Synthesis of new series of pyrimido[4,5-b][1,4] benzothiazines as 15-lipoxygenase inhibitors and study of their inhibitory mechanism

Article abstract of DOI:10.1007/s00044-013-0506-7

A series of 2-substituted 4-n-propyl pyrimido[4,5-b][1,4]benzothiazines were synthesized, and evaluated as soybean 15-lipoxygenase (SLO) inhibitors. Among the synthesized compounds, 2-(4-methyl piperazinyl) analog showed the best SLO inhibition activity (IC₅₀ = 8.9 ± 0.4 μM). To rationalize the inhibitory potency variation of the compounds, computer-assisted modeling of the enzyme-inhibitor, radical scavenging evaluation, and inhibitory mechanism analyses were performed. The results showed that in 4-alkyl pyrimido[4,5-b]benzothiazines with same 2-substituent, radical scavenging potency plays a key role in lipoxygenase inhibition.

Full text of DOI:10.1007/s00044-013-0506-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:5036–5043
DOI 10.1007/s00044-013-0506-7
ORIGINAL RESEARCH
Synthesis of new series of pyrimido[4,5-b][1,4] benzothiazines
as 15-lipoxygenase inhibitors and study of their inhibitory
mechanism
•
Mohsen Nikpour Mina Mousavian
•
•
•
Mahdieh Davoodnejad Maliheh Alimardani
Hamid Sadeghian
Received: 31 October 2012 / Accepted: 18 January 2013 / Published online: 6 February 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of 2-substituted 4-n-propyl pyrimido[4,5-
b][1,4]benzothiazines were synthesized, and evaluated as
soybean 15-lipoxygenase (SLO) inhibitors. Among the syn-
thesized compounds, 2-(4-methyl piperazinyl) analog showed
the best SLO inhibition activity (IC50 = 8.9 0.4 lM). To
rationalize the inhibitory potency variation of the compounds,
computer-assisted modeling of the enzyme-inhibitor, radical
scavenging evaluation, and inhibitory mechanism analyses
were performed. The results showed that in 4-alkyl pyrimi-
do[4,5-b]benzothiazines with same 2-substituent, radical
scavenging potency plays a key role in lipoxygenase inhibition.
Introduction
Mammalian lipoxygenases are a series of non-hem iron
containing enzymes which oxidize unsaturated fatty acids
and esters into hydroperoxide derivatives (Fig. 1).
The above mentioned proteins are non-homogeneous
enzymes that are widely found in plants and animals,
nominated mainly based on situation in which a key sub-
strate such as arachidonic acid or other 1,4-unsaturated
fatty acids is oxidized.
Nowadays, among the human lipoxygenases, 15-lipoxy-
genase (15-LO) is introduced as a novel therapeutic target for
the treatment of virus-associated asthma characterized by a
Th1 immune response to inhaled allergens (Jeon et al.,
2009).
Keywords Docking Á Radical scavenging Á DPPH Á
SAR Á DMAB
The effect of 15-LO inhibition on the treatment of car-
diovascular and atherosclerosis diseases has been discov-
ered. In atherosclerosis, the above mentioned enzyme
significantly expresses in macrophages (Cyrus et al., 1999;
Harats et al., 2002). 15-Lipoxygenase (15-LO) metabolites
influence the origination and progression of prostate cancer
(Kelavkar et al., 2002). The final products of arachidonic
acid peroxidation by 15-LO in eosinophils have been
introduced as one of the inflammation source (Feltenmark
et al., 2008).
M. Nikpour
Department of Chemistry, Ahvaz Branch, Islamic Azad
University, Ahvaz, Iran
M. Mousavian
Department of Biology, Mashhad Branch, Islamic Azad
University, Mashhad, Iran
Three known groups of lipoxygenase inhibitors have
been reported (Charlier and Michaux, 2003):
M. Davoodnejad Á M. Alimardani Á H. Sadeghian
Department of Laboratory Sciences, School of Paramedical
Sciences, Mashhad University of Medical Sciences, Mashhad,
Iran
(1) Reductive inhibitors or antioxidants interact with
peroxidation cycle of LO; (2) Iron chelating inhibitors; and
(3) Non-reductive competitive inhibitors which compete
with arachidonic acid or other unsaturated fatty acids for
bonding to active site of the enzyme.
H. Sadeghian (&)
Microbiology & Virology Research Center, Buali Research
Institute, Mashhad University of Medical Sciences,
91967-73117 Mashhad, Iran
One of the well-known synthetic heterocyclic com-
pounds which have been reported as lipoxygenase inhibitor,
is 2-substituted pyrimido[4,5-b][l,4]benzothiazines. The
e-mail: sadeghianh@mums.ac.ir
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Med Chem Res (2013) 22:5036–5043
5037
Fig. 1 Enzymatic peroxidation
cycle of unsaturated fatty acids
H
H
H
H
H
H
O
O
II
III
Fe
Fe
H
O
OH
O
O
H
O
H
O
H
H
H
H
O
O
II
II
Fe
Fe
aforementioned compounds were first reported as soybean
15-lipoxygenase (SLO) inhibitor by (Bakavoli et al., 2007).
After that, the inhibitory activity of 4-H analogs against
SLO, in comparison with 4-Me analogs, was determined.
The results showed that, by grafting of methyl group at
position 4 of 2-substituted pyrimido[4,5-b][l,4]benzothia-
zines, an increase in inhibitory activity occurs (Bakavoli
et al., 2008). In a recent research, it was found that the
difference mentioned between the inhibitory potency of 4-H
and 4-Me analogs might have arisen from their HOMO
energy level differences (Pooryaghoobi et al., 2013). For
further proofing of the aforementioned theory, we synthe-
sized the 4-propyl derivatives and their SLO inhibitory
potency compared with that of 4-H and 4-Me analogs. SAR
comparative study on the SLO inhibitory activities of the
aforementioned compounds using ab initio calculations
and the version of AutoDock (i.e. AutoDock 4.2) (http://
www.scripps.edu/pub/olson-web/doc/autodock/) was also
evaluated. DPPH bleaching activity of the synthetic com-
pounds was tested and compared to the SLO inhibitory
results to find a rational relationship between the radical
scavenging activity and lipoxygenase inhibitory potency. In
this study, the inhibitory mechanism of the most potent
inhibitor was furthermore studied.
[4,5-b][l,4]benzothiazines separately comply with the
estimated inhibitory constant (Ki), which had been deter-
mined via docking calculations. Further investigation
showed that the inhibitory potency differences between
each 4-H and 4-Me analogs might have emerged from their
HOMO energy level differences. Herein, to prove the afore-
mentioned hypothesis, 4-n-propyl analogs were synthesized,
and their inhibitory potencies were compared to 4-Methyl-2-
(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine (4-MMPB)
and 2-(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine
(2-MPB). 2-MPB and 4-MMPB were synthesized acc-
ording to the synthetic procedures reported in the previous
study (Bakavoli et al., 2007, 2008) via starting from uracil
and 6-methyl uracil, respectively. The structural confor-
mation of the synthetic compounds was determined by
comparing their melting points and IR spectra, which were
reported in the aforementioned literatures. Similarly,
compounds 8a–f were synthesized via starting from 5-
bromo-2,4-dichloro-6-propylpyrimidine (5) which had
been prepared according to the previous studies from 6-n-
propyl uracil (3) (Nikpour et al., 2012). Compound 3 was
produced via hydrolysis of 2, a condensation product of
thiourea and ethyl 3-oxohexanoate (1) (Scheme 1).
The inhibitory activity of the above mentioned com-
pounds toward SLO was determined by measuring the
amount of peroxide formation. The peroxide concentra-
tion was measured at 598 nm using a mixture of MBTH
(3-methyl-2-benzothiazolonhydrazone), DMAB (3-dim-
ethylaminobenzoic acid), and hemoglobin (Jabbari et al.,
2012).
Results and discussions
In a recent study, it was suggested that the inhibitory
potency of the 4-H and 4-Me analogs of pyrimido
123

5038
Med Chem Res (2013) 22:5036–5043
H
N
H
N
H
N
O
O
S
O
O
O
O
O
Cl
N
Cl
ClCH2COOH
Thiourea
POCl3
Br2 / H2O
O
HN
HN
HN
N
Br
Br
1
5
2
4
3
2-Aminothiophenol
NR2
N
N
S
NH2
NH2
NaNH2 / CH3CN
R2NH / Et3N
NR2
N
S
Cl
N
6
S
N
H
N
N
Br
Br
8a-f
7a-f
NH
Me
Ph
N
Me
N
O
NH
NH
NH
NH
b
N
H
f
e
c
a
d
N
N
N
N
S
N
N
S
2-MPB
4-MMPB
N
N
N
H
N
H
CH3
Scheme 1 General procedure for the synthesis of 4-propyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine and the chemical structure of
2-MPB and 4-MMPB
The IC50 values of the synthetic compounds were nearly
half those of the Methyl analogs’. Among the n-propyl
analogs, compound 8f with N-methyl piperazine moiety was
the most potent SLO inhibitor with an IC50 value of 8.9 lM.
To rationalize the observed variation in inhibitory potency,
computer-assisted modeling was performed on intermolec-
ular interactions of SLO-Inhibitor complex. The theoretical
inhibitory constant of the compounds was determined via
docking process. To fulfil this purpose, ab initio energy-
minimized 3D structures of the inhibitors were prepared
similar to the previous study. Structure optimization led to
formation of two possible enantiomers (Fig. 2). In Fig. 2, the
enantiomer shown in the right side was selected for the
docking process (Pooryaghoobi et al., 2013).
have had direct contact with the substrate, were allowed to be
rotatable. Docked conformers of the compounds were gen-
erated in ADT (Auto Dock Tools) software to acquire the
bonding affinity of the designed molecular structures toward
SLO. Among the docked conformers of each compound, one
that belonged to a cluster in which its sulfur portion was
vertically oriented toward the SLO Iron atom besides pos-
sessing the lowest Ki, was considered as ‘‘consensus struc-
ture’’ to study the structure–activity relationships (Fig. 3).
In the consensus structure, the thiazine ring is fixed with
two amino acids Leu565, Leu773, and the aliphatic moiety
of the pyrimidine ring is surrounded by the pocket of the
conserved amino acids Leu770, Val769, Asp766, Gln716,
Phe576, Ile572, and Gln514 (Sadeghian et al., 2009, 2008)
(Fig. 4).
The 3D structures of the inhibitors were docked into the
SLO (PDB entry: 1IK3) active site pocket while the side
chains of the hydrophobic amino acids Ile515, Ile557,
Leu565, Ile566, Ile572, Phe576, Leu770, and Ile773, which
Complementary studies on inhibitory mechanism
showed a competitive inhibitory mechanism for this series
of compounds (Fig. 5).
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Med Chem Res (2013) 22:5036–5043
5039
Fig. 2 Two enantiomers of 4-
MMPB derived from structural
optimization
no inhibitor
10 µM
0.30
0.25
0.20
0.15
0.10
0.05
0.00
20 µM
40 µM
0.00
0.01
0.02
0.03
0.04
0.05
1/[8f] µM
Fig. 5 Lineweaver–Burk plot of SLO inhibition by 8f. Lipoxygenase
activity was measured at 25, 50, 100, and 200 lM concentrations of
linoleic acid by MBTH–DMAB method. The Y-intercept average
(1/Vmax) of the plotted curves is 0.0655 0.0035 (Km = 20.19 lM)
Fig. 3 Stick view of consensus structures of 8f, 4-MMPB, and 2-
MPB with flexible side chain of hydrophobic residues in the SLO
active site pocket
Fig. 4 Consensus structure of 8f in the active site pocket of SLO in stick (left) and solvent surface (right) views. Fe atom is distinguished by pink
bull. Carbon, oxygen, nitrogen, sulfur, and hydrogen atoms are distinguished by gray, red, blue, yellow, and white colors, respectively
The enzyme assessment demonstrated that, in pyr-
imidobenzothiazine moiety, replacement of 4-methyl with
n-propyl led to twofold increase in inhibitory potency.
The radical quenching capacity of 8a–f, 4-MPB, and
4-MMPB was assayed using DPPH radical scavenging
method. In vitro bleaching method of DPPH free radicals is
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Med Chem Res (2013) 22:5036–5043
Table 1 Enzyme inhibitory,
DPPH bleaching assessment
data of the synthetic compounds
and docking analysis data of
consensus conformers
Compound
IC50 (lM) DPPH
bleaching
IC50 (lM) SLO
inhibition
DGb
Ki (lM)
Number of
conformers in
cluster
(Kcal mol-1
)
8a
310.9 5.1
620.7 4.3
599.3 6.1
668.7 5.9
357.3 4.4
17.3 1.1
69.6 1.8
141.6 3.9
611.0 19.8
121.2 4.2
86.3 2.4
726.4 15.9
403.7 8.9
8.9 0.4
7.81
8.42
8.60
7.76
8.08
9.15
9.13
9.11
1.845
0.662
0.486
2.011
1.163
0.192
0.198
0.204
28
35
23
33
24
22
37
39
8b
8c
8d
8e
8f
Ki estimated inhibition constant,
DGb estimated bonding free
energy
4-MMPB
2-MPB
19.5 0.9
41.7 1.5
a widely adopted assay to evaluate the scavenging potential
of stable free radicals. Among the test compounds, 8e
showed the best radical scavenging activity (Table 1). The
activity of 8e was twofold higher than 4-methyl analog
4-MMPB. It was interesting to note that the bleaching
activity variation was not in accordance with the lipoxy-
genase inhibitory potency. For example, the radical scav-
enging activity of 8b was higher than that of 8c, while the
SLO inhibitory potency of 8c was better.
considering 4-MMPB and 2-MPB, no such pattern
could be found. The observed deflection of 4-MMPB and
2-MPB could be explained by their HOMO energy level
which had been discussed in the previous literature
(Pooryaghoobi et al., 2013). HOMO energy level can dis-
pose the molecules for radical scavenging activity. By
calculating HOMO energy level of 4-MMPB, 2-MPB, and
8f via ab initio method, it was found that among the
aforementioned compounds, 8f with 4-n-propyl moiety has
the highest HOMO energy level which makes it the most
potent radical scavenger.
Both results of competitive inhibitory mechanism and
radical scavenging potency showed that the main factor in
lipoxygenase inhibitory activity of each 4-alkyl series of
primidobenzothiazines is their bonding affinity to the
enzyme active site pocket via intermolecular forces. But
when the position 4 substituent is exchanged, the role of
sulfur oxidation potency becomes predominant.
In summary, the results showed that in 4-alkyl pyrimi-
do[4,5-b]benzothiazine with fix substitution at position 2,
radical scavenging potency plays a key role in lipoxyge-
nase inhibition. On the other hand, It was confirmed that
among the above mentioned pyrimido[4,5-b]benzothia-
zines, 2-N-methyl piperazinyl analogs are the most potent
inhibitors of 15-lipoxygenase enzyme.
In Fig. 6, a good linear relationship between Log IC50
and Log Ki of 8a–f consensus structures is seen while by
5.5
Molecular modeling, docking and SAR study
Structure optimization
8f
5.0
Three dimensional structures of 2-MPB, 4-MMPB, and 8a–f
were simulated in Hyper Chem7.5 using MM? method
(RMS gradient = 0.1 kcal mol-1) (HyperChemÒ Release 7,
Hypercube Inc., http://www.hyper.com/). In the second opti-
mization, output files were minimized under Semiempirical
AM1 methods (Convergence limit = 0.01; Iteration
limit = 50; RMS gradient = 0.1 kcal mol-1; Polak–Ribiere
optimizer algorithm); then, the output files were minimized
under ab initio methods with 6-311G* basis set (Convergence
limit = le-5; Iteration limit = 50; RMS gradient = 0.1
kcal mol-1; Polak–Ribiere optimizer algorithm.
4-MMPB
4.5
2-MPB
8c
4.0
8b
3.5
8e
8a
8d
3.0
5.50
5.75
6.00
6.25
6.50
6.75
7.00
Crystal structure of Soybean lipoxygenase-3(arachi-
donic 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).
- Log Ki
Fig. 6 -Log IC50 versus -Log Ki for consensus structures of 8a–f,
4-MMPB, and 2-MPB
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Med Chem Res (2013) 22:5036–5043
Molecular docking
5041
(130 lL) were added to start the color formation. Further,
5 min later, 200 lL of a 2 % SDS solution was added to
terminate the reaction. The absorbance at 598 nm was
compared to control test.
Automated docking simulation was implemented to dock
2-MPB, 4-MMPB, and 8a–f into the active site of SLO
with flexiblizing of desired residues in AutoDock Tools
version 4.2 (revision 30) (Morris et al., 2009) using
Lamarckian genetic algorithm (Morris et al., 1998). The
aforementioned method has been previously shown to
produce bonding models similar to the experimentally
observed models (Seyedi et al., 2009; Nikpour et al., 2010;
Iranshahi et al., 2012). The torsion angles of the ligands
were identified; hydrogens were added to the macromole-
cule, bond distances were edited, and solvent parameters
were added to the enzyme 3D structure. Partial atomic
charges were then assigned to the macromolecule as well
as ligands (Gasteiger for the ligands and Kollman for the
protein) (Huey et al., 2006).
Chemistry
Melting points were recorded on an Electrothermal type
9100 melting point apparatus. The IR spectra were obtained
on a 4300 Shimadzu Spectrometer. The 1H NMR (500 MHz)
and 13C NMR (125 MHz) spectra were recorded on Bruker
Avance DRX-500 spectrometer. The mass spectra were
scanned on a Varian Mat CH-7 instrument at 70 eV. Ele-
mental analysis was obtained on a Thermo Finnigan Flash
EA microanalyzer.
The regions of interest of the enzyme were defined by
considering Cartesian chart 20.4, 3.5, and 20.5 as the central
of a grid size of 42, 42, and 56 points in X-, Y- and Z-axes.
Ile557, Ile566, Ile572, Ile515, Phe576, Leu770, and Ile773
were selected flexible. The docking parameter files were
generated using Genetic Algorithm and Local Search Param-
eters (GALS) while number of generations and maximum
number of energy evaluations were set to 256 and 2,500,000,
respectively.The256dockedcomplexeswereclusteredwitha
2-[(5-Bromo-2-chloro-6-propylpyrimidin-4-
yl)sulfanyl]aniline (6)
To a solution of 5-bromo-2,4-dichloro-6-propylpyrimidine
5 (2.72 g, 10 mmol) and triethylamine (1.2 g) in chloro-
form (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 res-
idue was washed with warm water and then crystallized
from ethanol (2.5 g, 80 % yield, mp 146 °C (dec). IR:
3300, 3,430 cm-1, 1H NMR: (CDCl3) d, 1.03 (t, 3H, CH3),
1.75 (sextet, 2H, CH2-CH2-CH3), 2.85 (t, 2H, CH2-
pyrimidine ring), 4.2 (broad, 2H, NH2), 6.8–7.4 (m, 4H),
13C NMR: (CDCl3) d, 13.9, 21.8, 30.5, 114.2, 117.3, 118.3,
120.1, 127.8, 128.4, 147.1, 157.1, 161.3, 162.5 MS m/z:
357, 359, 361.
˚
root-mean-square deviation tolerance of 0.2 A. The program
generated 256 dockedconformerscorrespondingto thelowest
energy structures. After docking procedure, docking results
were submitted to DS visualizer (http://www.accelrys.com/
products/discovery-studio) for further evaluations.
Experimental protocols
SLO screening assay
General procedure for the synthesis of pyrimido[4,5-
Linoleic acid and two assay solutions (A and B) were
prepared in advance. Solution A was 50 mM DMAB in a
l00 mM phosphate buffer (pH 7.0). Solution B was a
b][1,4]benzothiazines 8a–f
mixture of l0 mM MBTH (3 mL), hemoglobin (5 mg mL-1
,
A mixture of 2-[(5-bromo-2-chloro-6-propylpyrimidin-
4-yl)sulfanyl]aniline 6 (3.6 g, 10 mmol) and appropriate
secondary amine (30 mmol) in ethanol (20 mL) was heated
under reflux for 5 h. The water (40 mL) added to solution
and the precipitated viscose liquid (intermediate compounds
7a–f) were dried at 80 °C for an hour and immediately were
utilized without purification. To stirred solution of the
foregoing compounds in acetonitrile (30 mL), sodium amide
(1.2 g, 30 mmol) was added and then heated under reflux
condition (3 h). The solvent was removed under reduced
pressure, and a solution of 2 % acetic acid (20 mL) was
added to the residue and filtered off. Then the residue crys-
tallized from benzene to give 8a–f.
3 mL) in 50 mM phosphate buffer at pH 5.0 (25 mL). A
linoleic acid solution was prepared by mixing 5 mg of
linoleic acid with 0.5 mL ethanol and then diluting with
KOH 100 mM to a final volume of 5 mL. In the standard
assay, the sample in ethanol (25 lL), SLO (4,000 units/mL
in 50 mM phosphate buffer pH 7.0; 25 lL), and phosphate
buffer pH 7.0 (50 mM; 900 lL) was mixed in a test tube,
and preincubation was carried out for 5 min at room tem-
perature. A control test was done with the same volume of
ethanol. After the pre-incubation, linoleic acid solution
(50 lL) was added to start the peroxidation reaction, and,
7 min later, solution A (270 lL) and then solution B
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Med Chem Res (2013) 22:5036–5043
4-Propyl-2-(pyrrolidin-1-yl)-5H-pyrimido[4,5-
C, 70.76; H, 7.42; N, 12.38; S, 9.44. Found: C, 71.38; H,
7.19; N, 12.29; S, 9.35.
b][1,4]benzothiazine (8a)
This compound was obtained as a yellow powder in 55 %
1
2-(4-Phenylpiperazin-1-yl)-4-propyl-pyrimido[4,5-
yield, mp 166 °C; IR: 3,320 cm-1, H NMR: (CDCl3) d,
b][1,4]benzothiazine (8e)
1.00 (t, 3H, CH3), 1.77 (sextet, 2H, CH2-CH2-CH3), 1.98 (t,
4H, 2 ((CH2)2-CH2N), 2.89 (t, 2H, CH2-pyrimidine ring),
3.5 (t, 4H, 2(CH2N)), 6.9 (dd, 2H, C7H & C8H), 7.35 (d,
1H, C6H), 8.6 (d, 1H, C9H), 8.4 (s, 1H, NH), 13C NMR:
(CDCl3) d, 14.1, 20.9, 25.5, 39.4, 45.4, 94.4, 120.6, 122.8,
124.1, 131.1, 136.6, 141, 155.7, 159.3, 167.1; MS m/z: 312.
Anal. Calcd. For C17H20N4S: C, 65.35; H, 6.45; N, 17.93;
S, 10.26. Found: C, 65.67; H, 6.57; N, 17.78; S, 10.03.
This compound was obtained as a brown powder in 85 %
1
yield, mp 213 °C; IR: 3,350 cm-1, H NMR: (CDCl3) d,
1.0 (t, 3H, CH3), 1.78 (sextet, 2H, CH2-CH2-CH3), 2.85 (t,
2H, CH2-pyrimidine ring), 3.2 (t, 4H (CH2)2 NPh), 3.9 (t,
4H, 2(CH2N)), 6.8–7.4 (m, 8H), 8.2-8.4 (d, 2H, C9H &
NH); 13C NMR: (CDCl3) d, 14.1, 20.9, 39.4, 44.1, 50.3,
94.6, 115.9, 119.5, 120.4, 122.7, 124.1, 129.0, 131.3,
136.5, 141.1, 151.6, 155.6, 159.2, 167.2; MS m/z: 403.
Anal. Calcd. For C23H25N5S: C, 68.46; H, 6.24; N, 17.35;
S, 7.95. Found: C, 68.28; H, 6.39; N, 17.14; S, 7.72.
2-(Piperidin-1-yl)-4-propyl-5H-pyrimido[4,5-
b][1,4]benzothiazine (8b)
This compound was obtained as a yellow powder in 80 %
1
2-(4-Methylpiperazin-1-yl)-4-propyl-5H-pyrimido[4,5-
yield, mp 168 °C; IR: 3,300 cm-1, H NMR: (CDCl3) d,
b][1,4]benzothiazine (8f)
1.01 (t, 3H, CH3), 1.4–1.8 (m, 8H ((CH2)3-CH2N & CH2-
CH2-CH3), 2.89 (t, 2H, CH2-pyrimidine ring), 3.7 (t, 4H,
2(CH2N)), 6.9 (dd, 2H, C7H & C8H), 7.35 (d, 1H, C6H),
8.2–8.4 (d, 2H, C9H & NH); 13C NMR: (CDCl3) d, 14.1,
20.9, 25.2, 27.2, 39.4, 45.4, 94.3, 120.5, 122.7, 124.2,
131.1, 136.6, 141.0, 155.7, 159.3, 167.0; MS m/z: 326.
Anal. Calcd. For C18H22N4S: C, 66.22; H, 6.79; N, 17.16;
S, 9.82. Found: C, 65.94; H, 6.89; N, 17.36; S, 9.60.
This compound was obtained as a yellow powder in 50 %
1
yield, mp 167 °C; IR: 3,350 cm-1, H NMR: (CDCl3) d,
1.01 (t, 3H, CH3), 1.77 (sextet, 2H, CH2-CH2-CH3), 2.3 (m,
7H, CH3N(CH2)2), 2.89 (t, 2H, CH2-pyrimidine ring), 3.7
(t, 4H, 2(CH2N)), 6.9 (dd, 2H, C7H & C8H), 7.35(d, 1H,
C6H), 8.2–8.4 (d, 2H, C9H & NH); 13C NMR: (CDCl3) d,
14.1, 20.9, 39.4, 44.0, 46.7, 56.4, 94.7, 120.5, 122.7, 124.1,
131.1, 136.6, 141, 155.7, 159.5, 167.0; MS m/z: 341. Anal.
Calcd. For C18H23N5S: C, 63.31; H, 6.79; N, 20.51; S,
9.39. Found: C, 63.48; H, 6.89; N, 20.24; S, 9.17.
2-(4-Methylpiperidin-1-yl)-4-propyl-5H-pyrimido[4,5-
b][1,4]benzothiazine (8c)
Acknowledgments The authors are grateful to Iran National Sci-
ence Foundation (INSF) for financial support of this work (project
No. 89001368).
This compound was obtained as a viscose liquid in 50 %
1
yield, mp 174 °C; IR: 3,350 cm-1; H NMR: (CDCl3) d,
0.91–1.04 (m, 6H, CH3-(CH) & CH3CH2CH2), 1.2–1.8 (m,
7H, 2CH2, CH & CH2-CH2-CH3), 2.87 (t, 2H, CH2-
pyrimidine ring), 3.95 (t, 4H, 2(CH2N-Pyr.)), 6.9 (dd, 2H,
C7H & C8H), 7.35 (d, 1H, C6H), 8.2–8.4 (d, 2H, C9H &
NH); 14.1, 20.9, 22.6, 31.3, 35.6, 39.4, 44.7, 94.3, 120.5,
122.7, 124.2, 131.1, 136.6, 141, 155.7, 159.2, 167.0; MS m/
z: 340. Anal. Calcd. For C20H25N3S: C, 62.17; H, 6.14; N,
17.06; S, 9.76. Found: C, 62.38; H, 5.95; N, 17.29; S, 9.52.
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