)
showed a linear relationship with IC50 except for com-
pounds 4g–h which did not exhibit inhibitory effect up
to 200 lM concentration (Fig. 1). In the light of these
findings it can be assumed that among the three different
strategies of LO’s pathway inhibition, the antioxidant
strategy matched soybean 15-LO inhibition. As a matter
of fact:
4. 15-LO inhibition assessment
Lipoxygenase activity was measured in borate buffer
solutions (0.2 M, pH 9.00) using the method described
in previously published work,24 by the increase in absor-
bance at 234 nm from 30 to 90 s after addition of the en-
zyme (soybean 15-lipoxygenase), using linoleic acid
(134 lM) as substrate. The final enzyme concentration
was 167 U/mL. Test substances were added as ethanol
solutions (final ethanol concentration 1%); ethanol
alone was added in uninhibited control experiments.
(i) there is no linear relationship between docking data
(Ki, DGb, and DGd) and IC50; (ii) the S–Fe distance is
not in the range of sulfur–Fe2+/3+ chelation but the sul-
fur atom is situated toward the hydroxide moiety which
is bonded to the iron ion25 (Fig. 2 and 3); (iii) a linear

M. Bakavoli et al. / Bioorg. Med. Chem. 15 (2007) 2120–2126
2123
Figure 2. Docking result of 4-methyl-2-(4-methylpiperazin-1-yl)pyrim-
ido[4,5-b][1,4]benzothiazine 4d (ball and stick) in the active site of
soybean lipoxygenase-3 in ribbon view. Red ball represents iron atom.
Distance between iron and sulfur atom is shown by green line.
Figure 1. Diagrams of measured IC50 versus: (A) hydrophilicity (Hy),
(B) distance between sulfur of desired compounds and LO’s iron, and
(C) lipophilicity. The data of 4f were excluded from diagram C because
of high standard deviation. Compounds 4g and 4h were excluded from
diagram (B) and (C) because of lacking inhibitory response in the
range of 200 lM concentrations. In diagram A, 4g and 4h are shown to
demonstrate the suitable range of hydrophilicity which might be
necessary for inhibitory response.
Figure 3. Superimposition of the binding conformations of pyrimi-
do[4,5-b][1,4]benzothiazine analogues 4a–h (stick) in the active site of
˚
soybean lipoxygenase-3 within 8 A. The iron atom is presented in
black ball.
relationship was observed between S–Fe distance (which
was obtained from 3D presentation of ligand–enzyme
interaction by Weblab Viwerlite software, after docking
process) and IC50 (Fig. 1C).
O
S
N
N
KIO4/MeOH/0 oC - r. t.
N
S
N
N
N
N
N
N
H
N
H
CH3
4d
CH3
5d
As a conclusion, catalytic iron site of LOs enzymes
likely links to an oxygen molecule forming hydroperox-
ide via interaction to double bond of the fatty acid.26
Subsequently the sulfur atom of the pyrimidobenzothi-
azine ring undergoes oxidation to exert its inhibitory
potency upon 15-LO. To prove this hypothesis, com-
pound 4d was converted to its sulfoxide derivative 5d
upon oxidation with sodium metaperiodate according
to a literature procedure27 (Scheme 2). The result of this
experiment was compared with the one obtained
from oxidation of 4d with soybean 15-lipoxygenase
Scheme 2.
(100 U/mL enzyme, 1 mg/mL 4d, pH 9, time: 5 min)
using thin-layer chromatography technique (silica gel
60, F254) and CHCl3/EtOH (95:5) as the eluent. Both
compounds showed the same Rf values (0.375) on
TLC plate.

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M. Bakavoli et al. / Bioorg. Med. Chem. 15 (2007) 2120–2126
6. Experimental
2.42 (s, 3H, CH3), 3.95 (t, 4H, 2(CH2N–Pyr.)), 4.1 (s,
2H, NH2), 6.7–7.35 (m, 4H, aromatic); MS m/z: 392,
394.
6.1. Chemistry
Melting points were recorded on an Electrothermal type
9100 melting point apparatus. The IR spectra were ob-
tained on a 4300 Shimadzu Spectrometer. The 1H
NMR (100 MHz) spectra were recorded on a Bruker
AC 100 spectrometer. The mass spectra were scanned
on a Varian Mat CH-7 instrument at 70 eV. Elemental
analysis was performed on a Thermo Finnigan Flash
EA microanalyzer. All measurements of 15-lipoxyge-
nase activities were carried out using an Agilent 8453
spectrophotometer.
6.1.6. 2-(5-Bromo-6-methyl-2-(4-methylpiperazin-1-yl)pyr-
imidin-4- ylthio)benzenamine (3d). This compound was
obtained as a yellow powder in 65% yield, mp 98–
100 ꢁC; IR: 3330 and 3450 cmÀ1 (NH2); 1H NMR:
(CDCl3) d 2.25 (m, 7H, 2(CH2N)–CH3), 2.42 (s, 3H,
CH3), 3.47 (t, 4H, 2(CH2N–Pyr.)), 4.15 (s, 2H,
NH2), 6.7–7.35 (m, 4H, aromatic); MS m/z: 393,
395. Anal. Calcd for C16H20BrN5S: C, 48.73; H,
5.11; N, 17.76; S, 8.13. Found: C, 48.8; H, 5.05; N,
17.85; S, 8.03.
6.1.1. 2-(5-Bromo-2-chloro-6-methylpyrimidin-4-ylthio)ben-
zenamine (2). To a solution of 5-bromo-2,4-dichloro-6-
methylpyrimidine (2.44 g, 10 mmol) and triethylamine
(1.2 g) in chloroform (30 mL), 2-aminothiophenol
(1.25 g, 10 mmol) was added dropwise with vigorous
stirring over a minute. The solvent was removed under
reduced pressure and the brown residue was washed
with warm water and then crystallized form ethanol
2.98 g, 80% yield, mp 210 ꢁC (dec). IR: 3350,
6.1.7. 2-(5-Bromo-2-(4-ethylpiperazin-1-yl)-6-methyl-pyr-
imidin-4-ylthio)benzenamine (3e). This compound was
obtained as a yellow powder in 65% yield, mp 95 ꢁC;
1
IR: 3330 and 3450 cmÀ1 (NH2); H NMR: (CDCl3) d
1.05 (t, 3H, CH3–(CH2N)) 2.25 (m, 6H, 2(CH2N)–
CH2), 2.42 (s, 3H, CH3), 3.47 (t, 4H, 2(CH2N–Pyr.)),
4.15 (s, 2H, NH2), 6.7–7.35 (m, 4H, aromatic); MS m/
z: 407, 409. Anal. Calcd for C17H22BrN5S: C, 50.00;
H, 5.43; N, 17.15; S, 7.85. Found: C, 49.7; H, 5.55; N,
17.27; S, 7.66.
3430 cmÀ1 1H NMR: (CDCl3) d 2.5 (s, 3H, CH3),
;
4.2 (br, 2H, NH2), 6.8–7.4 (m, 4H), MS m/z: 329,
331, 333.
6.1.8. 2-(5-Bromo-6-methyl-2-(morpholin-4-yl)pyrimidin-
4-ylthio)benzenamine (3f). This compound was obtained
as a yellow powder in 70% yield, mp 128–130 ꢁC; IR:
6.1.2. General procedure for the reaction of 2-(5-bromo-2-
chloro-6-methylpyrimidin-4-ylthio)benzenamine (2) with
secondary amines. A mixture of 2-(5-bromo-2-chloro-6-
methylpyrimidin-4-ylthio)benzenamine (3.29 g, 10 mmol)
and appropriate secondary amine (30 mmol) in ethanol
(20 mL) was heated under reflux for 5 h. Water
(30 mL) was added to solution and the residue was
filtered off and recrystallized from ethanol and dried at
80 ꢁC to give 3a–h.
1
3360 and 3520 cmÀ1 (NH2); H NMR: (CDCl3) d 2.41
(s, 3H, CH3), 3.5 (m, 8H, CH2–(O and N)), 4.1 (s, 2H,
NH2), 6.7–7.35 (m, 4H, aromatic); MS m/z: 380, 382.
Anal. Calcd for C15H17BrN4OS: C, 47.25; H, 4.49; N,
14.69; S, 8.41. Found: C, 47.02; H, 4.55; N, 14.8; S, 8.35.
6.1.9. 2-(5-Bromo-2-(4-hydroxylpiperidin-1-yl)-6-methyl-
pyrimidin-4-ylthio)benzenamine (3g). This compound
was obtained as a yellow viscose liquid in 50% yield;
IR: 3360 and 3470 cmÀ1 (NH2); 1H NMR: (CDCl3),
1.2–1.7 (m, 4H, 2CH2), 2.4 (s, 3H, CH3), 3.02 (m, 1H,
CH–(OH)), 3.9 (t, 4H, 2(CH2N–Pyr.)), 4.1 (s, 3H,
NH2 and OH), 6.7–7.35 (m, 4H, aromatic); MS m/z:
394, 396.
6.1.3. 2-(6-Bromo-6-methyl-2-(pyrrolidin-1-yl)pyrimidin-
4-ylthio)benzenamine (3a). This compound was obtained
as a yellow powder in 75% yield, mp 123–126 ꢁC; IR:
1
3350 and 3450 cmÀ1 (NH2); H NMR: (CDCl3) d 1.8
(t, 4H, 2(CH2–CH2N)), 2.42 (s, 3H, CH3), 3.3 (t, 4H,
2(CH2N)), 4.2 (s, 2H, NH2), 6.7–7.35 (m, 4H, aromatic);
MS m/z: 364, 366. Anal. Calcd for C15H17BrN4S: C,
49.32; H, 4.69; N, 15.34; S, 8.78. Found: C, 49.52; H,
4.58; N, 15.2; S, 8.62.
6.1.10. 2-(5-Bromo-6-methyl-2-(4-phenylpiperazin-1-yl)pyr-
imidin-4- ylthio)benzenamine (3h). This compound was
obtained as a yellow powder in 77% yield, mp 122–
124 ꢁC; IR: 3350 and 3470 cmÀ1 (NH2); 1H NMR:
(CDCl3) d 2.42 (s, 3H, CH3), 3.1 (t, 4H, 2(CH2N–Ph)),
3.55 (t, 4H, 2(CH2N–Pyr.)), 4.15 (s, 2H, NH2), 6.7–
7.35 (m, 9H, aromatic); MS m/z: 455, 457. Anal. Calcd
for C21H22BrN5S: C, 55.26; H, 4.86; N, 15.34; S, 7.03.
Found: C, 55.50; H, 4.73; N, 15.2; S, 6.95.
6.1.4. 2-(5-Bromo-6-methyl-2-(piperidin-1-yl)pyrimidin-4-
ylthio)benzenamine (3b). This compound was obtained
as a yellow powder in 75% yield, mp 105–107 ꢁC; IR:
1
3380 and 3480 cmÀ1 (NH2); H NMR: (CDCl3) d 1.2–
1.7 (m, 6H, 3CH2), 2.42 (s, 3H, CH3), 3.39 (t, 4H,
2(CH2N–Pyr.)), 4.1 (s, 2H, NH2), 6.7–7.35 (m, 4H, aro-
matic); MS m/z: 378, 380. Anal. Calcd for C16H19BrN5S:
C, 50.66; H, 5.05; N, 14.77; S, 8.45. Found: C, 50.5; H,
5.1; N, 14.84; S, 8.4.
6.1.11. General procedure for the conversion of 3a–h to
pyrimido[4,5-b][1,4]benzothiazine derivatives. A solution
of compounds 3a–h (10 mmol), sodium amide
(30 mmol) in acetonitrile (20 mL) was heated under re-
flux for 90 min. The solvent was removed under reduced
pressure and a solution of acetic acid (0.7 g) in water
(20 ml) added to the residue and filtered off. Then the
residue was crystallized from ethanol to give 4a–h,
respectively.
6.1.5. 2-(5-Bromo-6-methyl-2-(4-methylpiperidin-1-yl)pyr-
imidin-4-ylthio)benzenamine (3c). This compound was
obtained as a yellow viscose liquid in 55% yield; IR:
1
3360 and 3470 cmÀ1 (NH2); H NMR: (CDCl3) d 0.85
(d, 3H, CH3–(CH)), 1.2–1.7 (m, 5H, 2CH2 and CH),

M. Bakavoli et al. / Bioorg. Med. Chem. 15 (2007) 2120–2126
2125
6.1.12. 4-Methyl-2-(pyrrolidin-1-yl)pyrimido[4,5-b][1,4]ben-
zothiazine (4a). This compound was obtained as a yellow
Pyr.)), 4.1 (s, 1H, OH), 6.9 (dd, 2H, C7H and C8H),
7.35(d, 1H, C6H), 8.2–8.4 (d, 2H, C9H and NH); MS
m/z: 314.
1
powder in 60% yield, mp 181 ꢁC; IR: 3350 cmÀ1; H
NMR: (CDCl3) d 1.95 (t, 4H, 2 ((CH2)–CH2N)), 2.37
(s, 3H, CH3), 3.5 (t, 4H, 2(CH2N)), 6.9 (dd, 2H, C7H
and C8H), 7.35 (d, 1H, C6H), 8.6 (d, 1H, C9H) 8.4 (s,
1H, NH), MS m/z: 284. Anal. Calcd for C15H16N4S:
C, 63.35; H, 5.67; N, 19.70; S, 11.28. Found: C, 63.23;
H, 5.58; N, 19.57; S, 11.08.
6.1.19. 4-Methyl-2-(4-phenylpiperazin-1-yl)pyrimido[4,5-
b][1,4]benzothiazine (4h). This compound was obtained
as a brown powder in 85% yield, mp 122 ꢁC; IR:
1
3350 cmÀ1; H NMR: (CDCl3) d 2.4 (s, 3H, CH3), 3.2
(t, 4H, (CH2)2NPh), 3.9 (t, 4H, 2(CH2N)), 6.8–7.4 (m,
8H), 8.2–8.4 (d, 2H, C9H and NH), MS m/z: 375. Anal.
Calcd for C21H21N5S: C, 67.17; H, 5.64; N, 18.65; S,
8.54. Found: C, 67.33; H, 5.69; N, 18.49; S, 8.35.
6.1.13. 4-Methyl-2-(piperidin-1-yl)pyrimido[4,5-b][1,4]ben-
zothiazine (4b). This compound was obtained as a yellow
1
powder in 75% yield, mp 157 ꢁC; IR: 3330 cmÀ1; H
NMR: (CDCl3) d 1.4–1.8 (m, 6H, ((CH2)–CH2N)),
2.35 (s, 3H, CH3), 3.7 (t, 4H, 2(CH2N)), 6.9 (dd, 2H,
C7H and C8H), 7.35 (d, 1H, C6H), 8.2–8.4 (d, 2H,
C9H and NH), MS m/z: 298. Anal. Calcd for
C16H18N4S: C, 64.40; H, 6.08; N, 18.78; S, 10.75.
Found: C, 64.52; H, 6.02; N, 18.89; S, 10.61.
6.1.20. 4-Methyl-2-(4-methylpiperazino)-5,10-dihydro- 10k4-
benzo[b]pyrimido[5,4-e][1,4]thiazin-10- one (5d). A solution
of potassium metaperiodate (1 mmol) in water (5 mL)
was added dropwise to a stirred solution of 4d (1 mmol)
and concd HCl (0.5 mL) in methanol (20 mL) at 0 ꢁC.
The stirring was continued for further a hour at this
temperature. Then the reaction mixture was stirred over-
night at room temperature. Finally the reaction mixture
was basified by adding Na2CO3 (5%), before it was
extracted with dichloromethane (2· 5 mL). The com-
bined extract was evaporated to dryness at reduced pres-
sure and the residue recrystallized from ethanol as
yellow powder (40% yield), mp 210 ꢁC; IR:
6.1.14. 4-methyl-2-(4-methylpiperidin-1-yl)pyrimido[4,5-
b][1,4]benzothiazine (4c). This compound was obtained
as a viscose liquid in 55% yield, IR: 3380 cmÀ1 1H
;
NMR: (CDCl3) d 0.9 (d, 3H, CH3–(CH)), 1.2–1.7 (m,
5H, 2CH2and CH), 2.42 (s, 3H, CH3), 3.95 (t, 4H,
2(CH2N–Pyr.)), 6.9 (dd, 2H, C7H and C8H), 7.35 (d,
1H, C6H), 8.2–8.4 (d, 2H, C9H and NH); MS m/z: 312.
1080(S@O), 3350 cmÀ1 1H NMR: (CDCl3) d 2.3–2.7
;
(m, 10H, CH3N(CH2)2 and CH3), 3.8 (t, 4H,
2(CH2N)), 7.1 (dd, 2H, C7H and C8H), 7.6 (d, 1H,
C6H), 8.7 (d, 2H, C9H), 12.1 (s, 1H, NH), MS m/z: 329.
6.1.15. 4-Methyl-2-(4-methylpiperazin-1-yl)pyrimido[4,5-
b][1,4]benzothiazine (4d). This compound was obtained
as a yellow powder in 55% yield, mp 198 ꢁC; IR:
3350 cmÀ1 1H NMR: (CDCl3) d 2.3–2.6 (m, 10H,
;
CH3N(CH2)2 and CH3), 3.7 (t, 4H, 2(CH2N)), 6.9 (dd,
2H, C7H and C8H), 7.35(d, 1H, C6H), 8.2–8.4 (d, 2H,
C9H and NH), MS m/z: 313. Anal. Calcd for
C16H19N5S: C, 61.31; H, 6.11; N, 22.34; S, 10.23.
Found: C, 61.39; H, 6.17; N, 22.18; S, 10.07.
Acknowledgments
We express our sincere gratitude to Dr. R. Jalal for
reading the manuscript and Mr. M. Riazi for UV stud-
ies. We are also grateful to Ferdowsi University of
Mashhad for financial support of this work.
6.1.16. 2-(4-Ethylpiperazin-1-yl)4-methylpyrimido[4,5-b][1,
4]benzothiazine (4e). This compound was obtained as a
yellow powder in 65% yield, mp 192 ꢁC; IR:
3380 cmÀ1 1H NMR: (CDCl3) d 1.1 (t, 3H, CH3–
;
References and notes
(CH2N)) 2.3 (m, 6H, 2(CH2N)–CH2), 2.42 (s, 3H,
CH3), 3.7 (t, 4H, 2(CH2N–Pyr.)), 6.9 (dd, 2H, C7H
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62.36; H, 6.46; N, 21.39; S, 9.79. Found: C, 62.60; H,
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