Synthesis, characterization and biological evaluation of a novel series of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazines containing an amide linkage

Article abstract of DOI:10.1002/jhet.1766

Two series of combinatorial library of 3,6-disubstituted-7H-1,2,4-triazolo- [3,4-b]-1,3,4-thiadiazines bearing an amide linkage were synthesized. All the newly synthesized compounds were characterized by spectral analyses. The newly synthesized compounds were screened for their cytotoxicity, anti-inflammatory, and analgesic activities. Among the tested compounds, the compound 9g (Ar = 4-(methoxybenzyl)piperazine) is the most promising molecule with half maximal inhibitory concentration (IC₅₀) value of 14.24 μM in (MCF-7) cells. Compounds 9f (Ar = 4-(chlorobenzyl)piperazine), 9g, and 9k (Ar = 2-(fluorophenyl)piperazine) exhibited excellent anti-inflammatory activity at a dose level of 50 mg/kg, almost comparable with the standard drug. In case of analgesic activity among the tested compounds, the compounds 9f, 9g, and 9k showed more potent and consistent activity in both 100 and 200 mg/kg/po doses with less ulcerogenic risk.

Full text of DOI:10.1002/jhet.1766

August 2014
Synthesis, Characterization and Biological Evaluation of a Novel Series of
E55
1,2,4-Triazolo-[3,4-b]-1,3,4-thiadiazines Containing an Amide Linkage
a
c
Pushpan Puthiyapurayil,a,b Boja Poojary, and Sunil Kumar Buridipad
*
aDepartment of Chemistry, Mangalore University, Mangalagangotri, 574199, Karnataka, India
bSyngene International Ltd, Biocon Park, Jigani, Bangalore, 560099, Karnataka, India
cDepartment of Pharmaceutical Chemistry, NET Pharmacy College, Raichur, 584103, Karnataka, India
*
E-mail: bojapoojary@gmail.com
Additional Supporting Information may be found in the online version of this article.
Received February 29, 2012
DOI 10.1002/jhet.1766
Published online 1 April 2014 in Wiley Online Library wileyonlinelibrary.com.
N
S
SH
N
N
N
S
N
N
N
N
N
N
O
N
O
N
O
N
O
N
NH
N
Ar
N
F
F
F
F
9a-k
F
F
F
F
F
N
N
N
N
N
N
N
N
N
Cl
Cl
N
N
O
Cl
H2N
S
S
SH
O
O
NH
13a-g
Ar
Two series of combinatorial library of 3,6-disubstituted-7H-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazines bearing
an amide linkage were synthesized. All the newly synthesized compounds were characterized by spectral
analyses. The newly synthesized compounds were screened for their cytotoxicity, anti-inflammatory, and
analgesic activities. Among the tested compounds, the compound 9g (Ar = 4-(methoxybenzyl)piperazine) is the most
promising molecule with half maximal inhibitory concentration (IC50) value of 14.24 mM in (MCF-7) cells.
Compounds 9f (Ar = 4-(chlorobenzyl)piperazine), 9g, and 9k (Ar = 2-(fluorophenyl)piperazine) exhibited
excellent anti-inflammatory activity at a dose level of 50 mg/kg, almost comparable with the standard drug.
In case of analgesic activity among the tested compounds, the compounds 9f, 9g, and 9k showed more
potent and consistent activity in both 100 and 200 mg/kg/po doses with less ulcerogenic risk.
J. Heterocyclic Chem., 51, E55 (2014).
INTRODUCTION
heterocycles with wide range of biological activities [22,23].
In recent decades, the literature has been enriched with
progressive findings about the synthesis and pharmacological
activities of 4-amino-1,2,4-triazole-5-thione ring, which is a
core structure in various synthetic pharmaceuticals displaying
a wide range of biological activities [24–33].
In our recent studies toward the synthesis of amino-
triazolothiones, we were interested to prepare various
3-substituted-4-amino-1,2,4-triazolo-5-thiones, which could
be further utilized for the construction of triazolothiadiazines
analogs. During our research, we realized that it would
be worthwhile to design and synthesize a novel class of
3-substituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines bearing
an amide linkage at the sixth position of the thiadiazines ring
system (Scheme 1), which could exhibit an augmented recep-
tor/enzyme interaction on some biological targets. In the
course of our studies directed toward the synthesis of amide,
Synthesis of nitrogen containing heterocyclic systems has
been attracting increasing interest over the past decades
because of their utility in various applications. Over the years,
a special attention has been given to N-bridged heterocyclic
systems derived from 1,2,4-triazoles owing to their promising
biological activities such as antihypertensive, antimicrobial,
anticonvulsant, antidepressant, analgesic, antitumor [1–13],
and so forth. Many pyrazole derivatives make up the core
structure of numerous biologically active molecules. The
pyrazole derivatives have long been well known for their
biological activities such as antiviral/antitumor [14–16],
antibacterial [17], anti-inflammatory [18], analgesic [19],
fungistatic, and anti-hyperglycemic activity [20,21].
The fused ring of triazole and thiadiazines, namely triazo-
lothiadiazines, represents an important class of N-bridged
© 2014 HeteroCorporation

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P. Puthiyapurayil, B. Poojary, and S. K. Buridipad
Vol 51
Scheme 1. Modification in the series.
F
N
N
N
N
Ar
N
S
N
S
Cl
N
N
Cl
Without an amide linkage
Without an amide linkage
NH
Ar
B
A
N
N
S
N
N
N
O
With an amide linkage
N
HN
Ar
F
F
F
9
we attempted several amide coupling methods. All the
coupling agents except T3PW (propylphosphonic anhydride)
were invariably associated with certain limitations in terms
of poor yield, substrate stability, and difficulty in purification.
On the basis of these observations, we therefore decided to syn-
thesize a novel class of 4-amino-3-substituted-1,2,4-triazole-5-
thiones (6 and 10) and their corresponding combinatorial
library of condensed 3,6-disubstituted-7H-1,2,4-triazolo-[3,4-
b]-1,3,4-thiadiazine derivatives bearing an amide linkage, their
cytotoxicity, anti-inflammatory, and analgesic properties.
ethanol for 12 h to yield the corresponding 4-amino-5-[1-
methyl-5-(4-trifluoromethylphenyl)-1H-pyrazol-3-yl]-4H-
1,2,4-triazole-3-thiol (6). The derivatives (6) and (10) were
heated to reflux with ethyl bromo pyruvate in ethanol to
obtain the intermediate 3-[1-methyl-5-(4-trifluoromethyl-
phenyl)-1H-pyrazol-3-yl]-7H-1,2,4-triazolo-[3,4-b]-1,3,
4-thiadiazine-6-carboxylic acid ethyl ester (7) and 3-[1-(4-
chlorophenyl)-1-methyl-thyl]-7H-1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazine-6-carboxylic acid ethyl ester (11). The intermediates
(7) and (11) were hydrolysed using lithium hydroxide
monohydrate in methanol/water at 0ꢀC for 1 h to obtain the
corresponding carboxylic acids (8) and (12). Finally, the title
compounds (9a–k and 13a–g) were obtained in good yields
by treating carboxylic acid with different substituted amines,
T3PW, and N,N-diisopropylethylamine in dichloromethane
(DCM). The crude products were purified by flash column
chromatography (230–400 size mesh) using methanol in
dichloromethane to afford the pure products. The newly
CHEMISTRY
The synthetic methods for target compounds 9 and 13
were illustrated in Scheme 2 and 3. The starting compound
4-trifluoromethyl acetophenone was treated with sodium
methoxide and dimethyl oxalate in methanol at RT to yield
compound (1) by the reported procedure [34], which upon
cyclization using N-methyl hydrazine sulfate in glacial acetic
acid at 105ꢀC yielded the corresponding N-methyl-4-
(trifluoromethyl)phenyl pyrazole carboxylic acid methyl
esters (two isomers). The major isomer (2) was then hydrazi-
nolyzed using hydrazine hydrate in methanol at 100ꢀC to
obtain aryl hydrazides (4). The compound (4) on reaction
with carbon disulphide in methanolic potassium hydroxide
under reflux condition yielded corresponding 5-substituted-
1,3,4-oxadiazole-2-(3H)-thione (5); and intermediate 10
was synthesized according to reported procedure [33]. Inter-
mediate 5 was heated at 85ꢀC with hydrazine hydrate in
1
synthesized compounds were characterized by IR, H-NMR,
13C-NMR, LC–MS, and elemental analysis data. The spectral
data of newly synthesized intermediates and title compounds
are given in Experimental Section. The chemical profiles of
the compounds (9a–k and 13a–g) are given in Table 1.
BIOLOGICAL ACTIVITY
In vitro cytotoxic evaluation and time course MTT assay.
In vitro cytotoxicity of the synthesized compounds was
assessed by standard MTT bioassay in different cancer
Journal of Heterocyclic Chemistry
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1,2,4-Triazolo-[3,4-b]-1,3,4-thiadiazines Containing an Amide Linkage
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Scheme 2. Reagents and conditions: (i) Dimethyl oxalate, NaOMe, MeOH, RT; (ii) Methyl hydrazine hydrate, glacial AcOH, 105ꢀC; (iii) N2H4.H2O,
MeOH, 100ꢀC; (iv) CS2, KOH, MeOH, reflux; (v) N2H4.H20, EtOH, reflux; (vi) Ethyl bromo pyruvate, EtOH, reflux; (vii) LiOH.H2O, MeOH, H2O, 0ꢀC;
and (viii) T3P, diisopropylethylamine, amine, DCM, RT.
O
O
O
O
O-Na+
O
O
N
N
O
(ii)
(i)
N
N
+
F
F
F
F
O
F
F
F
F
F
F
F
F
(3) (Minor isomer)
(1)
(2) (Major isomer)
(iii)
SH
NH2
NH
N
SH
N
N
O
N
N
N
N
N
O
NH2
N
(iv)
(v)
N
F
N
F
F
F
F
F
F
(6)
(4)
F
F
(5)
(vi)
N
S
N
S
N
S
N
N
N
N
N
N
N
O
N
O
N
N
O
N
(vii)
(viii)
HO
N
O
N
HN
N
N
Ar
F
F
F
F
F
F
F
F
F
(7)
(8)
(9)
Scheme 3. Reagents and conditions: (i) Ethyl bromo pyruvate, EtOH, reflux; (ii) LiOH.H2O, MeOH, H2O, 0ꢀC; and (iii) T3P, diisopropylethylamine,
amine, DCM, RT.
Journal of Heterocyclic Chemistry
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P. Puthiyapurayil, B. Poojary, and S. K. Buridipad
Vol 51
Table 1
Characterization data of 3,6-disubstituted-7H-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazine-6-carboxylic acid amides (9a-k, 13a-g).
Compound
Ar
Mol. formula
Mol. weight
m.p. (ꢀC)
Yield (%)
9a
9b
9c
9d
9e
9f
9g
9h
9i
3-CF3-C6H4-
4-CH3-O-C6H4-CH2-
(R)-1-(4-Cl-C6H4)-(CH)CH3-
4-F-C6H4-CH2-
(S)-1-(4-Cl-C6H4)-(CH)CH3-
4-Cl-C6H4-CH2-N(CH2-CH2)2-
4-CH3-O-C6H4- CH2-N(CH2CH2)2-
(R)-2-CH3-O,a-CH3-C6H4-CH2-
4Cl-C6H4-
C23H15F6N7OS
C24H20F3N7O2S
C24H19ClF3N7OS
C23H17F4N7OS
C24H19ClF3N7OS
C27H24ClF3N8OS
C28H27F3N8O2S
C25H22F3N7O2S
C22H15ClF3N7OS
C21H21F3N8OS
C26H22F4N8OS
C22H21Cl2N5OS
C22H21Cl2N5OS
C25H26Cl2N6OS
C26H29ClN6O2S
C24H24ClFN6OS
C23H24ClN5O2S
C23H24ClN5O2S
551.48
527.53
545.98
515.50
545.98
601.06
596.64
541.56
517.92
490.51
570.58
474.42
474.42
529.50
525.08
499.01
470.00
470.00
190.0-192.1
185.3-187.3
223.6-225.5
194.2-197.3
100.0-104.3
100.0-104.8
92.0-95.8
112.0-117.0
216.0-219.2
90.1-95.0
103.0-107.2
69.1-71.9
69.5-72.8
76.3-80.9
74.8-79.3
64
65
70
70
86
73
77
64
78
63
72
85
92
84
85
95
85
89
9j
N-CH3-N(CH2-CH2)2-
9k
13a
13b
13c
13d
13e
13f
13g
2-F-C6H4-N(CH2-CH2)2-
(R)-1-(4-Cl-C6H4)-(CH)CH3-
(S)-1-(4-Cl-C6H4)-(CH)CH3-
4-Cl-C6H4-CH2-N(CH2-CH2)2-
4-CH3-O-C6H4- CH2-N(CH2-CH2)2-
2-F-C6H4-N(CH2-CH2)2-
(R)-2-CH3-O,a-CH3-C6H4-CH2-
3-CH3-O-C6H4- CH2-N(CH3)-
73.8-78.3
71.1-74.5
55.1-60.0
cells at 24 h of drug administration. In Ehrlich ascites
carcinoma (EAC) cells, the compounds carrying N-methyl-
(4-trifluoromethylphenyl)pyrazole (9a–k) were found to be
more effective when compared with the compounds
carrying 3-[1-(4-chlorophenyl)-1-methylethyl] (13a–g). To
assess the efficacy and to select the promising compounds
human breast cancer cell line, MCF-7, and alveolar
adenocarcinoma cell line, A549, were used.
acetylsalicylic acid, was used as a positive control. It caused
severe bleeding at a dose level of 100 mg/kg.
RESULTS AND DISCUSSION
Anticancer activity. In MCF-7 cells, the IC50 values for
the compounds carrying N-methyl-(4-trifluoromethylphenyl)-
pyrazole (9a–k) and 3-[1-(4-chlorophenyl)-1-methylethyl]
(13a–g) were in the range of 14.24–61.21 and 24.54–81.22 mM,
respectively (Table 2). Similarly, in A549 cells, the IC50
values for (9a–k and 13a–g) were found in the range of
39.21–85.11 and 64.22–72.11 mM, respectively. In MCF-7
cells, the compounds 6, 10, and 13f did not show 50%
inhibition even at a concentration of 100 mM; hence, they
were not evaluated in A549 cells. Present study reveals that
among the human cancer cell lines tested, MCF-7 cells were
more sensitive to all the tested compounds than A549 cells.
Many anticancer drugs are effective against MCF-7 and
A549 cells, by causing apoptosis through the expression of
caspase-3, generating reactive oxygen species and damaging
DNA [37]. Chemotherapeutic agents such as doxorubicin
and mitoxantrone cause cytotoxicity by generating reactive
oxygen species [38]. Hence, like the cytotoxic drugs, the
synthesized compounds may act as effective anticancer drug
by similar mechanism. The results of the in vitro
cytotoxicity revealed that compounds 6 and 10 having a free
mercapto and amino groups showed poor activity in both
EAC and MCF-7 cells. To draw a structure activity
relationship studies, we considered various substituted
amines for the synthesis of title compounds. Results from
Table 2 clearly indicates that compounds having free amine
and mercapto groups showed no activity even at a
In vivo anti-inflammatory and analgesic activity. In the
pharmacology study, we investigated anti-inflammatory and
analgesic activities as well as the ulcerogenic risk on acute
administration. Both starting compounds 6 and 10 and their
corresponding condensed derivatives (9a–k and 13a–g) were
evaluated. All the synthesized compounds were tested for
their anti-inflammatory activity using carregeenan-induced
rat hind paw edema method of Kasahara et al. [35]. The
analgesic activity of the compounds was studied by using the
acetic acid-induced writhing test in mice [36]. In both
screening tests, 100 mg/kg (body weight) doses of the test
drug were orally administered to the animals as a first step.
The compounds having more than 20% effectiveness in
relieving symptoms, even some of them are not be significant
statistically, were considered for further evaluation, and the
tests were repeated in two different dose levels (50 and
200 mg/kg). The analgesic activity was expressed as mean
increase in latency after drug administration ꢁ SEM relative
to controls, whereas the anti-inflammatory activity was
expressed as mean increase in paw volume ꢁ SEM, in terms
of millimeter and percentage inhibition in paw volume by
different doses of the compounds. Ulcerogenic effect on
acute administration was studied to assess the safety of
compounds at high dose levels (200 mg/kg). For the purpose
of comparison, a nonselective cyclooxygenase inhibitor,
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1,2,4-Triazolo-[3,4-b]-1,3,4-thiadiazines Containing an Amide Linkage
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Table 2
In-vitro cytotoxic activity of compounds (6, 9a-k, 10, 13a-g).
IC50 (mM)a
Compounds
EAC
MCF-7
A549
6
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
10
13a
13b
13c
70.25 ꢁ 0.02
25.48 ꢁ 0.03
28.70 ꢁ 0.005
36.54 ꢁ 0.0039
38.24 ꢁ 0.004
75.44 ꢁ 0.022
23.22 ꢁ 0.03
18.54 ꢁ 0.009
78.44 ꢁ 0.022
38.24 ꢁ 0.003
33.18 ꢁ 0.011
33.12 ꢁ 0.011
81.25 ꢁ 0.022
46.24 ꢁ 0.022
68.18 ꢁ 0.003
38.12 ꢁ 0.044
28.14 ꢁ 0.009
53.22 ꢁ 0.012
>100
>100
NTb
25.51 ꢁ 0.02
29.80 ꢁ 0.044
27.51 ꢁ 0.011
29.24 ꢁ 0.012
61.21 ꢁ 0.0039
23.12 ꢁ 0.0069
14.24 ꢁ 0.011
43.12 ꢁ 0.022
29.21 ꢁ 0.013
39.20 ꢁ 0.0039
41.12 ꢁ 0.009
>100
39.81 ꢁ 0.011
59.18 ꢁ 0.044
36.22 ꢁ 0.022
24.54 ꢁ 0.011
61.12 ꢁ 0.022
>100
59.01 ꢁ 0.025
43.02 ꢁ 0.18
44.28 ꢁ 0.017
52.18 ꢁ 0.022
85.11 ꢁ 0.012
48.12 ꢁ 0.022
39.21 ꢁ 0.012
>100
64.22 ꢁ 0.022
72.22 ꢁ 0.0011
>100
NTb
64.22 ꢁ 0.011
>100
68.12 ꢁ 0.022
72.11 ꢁ 0.009
>100
13d
13e
13f
NTb
13g
68.12 ꢁ 0.003
48.22 ꢁ 0.0013
>100
3.28 ꢁ 0.18
Doxorubicinc
5.22 ꢁ 0.03
2.82 ꢁ 0.0176
aEach experiment was independently performed three times and expressed as means ꢁ SD.
bNT not tested.
cDoxorubicin was used as reference drug.
Anti-inflammatory activity.
The investigation of anti-
inflammatory activity revealed that all compounds except
compounds 6, 9c, 9h, 9i, 10, 13a, 13e, and 13f showed
reduction in edema volume when compared with control
group at 100 mg/kg dose, po in any of the measurement
intervals. Among the tested compounds, the compounds 9e,
9g, 9f, and 9k showed excellent activity, whereas others were
less active at 100 mg/kg/po dose levels. Although generally
all of the compounds showed either increased (compounds
9a, 9d, 9f, 9j, 9k, and 13c) or similar anti-inflammatory
activity (compounds 9e, 9g, 13b, 13d, and 13g) to that
of 100 mg/kg dose at 200 mg/kg dose level, few compounds
(9f, 9g, and 9k) showed excellent activity at dose level of
concentration of 100 mM. The results are conclusive in showing
that there is a noticeable increase in cytotoxicity when the 3-
substituted-4-amino-5-mercapto-1,2,4-traizoles are condensed
with 1,3,4-thiadiazine ring. Among the testedcompounds, the
compounds (9a, 9f, 9g, and 13d) bearing 3-
trifluoromethylphenyl amide, [4-(4-chlorobenzyl)piperazine
amide], and [4-(4-methoxybenzyl)piperazine amide] at the sixth
position of the triazolothiadiazine ring are more potent than
the other analogs. The increased activity of these compounds
could be attributed to the presence of biologically active groups
such as trifluoromethylphenyl, 4-(4-chlorobenzyl)piperazine,
and 4-(4-methoxybenzyl)piperazine groups on the amide
linkage. We have also observed that compounds from
the first series (9a–k) were more active than the second
series (13a–g). These results further revealed that
compounds carrying N-methyl-(4-trifluoromethylphenyl)
pyrazole (9a–k) moiety were also responsible for the
enhanced cytotoxicity.
Table 3
In-vitro cytotoxic activity of compounds (9a, 9f, 9g and 13d) in MCF-7
cells at different time points of drug exposure by MTT assay.
In the MTT time course study, the selected compounds
(9a, 9f, 9g, and 13d) showed dose-dependent and time-
dependent activities. Experiments revealed that (Table 3)
there was substantial increase in cytotoxicity in MCF-7 cells
with increasing exposure to drug concentration. The IC50
values of the promising compounds at 48 and 72 h were sig-
nificantly reduced in comparison with 24 h values. The most
promising compound (9g) causes 1.45 and 2.85 times reduc-
tion in IC50 values at 48 and 72 h.
IC50 (mM)a
Compounds
24 h
48 h
72 h
9a
9f
9g
13d
25.51 ꢁ 0.02
20.80 ꢁ 0.011 14.11 ꢁ 0.013
23.12 ꢁ 0.0069 18.24 ꢁ 0.022 12.25 ꢁ 0.003
14.24 ꢁ 0.011
9.80 ꢁ 0.012
4.99 ꢁ 0.022
18.24ꢁ 0.0012
24.54 ꢁ 0.011 22.23 ꢁ 0.005
aEach experiment was independently performed three times and expressed
as means ꢁ SD.
Journal of Heterocyclic Chemistry
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P. Puthiyapurayil, B. Poojary, and S. K. Buridipad
Vol 51
50 mg/kg. In general, in case of anti-inflammatory activity,
compounds carrying N-methyl-[4-(trifluoromethyl)phenyl]
pyrazole (9a–k) moiety at the third position of the fused ring
are more potent than the other analogs carrying [1-
(4-chlorophenyl)-1-methylethyl] (13a–g) moiety at the third
position. Among all the compounds, compounds 9f, 9g, and
9k having piperazine bridge between the thiadiazine ring and
phenyl group of the amide at the sixth position of the fused
ring possessed the most prominent and consistent activity.
The results are tabulated in Table 4.
Table 4
Dose-dependent anti-inflammatory effects of test compounds (6, 9a-k, 10, 13a-g) against carrageenan-induced hind paw edema model in mice in different
doses.
Swelling in thickness (x 10-2 mm) ꢁ SEM (% inhibition)
Compounds
Dose mg/kg (per os)
90 min
180 min
270 min
360 min
6
100
50
200
100
50
200
100
50
200
100
100
50
200
100
50
200
100
50
200
100
50
200
100
100
100
50
200
100
50
200
100
100
100
100
50
200
100
50
200
100
100
100
50
42.4ꢁ 4.5 (21.0)
29.2 ꢁ 4.0
58.6 ꢁ 3.1 (24.3)
44.5 ꢁ 6.5
54.8 ꢁ 2.1
39.0 ꢁ 2.8 (18.5)
24.6 ꢁ 2.1** (29.6)
37.5 ꢁ 1.9 (21.2)
27.5 ꢁ 4.4** (42.7)
39.1 ꢁ 3.7 (20.4)
28.2 ꢁ 3.5* (41.7)
26.8 ꢁ 4.3 **
51.8 ꢁ 4.5 (20.9)
59.0 ꢁ 4.5
40.1 ꢁ 3.0
76.8 ꢁ 8.5
9a
9b
40.1 ꢁ 1.9
55.2 ꢁ 2.9
44.8 ꢁ 2.0* (35.8)
60.2 ꢁ 2.8
43.1 ꢁ 3.4
61.8 ꢁ 3.9
39.5 ꢁ 4.1
54.2 ꢁ 5.2
41.9 ꢁ 5.0* (39.3)
42.8 ꢁ 2.5*
39.8 ꢁ 1.9
57.4 ꢁ 3.0
42.5 ꢁ 3.2
61.9 ꢁ 3.8
62.5 ꢁ 2.9
39.4 ꢁ 3.5 *
40.1 ꢁ 4.4
53.0 ꢁ 5.0 (26.6)
6.12 ꢁ 3.3
42.5 ꢁ 5.4* (40.1)
68.9 ꢁ 3.3
28.5 ꢁ 3.5 (41.7)
55.4 ꢁ 3.2
9c
9d
40.7 ꢁ 1.9
40.1 ꢁ 3.0
43.1 ꢁ 7.0 (34.1)
60.7 ꢁ 3.1
41.2 ꢁ 4.1** (42.1)
58.4 ꢁ 4.4*
36.8 ꢁ 2.1
40.5 ꢁ 2.8
49.5 ꢁ 2.5
33.1 ꢁ 3.0 (24.5)
41.8 ꢁ 3.9
31.2 ꢁ 2.5* (50.8)
44.8 ꢁ 7.6
32.5 ꢁ 4.0** (50.0)
39.8 ꢁ 4.8* (41.0)
50.8 ꢁ 2.0
28.5 ꢁ 2.6** (42.5)
42.8 ꢁ 3.8* (51.2)
38.2 ꢁ 3.0 (21.4)
40.1 ꢁ 3.6** (51.0)
28.0 ꢁ 1.6** (48.0)
38.1 ꢁ 3.4
9e
9f
46.2 ꢁ 2.1
61.8 ꢁ 3.0 (29.8)
46.1 ꢁ 3.4 (29.8)
37.2 ꢁ 2.6** (51.8)
61.4 ꢁ 3.0 (29.8)
46.4 ꢁ 3.4 (49.8)
37.2 ꢁ 2.6*** (53.4)
58.2 ꢁ 2.8* (29.1)
32.2 ꢁ 2.6* (50.1)
62.0 ꢁ 3.0
37.6 ꢁ 3.6
38.2 ꢁ 4.4* (44.8)
37.4 ꢁ 1.8** (51.3)
54.8 ꢁ 2.0 (32.1)
38.2 ꢁ 4.3* (55.0)
37.2 ꢁ 1.8** (49.5)
59.0 ꢁ4.2* (32.1)
33.1 ꢁ 4.1** (49.5)
65.0 ꢁ 2.8
23.0 ꢁ 1.2* (51.0)
44.8 ꢁ 2.4 (28.2)
37.4 ꢁ 3.2
41.4 ꢁ 3.4* (51.8)
28.0 ꢁ 1.4** (51.4)
48.2 ꢁ 2.6 (27.4)
28.9 ꢁ 2.6 (52.3)
65.0 ꢁ 2.4
9g
23.0 ꢁ 1.2** (41.8)
41.1 ꢁ 2.6* (31.2)
30.1 ꢁ 3.0
9h
9i
9j
40.8 ꢁ 1.8
41.1 ꢁ 2.0
60.8 ꢁ 2.4
67.2 ꢁ 3.4
58.2 ꢁ 3.0
30.9 ꢁ 3.4 (26.0)
37.2 ꢁ 3.4
41.8 ꢁ 3.6* (34.9)
50.8 ꢁ 5.0 (20.6)
33.4 ꢁ 3.6** (50.8)
37.0 ꢁ 2.8** (50.9)
60.5 ꢁ 3.0 (39.8)
46.0 ꢁ 3.0
54.4 ꢁ 4.0 (21.4)
58.0 ꢁ 3.0
39.0 ꢁ 4.4
52.4 ꢁ 2.6
32.8 ꢁ 4.0 (23.6)
24.2 ꢁ 1.2*
44.1 ꢁ 2.4
46.0 ꢁ 4.0* (32.0)
37.2 ꢁ 1.6* (47.5)
52.8 ꢁ 2.1 (34.2)
39.1 ꢁ 4.2** (55.0)
68.0 ꢁ 3.1
32.9 ꢁ 4.1* (43.3)
28.2 ꢁ 1.8** (42.0)
39.1 ꢁ 3.0 (30.8)
41.6 ꢁ 3.8* (51.0)
45.2 ꢁ 3.4
9k
37.0 ꢁ 3.0
10
40.7 ꢁ 1.6
60.8 ꢁ 3.0
13a
13b
13c
41.5 ꢁ 1.8
60.9 ꢁ 2.8
59.2 ꢁ 1.8
46.2 ꢁ 3.0
37.6 ꢁ 2.8
42.8 ꢁ 3.4
39.0 ꢁ 3.2* (41.0)
50.1 ꢁ 3.1 (21.6)
62.5 ꢁ 2.7
39.8 ꢁ 3.0* (41.2)
31.0 ꢁ 2.4* (36.5)
45.4 ꢁ 4.4
41.5 ꢁ 4.0
50.1 ꢁ 3.2
39.0 ꢁ 3.0
60.5 ꢁ 2.0
31.2 ꢁ 1.8 (36.6)
44.8 ꢁ 3.2
44.2 ꢁ 1.8 (37.4)
50.0 ꢁ 4.0
45.0 ꢁ 1.5* (33.9)
44.9 ꢁ 2.6 (34.9)
58.7 ꢁ 3.6
30.0 ꢁ 1.8* (37.0)
31.5 ꢁ 2.2* (36.5)
38.0 ꢁ 4.0
13d
39.0 ꢁ 2.6
60.4 ꢁ 3.1
37.6 ꢁ 3.0
46.7 ꢁ 3.7 (33.5)
61.0 ꢁ 2.6
42.4 ꢁ 4.2 (32.2)
50.2 ꢁ 1.6
30.0 ꢁ 1.8* (37.5)
44.8 ꢁ 2.8
13e
13f
13g
41.8 ꢁ 1.8
41.5 ꢁ 1.8
61.0 ꢁ 2.8
52.2 ꢁ 1.8
46.2 ꢁ 2.8
20.2 ꢁ 1.8*
44.1 ꢁ 2.0
36.0 ꢁ 2.2* (36.4)
60.0 ꢁ 2.8 (39.8)
44.4 ꢁ 3.2
36.2 ꢁ 1.8*
30.1 ꢁ 1.8
50.8 ꢁ 2.8
39.1 ꢁ 3.0
200
34.2 ꢁ 2.8
40.0 ꢁ 3.2** (40.0)
59.0 ꢁ 6.2
40.4 ꢁ 3.2* (48.0)
55.0 ꢁ 4.0
Control
Stda
40.0 ꢁ 4.4
56.4 ꢁ 7.0
100
24.8 ꢁ 1.1* (51.4)
37.4 ꢁ 2.4*** (53.2)
37.6 ꢁ 1.8** (56.0)
28.6 ꢁ 1.2** (51.5)
*p < 0.05;
**p < 0.01;
***p < 0.001;
Significant from control (n = 4-5)
aIndomethacin.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

August 2014
1,2,4-Triazolo-[3,4-b]-1,3,4-thiadiazines Containing an Amide Linkage
E61
Table 5
Table 5
(Continued)
Dose-dependent analgesic effects of the test compounds (6, 9a-k, 10,
13a-g) on acetic acid-induced writhing response and ulcer score in mice.
Dose mg/
kg (per os)
Ulcer
score
Analgesic activity meanꢁ
Compounds
SEM (% inhibition)
Dose mg/
kg (per os)
Ulcer
score
Analgesic activity meanꢁ
Compounds
SEM (% inhibition)
200
100
2/4
0/4
4/5
54.2 ꢁ 1.18* (31.0)
68 ꢁ 1.12
Control
ASP
6
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
200
100
50
51.2 ꢁ 0.08** (20.2)
60.1 ꢁ 0.04* (21.2)
56.9 ꢁ 1.7** (24.2)
39.2 ꢁ 1.08* (56.0)
60.2 ꢁ 2.0 (15.0)
52.4 ꢁ 0.9*** (68.4)
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
3/4
0/4
0/4
2/4
3/4
3/4
3/4
2/4
2/4
3/4
*p < 0.05;
**p < 0.01;
***p < 0.001;
9a
38.0 ꢁ 2.0*** (56.1)
32.5 ꢁ 2.0* (57.0)
69.0 ꢁ 10.0 (20.0)
44.3 ꢁ 1.08* (36.0)
55.0 ꢁ 1.08* (20.2)
61.1 ꢁ 0.04 (21.8)
50.8 ꢁ 1.6** (23.2)
41.9 ꢁ 2.0* (52.0)
60.1 ꢁ 2.0* (23.1)
40.3 ꢁ 2.0** (53.0)
28.1 ꢁ 1.01* (40.1)
70.1 ꢁ 0.08* (25.2)
43.2 ꢁ 1.14* (41.2)
28.1 ꢁ 1.12* (60.2)
55.4 ꢁ 1.08* (30.0)
43.2 ꢁ 1.5* (58.2)
40.8 ꢁ 1.0*** (62.1)
58.5 ꢁ 5.0* (28.0)
38.6 ꢁ 1.12** (64.2)
40.4 ꢁ 1.0* (45.0)
68.0 ꢁ 1.12* (25.0)
40.3 ꢁ 1.08* (39.0)
28.2 ꢁ 0.05* (30.2)
58.2 ꢁ 1.01* (15.0)
40.1 ꢁ 1.14* (30.2)
43.0 ꢁ 1.6* (50.2)
60.9 ꢁ 2.6* (28.5)
39.8 ꢁ 1.4 (51.2)
Significant from control (n = 4-5).
9b
9c
Analgesic activity. Analgesic screening study revealed
that the compounds 9a, 9b, 9d, 9f, 9g, 9j, and 9k had either
higher or similar analgesic activity to that of aspirin at the
100 mg/kg dose level, whereas others showed less activity
when compared for percentage inhibition values. When
experiments were repeated at 200 mg/kg/po dose, the
compounds 9a, 9d, 9f, 9g, 9j, and 9k exhibited similar
activity profile to that of a 100 mg/kg dose level, whereas
compounds 9b, 9h, and 13b showed less percentage
inhibition. Among all the tested compounds, except 9k,
when administered in decreasing dose (50 mg/kg), the
activity was found to be decreased. The compound 9k
exhibited a higher percentage inhibition than aspirin in both
100 and 200 mg/kg/po doses among the set of compounds.
In general, in case of analgesic activity, compounds carrying
N-methyl-[4-(trifluoromethyl)phenyl]pyrazole (9a–k) moiety
are more potent than the other analogs carrying [1-
(4-chlorophenyl)-1-methylethyl] (13a–g) moiety at the third
position of triazolothiadiazine ring. Among the set of title
compounds, the compounds (9f, 9g, and 9k) having piperazine
bridge between triazolothiadiazine ring and phenyl groups at
the sixth position of the amide linkage were more potent and
displayed consistent activity. The results are given in Table 5.
Acute ulcerogensis. The compounds that were screened
for analgesic activity were further studied for their acute
ulcerogenic risk. As a general consideration, all compounds
except 9c, 9e, 9i, 13a, 13b, 13c, and 13f were found to be
safer as gastric lesion risks at high dose (200 mg/kg/po)
when compared with aspirin. Among the tested compounds,
compounds 6, 9a, 9b, 9g, 9j, and 9k induced no ulcerogenic
effect at high dose level, whereas aspirin caused severe
bleeding with 4/5 score at the same dose. It could be
hypothesized that the compounds causing lesser gastric
lesions at 200 mg/kg dose when compared with aspirin, may
display decreased ulcerogenic risk at lower doses.
9d
9e
9f
9g
9h
9i
9j
9k
10
40.2 ꢁ 1.18** (64.2)
59.5 ꢁ 2.2* (60.1)
39.9 ꢁ 0.09* (65.2)
61.4 ꢁ 0.08* (18.0)
65.4 ꢁ 0.04** (9.5)
44.5 ꢁ 1.17* (8.5)
46.1 ꢁ 1.01* (20.1)
57.2 ꢁ 0.9* (12.0)
44.2 ꢁ 1.11* (21.2)
52.1 ꢁ 2.0* (30.2)
65.2 ꢁ 1.18** (15.4)
46.8 ꢁ 1.12** (16.0)
38.2 ꢁ 0.09* (40.1)
45.2 ꢁ 2.3* (20.2)
46.4 ꢁ 3.2* (42.3)
43.0 ꢁ 1.6* (42.4)
63.2 ꢁ 2.5* (22.1)
39.8 ꢁ 1.5*** (44.5)
28.1 ꢁ 0.5** (46.5)
41.2 ꢁ 2.2** (24.0)
45.8 ꢁ 1.8* (48.0)
41.0 ꢁ 1.0* (31.4)
56.0 ꢁ 1.18* (15.0)
4 6.2 ꢁ 1.12* (29.2)
38.2 ꢁ 3.2* (29.2)
60.2 ꢁ 1.2* (12.0)
13a
13b
13c
13d
13e
13f
13g
200
100
50
200
100
50
200
100
50
200
100
50
CONCLUSION
We have synthesized two 4-amino-3-substituted-1,2,4-
triazole-5-thiones (6 and 10) and their corresponding con-
densed derivatives 3,6-disubstituted-7H-1,2,4-triazolo-[3,
4-b]-1,3,4-thiadiazine (9a–k and 13a–g) bearing an amide
linkage with the objective of developing better cytotoxic,
(Continued)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E62
P. Puthiyapurayil, B. Poojary, and S. K. Buridipad
Vol 51
of (0.5M) sodium methoxide (8.6 g, 435mL, 159 mmol). Reaction
mass was allowed to stir at RT for 1 h. A solution of dimethyl
oxalate (18.8g, 159mmol) in anhydrous methanol (20mL) was
added dropwise and allowed to stir at RT for 24h. The progress of
the reaction was monitored by TLC. Upon completion of the
reaction, the precipitate obtained was isolated and washed with
chilled methanol followed by diethyl ether and dried in vacuum to
obtain the pure product as sodium salt.
analgesic, and anti-inflammatory molecules with minimum
ulcerogenic risk. Among the tested compounds, the
compounds (9a, 9f, 9g, and 13d) showed good activity in
MCF-7 cells with IC50 values of the range from 14.24
to 25.51 mM. In case of analgesic and anti-inflammatory activ-
ities, several derivatives have been evaluated for potential
analgesic/anti-inflammatory activity devoid of ulcerogenic
risk. The results are conclusive in showing that there is a
noticeable increase in analgesic and anti-inflammatory activ-
ity when 3-substituted-4-amino-5-mercapto-1,2,4-triazoles
are condensed with 1,3,4-thiadiazine ring. The anti-inflamma-
tory study revealed that compounds 9a, 9d, 9f, 9j, 9k, and 13c
showed increased activity at 200 mg/kg dose level. Com-
pounds having piperazine (9f, 9g, and 9k) bridge between the
triazolothiadiazine ring and phenyl group of the amide linkage
showed excellent activity at a dose level of 50 mg/kg. In case of
analgesic activity among the tested compounds, the compounds
9f, 9g, and 9k having a piperazine bridge between the core tria-
zolothiadiazine ring and phenyl group of the amide linkage car-
rying 4-chloride, 4-methoxy, and 2-fluoride substituents
showed more potent and consistent activity in both 100 and
200 mg/kg/po doses and less ulcerogenic risk. These results
are in parallel to those of Prasad et al. [30] and S.P. Aytac
et al. [39]. In general, the analgesic and anti-inflammatory
studies revealed that compounds carrying N-methyl-[4-(trifluor-
omethyl)phenyl]pyrazole (9a–k) are more potent than the other
analogs carrying [1-(4-chlorophenyl)-1-methylethyl] moiety
(13a–g). Further studies may reveal the exact mechanism re-
sponsible both for the analgesic/anti-inflammatory and gastric
toxicity. In conclusion, these types of compounds with promis-
ing cytotoxic, analgesic/anti-inflammatory activity profile
make them certain lead molecules for further lead optimization
in the development of novel anticancer, analgesic, and anti-
inflammatory agents.
1
Yellow color semisolid. Yield: 11.3 g (48%). H-NMR (400 MHz,
DMSO-d6): d 7.94 (d, J=8.0 Hz, 2H), 7.88 (d, J=8.0 Hz, 1.3H), 7.74
(d, J=8.2 Hz, 2H), 7.66 (d, J=8.1 Hz, 1.3H), 6.26 (s,1H), 5.30 (s,
0.7H), 3.65 (s, 3H), 3.57 (s, 2H). MS (MM-ES + APCI) 273.0 (M ꢂ H)+.
Synthesis of 1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-
pyrazole-3-carboxylate acid methyl ester (2). To a mixture
of compound (1) (11.3g, 38.1 mmol) and methyl hydrazine sulfate
(8.25 g, 57.2 mmol), glacial acetic acid (152mL) was added under
N2 atmosphere and heated at 105ꢀC for 1 h. The completion of the
reaction was confirmed by TLC. Upon completion of the reaction,
the solvent was removed in vacuum. Residue obtained was diluted
with water (100mL) and extracted in ethyl acetate (3 ꢃ 150 mL).
The combined extract was washed with 10% sodium bicarbonate
solution, water followed by brine and dried over anhydrous
sodium sulfate. The solvent was removed under reduced
pressure, and the crude product was purified by flash column
chromatography (230–400 size mesh) using 18–19% ethyl
acetate in pet ether to afford the minor isomer (compound 3)
and 28–29% ethyl acetate in pet ether to afford the major
isomer (compound 2).
Yield (6.4 g, 63%), off white solid, mp = 108.5–109.9ꢀC. Found: C,
55.23; H, 4.15; N, 10.05. Calcd for C13H11F3N2O2: C, 54.93; H, 3.90;
N, 9.86. IR (KBr) υ/cmꢂ1: 3132, 3081 (Ar—H), 3023, 2954 (C—H),
1712 (C═O), 1618 (C═N), 1474 (C═C). 1H-NMR (400 MHz,
CDCl3): d 7.76 (d, J=8.1 Hz, 2H), 7.57 (d, J=8.0 Hz, 2H), 6.91 (s,
1H), 3.97 (s, 3H), 3.95 (s, 3H). MS (MM-ES + APCI) 285.2 (M + H)+.
Procedure for the synthesis of 1-methyl-5-[4-(trifluoromethyl)
phenyl]-1H-pyrazole-3-carbohydrazide (4).
To
a
stirred
solution of compound (2) (1 mmol) in methanol (2 mL),
hydrazine hydrate (2 mmol) was added at RT under N2
atmosphere. The reaction mass was allowed to heat at 100ꢀC for
2 h. After the completion of the reaction as monitored by TLC,
solvent was removed in vacuum. The residue obtained was
diluted with ethyl acetate, washed with water, brine and dried
over anhydrous sodium sulfate. The solvent was removed under
reduced pressure, and the crude product was recrystallised from
methanol to afford the pure product (4).
EXPERIMENTAL
General.
1H-NMR and 13C-NMR spectra were recorded on
400 and 100 MHz Bruker spectrometer, respectively, and elemental
analysis was performed on a Thermo Finnigan FLASH EA 1112
carbon, hydrogen and nitrogen (CHN) analyzer. Melting points were
recorded (uncorrected) on Buchi melting B-545 instrument. Infrared
spectra were recorded on a Thermo scientific Nicolet 6700 FTIR
spectrometer. Coupling constants were reported whenever it was
necessary in hertz (Hz). The mass spectra were recorded on Agilent
LC/MSD SL 1100 instrument. Reactions were carried out in an
oven dried three necked round bottomed flask. Yields in table refer
to isolated yields of the title compounds with purity >95% as
determined by LC–MS analysis. All the reagents and solvents used
were of analytical grade and were used as supplied, unless otherwise
stated. TLC was performed on aluminum plates coated with silica
gel (Merck) for monitoring the reactions.
Yield (90%), off white solid, mp = 142.1–144.8ꢀC. Found:
C, 51.03; H, 4.15; N, 19.91. Calcd for C12H11F3N4O: C, 50.71;
H, 3.90; N, 19.71. IR (KBr) υ/cmꢂ1: 3315 (—NH—), 3265,
3204 (—NH2), 3059 (Ar—H), 2935 (C—H), 1598 (C═O), 1537
(C═C). 1H-NMR (400 MHz, DMSO-d6): d 9.44 (s, 1H), 7.88
(d, J= 8.3 Hz, 2H), 7.82 (d, J =8.2 Hz, 2H), 6.90 (s, 1H), 4.4
(s, 2H), 3.92 (s, 3H). MS (MM-ES + APCI) 285.2 (M + H)+.
Procedure for the synthesis of 5-{5-[4-(trifluoromethyl)phenyl]-
1-methyl-1H-pyrazole-3-yl}-1,3,4-oxadiazole-2-thione (5).
To a
stirred solution of compound (4) (1 mmol) in anhydrous
methanol (5 mL), potassium hydroxide (1.5 mmol) was added
followed by carbon disulphide (1.5 mmol) at 0ꢀC under N2
atmosphere. Reaction mass was heated at 65ꢀC for overnight
period. Starting material consumption was monitored by TLC, upon
completion of the reaction, solvent was removed in vacuum.
Residue obtained was diluted with water and extracted in ethyl
acetate. Organic phase was washed with water, brine solution and
Synthesis of sodium; (Z)-3-methoxycarbonyl-3-oxo-1-
(4-trifluoromethyl)phenylpropen-1-olate (1).
To a stirred
solution of 4-trifluoromethyl acetophenone (15 g, 79.7 mmol) in
anhydrous methanol (15 mL), cooled at 0ꢀC was added a solution
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

August 2014
1,2,4-Triazolo-[3,4-b]-1,3,4-thiadiazines Containing an Amide Linkage
E63
dried over anhydrous sodium sulfate. The solvent was removed under
reduced pressure, and the crude product was purified by flash
column chromatography (60–120 size mesh) using 35–36% ethyl
acetate in pet ether to afford the pure product (5).
N, 15.36. IR (KBr) υ/cmꢂ1: 2984, 2930 (C—H), 1717 (C═O),
1467 (C═C), 824 (C—Cl). H-NMR (300 MHz, CDCl3): d 7.22
1
(s, 4H), 4.34 (q, J = 7.1 Hz, 2H), 3.75 (s, 2H), 1.86 (s, 6H), 1.38
(t, J = 7.1 Hz, 3H) MS (MM-ES + APCI) 365.2 (M+ H)+.
Yield (91%), off white solid, mp= 226.4–227.8ꢀC. Found:
C, 48.05; H, 3.00; N, 17.32. Calcd for C13H9F3N4OS: C, 47.85;
H, 2.78; N, 17.17. IR (KBr) υ/cmꢂ1: 3113 (Ar—H), 2947 (C—H),
2605 (—SH), 1622 (C═N), 1440 (C═C), 1318 (—N—N═C).
1H-NMR (400 MHz, DMSO-d6): d 14.77 (br, s, 1H), 7.91
(d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.14 (s, 1H), 3.99
(s, 3H). 13C-NMR (100 MHz, DMSO-d6): d 176.7, 153.2, 148.2,
135.6, 128.4, 127.7, 125.7, 122.8, 106.6, 39.0. MS (MM-ES +
APCI) 325.0 (M+ H)+
General procedure for the synthesis of 3-[5-(4-
trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-1,2,4-triazolo
[3,4-b]-1,3,4-thiadiazine-6-carboxylic acid (8) and 3-[1-(4-
chlorophenyl)-1-methylethyl]-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-
6-carboxylic acid (12). To a stirred solution of 1,2,4-triazolo-
[3,4-b]-1,3,4-thiadiazine-6-carboxylic acid ethyl ester (1 mmol)
in methanol/water (4:1), lithium hydroxide monohydrate
(2 mmol) was added at 0ꢀC. The reaction mass was allowed to
stir at 0ꢀC for 1 h. After the completion of the reaction as
monitored by TLC, solvent was removed in vacuum. The
residue obtained was acidified with saturated solution of citric
acid and extracted in ethyl acetate (3 ꢃ 75 mL). Combined
organic layer was washed with water, brine solution and dried
over anhydrous Na2SO4. The crude product obtained was
purified by the column chromatography (60–120 size mesh)
using 4.0–5.0% methanol in dichloromethane to afford the
pure product.
3-{5-[4-(Trifluoromethyl)phenyl]-1-methyl-1H-pyrazol-3-yl}-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic acid (8). Yield
(64%), off white solid, mp = 162.0–164.5ꢀC. Found: C, 47.28; H,
2.95; N, 20.83. Calcd for C16H11F3N6O2S: C, 47.06; H, 2.72; N,
20.58. IR (KBr) υ/cmꢂ1: 3140 (Ar—H), 2952, 2918 (C—H), 1649
(C═O), 1619 (C═N), 1402 (C═C), 1321 (C—O). 1H-NMR
(400 MHz, DMSO-d6): d 14.0 (br, s, 1H), 7.90 (d, J=8.3 Hz, 2H),
7.85 (d, J=8.2 Hz, 2H), 7.06 (s, 1H), 4.12 (s, 2H), 3.98 (s, 3H). MS
(MM-ES + APCI) 409.0 (M + H)+.
Synthesis of 4-amino-5-{5-[4-(trifluoromethyl)phenyl]-1-
methyl-1H-pyrazol-3-yl}-4H-1,2,4-triazole-3-thiol (6).
To a
stirred solution of compound 5 (11.3 g, 34.6 mmol) in ethanol
(173mL), hydrazine hydrate was added and heated at 85ꢀC for
12h. The progress of the reaction was monitored by TLC, upon
completion of the reaction, solvent was removed in vacuum.
Residue obtained was diluted with ethyl acetate (200mL), washed
with water (2ꢃ 100 mL), brine solution and dried over anhydrous
Na2SO4. Crude product obtained was purified by column
chromatography (60–120 size mesh) using 45–50% ethyl acetate
in pet ether to afford the pure product (6).
Yield: 10.1 g (84%), off white solid. mp = 197.8–198.9ꢀC. Found:
C, 46.20; H, 3.48; N, 24.87. Calcd for C13H11F3N6S: C, 45.88;
H, 3.26; N, 24.69. IR (KBr) υ/cmꢂ1: 3401 (—NH2— anti), 3250
(—NH2— sym), 3178 (Ar—H), 2987, 2946 (C—H), 1621 (C═N),
1484 (C═C). 1H-NMR (400 MHz, DMSO-d6): d 13.89 (s, 1H),
7.91 (d, J=8.2 Hz, 2H), 7.86 (d, J= 8.3 Hz, 2H), 7.16 (s, 1H), 5.89
(s, 2H), 3.98 (s, 3H). MS (MM-ES+ APCI) 341.0 (M + H)+.
3-[1-(4-Chlorophenyl)-1-methylethyl]-7H-1,2,4-triazolo[3,4-b]-1,3,4-
General procedure for the synthesis of 3-[5-(4-
trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-1,2,4-triazolo
[3,4-b]-1,3,4-thiadiazine-6-carboxylic acid ethyl ester (7) and 3-[1-
(4-chlorophenyl)-1-methylethyl]-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-
6-carboxylic acid ethyl ester (11). To a stirred solution of 4-amino-
5-substituted-4H-1,2,4-triazole-3-thiol (1 mmol) in absolute
alcohol (10 mL), ethyl bromo pyruvate (1.2 mmol) was added
at RT under N2 atmosphere. Reaction mixture was heated to
reflux for 6 h. Upon completion of the reaction as monitored by
the TLC, solvent was removed in vacuum. Residue obtained
was diluted with water and extracted in ethyl acetate
(3 ꢃ 100 mL). The combined extract was washed with 10%
sodium bicarbonate solution, water followed by brine and dried
over anhydrous Na2SO4. The solvent was removed under
reduced pressure, and the crude product was purified by flash
column chromatography (230–400 size mesh) using 0.5–0.6%
methanol in dichloromethane to afford the pure product.
thiadiazine-6-carboxylic acid (12).
Yield (83%), off white solid,
mp = 162.4–164.8ꢀC. Found: C, 50.05; H, 3.69; N, 16.71. Calcd for
C14H13ClN4O2S: C, 49.93; H, 3.89; N, 16.83. IR (KBr) υ/cmꢂ1: 2983,
2928 (C—H), 1722 (C═O), 1618 (C═N), 1466 (C═C), 1295 (C—O).
1H-NMR (400 MHz, DMSO-d6): d 14.08 (br, s, 1H), 7.32 (d,
J=8.5 Hz, 2H), 7.20 (d, J=8.5 Hz, 2H), 4.02 (s, 2H), 1.73 (s, 6H).
MS (MM-ES + APCI) 337.0 (M + H)+.
General procedure for the synthesis of 1,2,4-triazolo-[3,4-b]-
1,3,4-thiadiazine-6-carboxylic acid amides (9a–k, 13a–g). To a
stirred solution of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazine-
6-carboxylic acid (1mmol) in anhydrous dichloromethane (5 mL),
1-propane-phosphonic acid cyclic anhydride (50% solution in
EtOAc, 2.5 mmol) was added dropwise at 0ꢀC under N2
atmosphere followed by diisopropyl ethyl amine (3.0mmol) and
allowed to stir at 0ꢀC for 10min. Substituted amine (1.1 mmol)
was added and allowed to stir at RT for 3–4 h. Starting material
consumption was monitored by TLC, upon completion of the
reaction, reaction mass was diluted with dichloromethane. Organic
phase was washed with 10% sodium bicarbonate solution, water,
brine solution and dried over anhydrous Na2SO4. The crude
product obtained was purified by flash column chromatography
(230–400 size mesh) using 1.0–2.5% methanol in dichloromethane
to afford the pure product.
3-[5-(4-Trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic acid ethyl
ester (7).
Yield (71%), off white solid, mp = 178.0–179.3ꢀC.
Found: C, 47.74; H, 3.34; N, 19.48. Calcd for C18H15F3N6O2S: C,
47.54; H, 3.46; N, 19.26. IR (KBr) υ/cmꢂ1: 3162 (Ar—H), 2980,
1
2910 (C—H), 1718 (C═O), 1617 (C═N), 1457 (C═C). H-NMR
(400 MHz, DMSO-d6): d 7.91 (d, J= 8.3 Hz, 2H), 7.85 (d,
J= 8.2 Hz, 2H), 7.09 (s, 1H), 4.36 (q, J =7.0 Hz, 2H), 4.21 (s, 2H),
4.02 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H). MS (MM-ES + APCI) 437.2
(M + H)+.
3-[5-(4-Trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic
trifluoromethylphenyl)amide (9a). Yield (0.10 g, 64%), off
white solid, Found: C, 50.34; H, 2.97; N, 18.08. Calcd for
C23H15F6N7OS: C, 50.09; H, 2.74; N, 17.78. IR (KBr) υ/cmꢂ1
3246 (amide —NH—), 3154, 3083 (Ar—H), 2998 (C—H), 1666
(amide C═O), 1607 (C═N), 1450 (C═C). H-NMR (400 MHz,
acid-(3-
3-[1-(4-Chlorophenyl)-1-methylethyl]-7H-1,2,4-triazolo[3,4-b]-
1,3,4-thiadiazine-6-carboxylic acid ethyl ester (11).
Yield
:
(64%), off white solid, mp= 122.0–126.5ꢀC. Found: C, 52.89;
1
H, 4.90; N, 15.54. Calcd for C16H17ClN4O2S: C, 52.67; H, 4.70;
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E64
P. Puthiyapurayil, B. Poojary, and S. K. Buridipad
Vol 51
CDCl3): d 9.59 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.94 (s, 1H), 7.79
(d, J = 7.8Hz, 2H), 7.63 (d, J = 7.9Hz, 2H), 7.57 (t, J = 7.9 Hz, 1H),
7.48 (d, J = 7.6 Hz, 1H), 7.05 (s, 1H), 4.09 (s, 2H), 4.03 (s, 3H). 13C-
NMR (100 MHz, DMSO-d6): d 160.4, 150.4, 147.2, 142.6, 142.1,
138.5, 136.5, 133.2, 130.1, 129.6, 129.2, 128.8, 125.7, 125.3,
124.0, 122.6, 120.9, 116.7, 108.0, 38.4, 21.6. MS (MM-
ES+ APCI) 552.0 (M + H)+.
[4-(4-Chlorobenzyl)piperazine-1-yl]-{3-[5-(4-trifluoromethylphenyl)-
1-methyl-1H-pyrazol-3-yl]-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-
yl}methanone (9f). Yield (0.26 g, 78%), off white solid, Found:
C, 54.08; H, 4.17; N, 18.86. Calcd for C27H24ClF3N8OS:
C, 53.96; H, 4.02; N, 18.64. IR (KBr) υ/cmꢂ1: 2919, 2811
(C—H), 1641 (amide C═O), 1631 (C═N), 1448 (C═C), 1326
(tert-amine). 1H-NMR (400 MHz, CDCl3):
d
7.79 (d,
3-[5-(4-Trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-
J = 8.2 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz,
2H), 7.27 (d, J = 8.0 Hz, 2H), 6.99 (s, 1H), 4.01 (br, s, 2H),
3.97 (br, s, 2H), 3.96 (s, 3H), 3.79 (br, s, 2H), 3.50 (br, s, 2H),
2.64 (br, s, 4H). 13C-NMR (100 MHz, CDCl3): d 161.7, 149.6,
147.9, 143.3, 141.8, 137.7, 135.8, 133.2, 131.3, 130.2, 129.1,
128.5, 125.9, 125.1, 122.4, 107.3, 61.9, 53.3, 52.4, 47.2, 42.8,
38.1, 23.6. MS (MM-ES + APCI) 601.0 (M + H)+.
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic
acid-4-
methoxybenzylamide (9b). Yield (0.16 g, 65%), off white solid,
Found: C, 54.44; H, 3.67; N, 18.34. Calcd for C24H20F3N7O2S: C,
54.64; H, 3.82; N, 18.59. IR (KBr) υ/cmꢂ1: 3346 (amide —NH—
), 2934, 2838 (C—H), 1677 (amide C═O), 1615 (C═N), 1457
1
(C═C). H-NMR (300 MHz, CDCl3): d 7.77 (d, J= 8.1 Hz, 2H),
7.54 (d, J=8.1 Hz, 2H), 7.30 (d, J =8.3 Hz, 2H), 6.94 (s, 1H),
6.89 (d, J=8.6 Hz, 2H), 4.56 (d, J =5.6 Hz, 2H), 4.05 (s, 2H),
3.85 (s, 3H), 3.79 (s, 3H). 13C-NMR (75.4 MHz, CDCl3): d 159.9,
159.3, 148.1, 147.7, 143.4, 141.9, 137.6, 133.5, 131.4, 129.1,
129.0, 125.9, 125.8, 114.1, 107.3, 55.2, 43.5, 38.0, 20.3. MS
(MM-ES + APCI) 528.0 (M + H)+.
[4-(4-Methoxybenzyl)piperazine-1-yl]-{3-[5-(4-trifluoromethylphenyl)-1-
methyl-1H-pyrazol-3-yl]-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-
6-yl}methanone (9g). Yield (0.28 g, 77%), off white solid, Found:
C, 56.17; H, 4.33; N, 18.63. Calcd for C28H27F3N8O2S: C, 56.37; H,
4.56; N, 18.78. IR (KBr) υ/cmꢂ1: 2934, 2807 (C—H), 1641 (amide
C═O), 1632 (C═N), 1449 (C═C), 1326 (tert-amine). 1H-NMR
(400 MHz, CDCl3): d 7.79 (d, J=8.1 Hz, 2H), 7.59 (d, J=8.1 Hz,
2H), 7.23 (d, J=7.4 Hz, 2H), 6.99 (s, 1H), 6.87 (d, J=8.4 Hz, 2H),
3.97 (br, s, 2H), 3.90 (s, 5H), 3.81 (s, 5H), 3.50 (br, s, 2H), 2.64
(br, s, 4H). 13C-NMR (75.4 MHz, CDCl3): d 161.8, 158.9, 149.5,
147.9, 143.2, 141.7, 137.6, 133.0, 131.2, 130.1, 129.0, 125.0,
113.6, 107.3, 62.0, 55.1, 53.2, 52.3, 47.2, 42.8, 38.0. MS (MM-
ES + APCI) 597.3 (M + H)+.
3-[5-(4-Trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic
chlorophenyl)ethyl]amide (9c). Yield (0.18 g, 70%), off white
solid, Found: C, 53.15; H, 3.76; N, 18.20. Calcd for
C24H19ClF3N7OS: C, 52.80; H, 3.51; N, 17.96. IR (KBr) υ/cmꢂ1
acid-[(R)-1-(4-
:
3215 (amide —NH—), 3047 (Ar—H), 2974, 2923 (C—H), 1666
1
(amide C═O), 1621 (C═N), 1456 (C═C). H-NMR (300 MHz,
CDCl3): d 7.80 (d, J = 8.1Hz, 2H), 7.65 (br, s, 1H), 7.59
(d, J = 8.1 Hz, 2H), 7.38–7.28 (m, 4H), 6.98 (s, 1H), 5.23
(q, J = 4.5 Hz, 1H), 4.10–3.96 (m, 2H), 3.90 (s, 3H), 1.63
(d, J = 6.9 Hz, 3H). 13C-NMR (100MHz, CDCl3): d 159.5, 148.0,
147.7, 143.5, 141.9, 140.8, 137.8, 133.6, 132.9, 129.0, 128.9,
127.5, 126.0, 107.4, 49.1, 38.1, 21.7, 20.3. MS (MM-ES + APCI)
546.0 (M + H)+.
3-[5-(4-Trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic acid-[(R)-1-
(2-methoxyphenyl)ethyl]amide (9h).
white solid, Found: C, 55.75; H, 4.37; N, 18.36. Calcd for
C25H22F3N7O2S: C, 55.45; H, 4.09; N, 18.10. IR (KBr) υ/cmꢂ1
Yield (0.21 g, 64%), off
:
3395 (amide —NH—), 2976, 2928, 2839 (C—H), 1677 (amide
1
C═O), 1621 (C═N), 1457 (C═C). H-NMR (400 MHz, CDCl3):
3-[5-(4-Trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-
d 8.15 (d, J = 8.6 Hz, 1H), 7.79 (d, J = 8.1Hz, 2H), 7.59
(d, J = 8.0 Hz, 2H), 7.25–7.20 (m, 2H), 6.98 (s, 1H), 6.93
(t, J = 7.4 Hz, 1H), 6.77 (d, J = 8.2Hz, 1H), 5.42–5.35 (m, 1H),
4.10–3.98 (m, 2H), 3.97 (s, 3H), 3.67 (s, 3H), 1.56 (d, J = 6.8 Hz,
3H). 13C-NMR (100MHz, CDCl3): d 159.0, 156.8, 148.6, 147.7,
143.3, 142.1, 137.7, 133.1, 131.3, 130.0, 129.0, 128.8, 127.5,
126.1, 125.1, 122.4, 121.0, 111.2, 107.3, 55.2, 47.4, 38.2, 21.3,
20.5. MS (MM-ES+ APCI) 542.2 (M +H)+.
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic
acid-4-
fluorobenzylamide (9d). Yield (0.19g, 70%), off white solid,
Found: C, 53.84; H, 3.54; N, 19.25. Calcd for C23H17F4N7OS: C,
53.59; H, 3.32; N, 19.02. IR (KBr) υ/cmꢂ1: 3378 (amide —NH—
), 3063 (Ar—H), 2925 (C—H), 1677 (amide C═O), 1604
(C═N), 1458 (C═C). 1H-NMR (400MHz, CDCl3): d 7.78
(d, J = 8.0Hz, 2H), 7.67 (t, J = 5.4 Hz, 1H), 7.56 (d, J = 8.1 Hz,
2H), 7.36 (dd, J = 8.5, 5.3 Hz, 2H), 7.05 (t, J = 8.6 Hz, 2H), 6.97
(s, 1H), 4.60 (d, J = 5.7 Hz, 2H), 4.05 (s, 2H), 3.86 (s, 3H). 13C-
NMR (100MHz, DMSO-d6): d 162.3, 161.0, 159.9, 150.5, 147.0,
142.5, 142.0, 136.4, 134.8, 133.2, 129.3, 128.9, 125.7, 125.3,
122.6, 115.0, 114.8, 107.9, 41.7, 38.3, 21.4. MS (MM-
ES+ APCI) 516.0 (M+ H)+.
3-[5-(4-Trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic
acid-(4-
chlorophenyl)amide (9i). Yield (0.25 g, 78%), off white solid,
Found: C, 51.28; H, 3.28; N, 18.73. Calcd for C22H15ClF3N7OS:
C, 51.02; H, 2.92; N, 18.93. IR (KBr) υ/cmꢂ1: 3264 (amide —
NH—), 2919 (C—H), 1677 (amide C═O), 1622 (C═N), 1461
(C═C). 1H-NMR (400 MHz, CDCl3): d 9.42 (s, 1H), 7.80 (d,
J = 8.0Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H),
7.39 (d, J = 8.6Hz, 2H), 7.07 (s, 1H), 4.09 (s, 2H), 4.03 (s, 3H).
13C-NMR (100MHz, DMSO-d6): d 160.1, 150.6, 147.2, 142.6,
142.2, 136.6, 133.2, 129.3, 128.8, 128.3, 125.8, 125.3, 122.6,
122.0, 107.8, 38.4, 21.7. MS (MM-ES + APCI) 518.0 (M + H)+.
(4-Methylpiperazin-1-yl)-{3-[5-(4-trifluoromethylphenyl)-1-
methyl-1H-pyrazol-3-yl]-7H-1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazine-6-yl}methanone (9j). Yield (0.16 g, 63%), off white
solid, Found: C, 51.68; H, 4.70; N, 23.04. Calcd for C21H21F3N8OS:
C, 51.42; H, 4.32; N, 22.84. IR (KBr) υ/cmꢂ1: 3428 (amide —NH—
), 2938(C—H), 1641(amideC═O), 1632 (C═N), 1450 (C═C). 1H-
NMR (400MHz, CDCl3): d 7.79 (d, J = 8.1 Hz, 2H), 7.61
(d, J = 8.0 Hz, 2H), 7.00 (s, 1H), 4.04 (br, s, 2H), 3.98 (s, 3H), 3.92
3-[5-(4-Trifluoromethylphenyl)-1-methyl-1H-pyrazol-3-yl]-7H-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine-6-carboxylic
chlorophenyl)ethyl]amide (9e). Yield (0.23 g, 86%), off white
solid, Found: C, 53.10; H, 3.71; N, 18.11. Calcd for
C24H19ClF3N7OS: C, 52.80; H, 3.51; N, 17.96. IR (KBr) υ/cmꢂ1
acid-[(S)-1-(4-
:
3381 (amide —NH—), 3047 (Ar—H), 2979, 2923 (C—H), 1666
1
(amide C═O), 1621 (C═N), 1456 (C═C). H-NMR (400 MHz,
CDCl3): d 7.80 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 7.7 Hz, 1H), 7.59
(d, J = 8.0Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.4 Hz,
2H), 7.01 (s, 1H), 5.21 (q, J = 7.0 Hz, 1H), 4.08–3.92 (m, 2H),
3.91 (s, 3H), 1.63 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz,
CDCl3): d 159.6, 148.1, 147.6, 143.5, 141.9, 140.9, 137.7, 133.5,
132.8, 131.4, 129.0, 128.9, 127.6, 126.0, 125.0, 122.3, 107.4,
49.2, 38.1, 21.7, 20.4. MS (MM-ES+ APCI) 546.0 (M+ H)+.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

August 2014
1,2,4-Triazolo-[3,4-b]-1,3,4-thiadiazines Containing an Amide Linkage
E65
(s, 2H), 3.84 (br, s, 2H), 2.57 (br, s, 4H), 2.37 (s, 3H). 13C-NMR
(100MHz, DMSO-d6): d 161.9, 151.7, 147.1, 142.6, 141.6, 136.8,
133.1, 129.4, 129.2, 128.9, 125.7, 125.3, 122.6, 107.2, 54.8, 53.9,
46.3, 45.3, 41.7, 38.3, 23.2. MS (MM-ES + APCI) 491.2 (M+ H)+.
[4-(2-Fluorophenyl)piperazin-1-yl]-{3-[5-(4-trifluoromethylphenyl)-
1-methyl-1H-pyrazol-3-yl]-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-
yl}methanone (9k). Yield (0.24 g, 72%), off white solid, Found: C,
55.05; H, 4.19; N, 19.85. Calcd for C26H22F4N8OS: C, 54.73; H, 3.89;
N, 19.64. IR (KBr) υ/cmꢂ1: 2912, 2820 (C—H), 1635 (amide C═O),
1444 (C═C). 1H-NMR (300 MHz, CDCl3): d 7.74 (d, J=8.2 Hz, 2H),
7.57 (d, J=8.0 Hz, 2H), 7.07–7.02 (m, 3H), 6.99 (s, 1H), 6.96–6.93
(m, 1H), 4.20 (t, J=4.4 Hz, 2H), 4.00 (t, J=4.9 Hz, 2H), 3.95 (s, 2H),
3.94 (s, 3H), 3.22 (br, s, 4H). 13C-NMR (75.4 MHz, CDCl3): d
161.7, 157.2, 154.0, 149.4, 147.9, 143.3, 141.7, 139.1, 137.6, 132.9,
130.8, 129.0, 125.8, 124.6, 123.5, 119.1, 116.4, 107.2, 51.5, 50.0,
47.5, 43.0, 38.0, 23.5. MS (MM-ES + APCI) 571.2 (M + H)+.
158.9, 147.6, 144.6, 132.2, 130.2, 129.0, 128.6, 126.8,
113.7, 61.9, 55.2, 52.7, 51.9, 46.2, 42.4, 40.0, 28.0, 23.4. MS
(MM-ES+ APCI) 525.2 (M+).
{3-[1-(4-Chlorophenyl)-1-methylethyl])-7H-1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazine-6-yl}-[4-(2-fluorophenyl)piperazin-1-yl]
methanone (13e). Yield (0.53 g, 95%), off white solid, Found:
C, 58.01; H, 5.10; N, 17.14. Calcd for C24H24ClFN6OS: C, 57.77;
H, 4.85; N, 16.84. IR (KBr) υ/cmꢂ1: 2978, 2921, 2825 (C—H),
1
1641 (amide C═O), 1457 (C═C). H-NMR (400 MHz, CDCl3):
d 7.26 (d, J = 8.6 Hz, 2H), 7.13–7.00 (m, 5H), 6.92 (t, J = 8.5 Hz,
1H), 3.81 (t, J = 5.0 Hz, 2H), 3.75 (s, 2H), 3.12 (t, J = 4.8 Hz,
2H), 3.07 (t, J = 5.1 Hz, 2H), 2.71 (t, J = 5.1 Hz, 2H), 1.83
(s, 6H). 13C-NMR (75.4 MHz, CDCl3): d 161.5, 158.6, 157.2,
154.0, 147.6, 144.7, 141.8, 139.0, 132.3, 128.6, 126.9, 124.4,
123.4, 119.1, 116.4, 116.1, 50.6, 49.9, 46.4, 42.6, 40.1, 28.0,
23.5. MS (MM-ES + APCI) 499.2 (M+).
3-[1-(4-Chlorophenyl)-1-methylethyl]-7H[1,2,4-triazolo[3,4-b]-
3-[1-(4-Chlorophenyl)-1-methylethyl]-7H-1,2,4-triazolo[3,4-b]-
1,3,4-thiadiazine-6-carboxylic acid-[(R)-1-(4-chlorophenyl)ethyl]
1,3,4-thiadiazine-6-carboxylic
acid-[(R)-1-(2-methoxyphenyl)
ethyl]amide (13f). Yield (0.45g, 85%), off white solid, Found:
C, 59.0; H, 5.38; N, 15.15. Calcd for C23H24ClN5O2S: C, 58.78; H,
5.15; N, 14.90. IR (KBr) υ/cmꢂ1: 3402 (amide —NH—), 2974,
2929 (C—H), 1676 (amide C═O), 1459 (C═C). 1H-NMR
(400 MHz, CDCl3): d 7.35 (t, J=8.3 Hz, 1H), 7.13–7.06 (m, 5H),
7.03 (t, J=7.4 Hz, 1H), 6.97 (d, J=8.2 Hz, 1H),
6.88 (d, J=8.3 Hz, 1H), 5.26 (q, J=7.1 Hz, 1H), 3.89 (s, 3H),
3.88–3.72 (m, 2H), 1.85 (d, J=9.6 Hz, 6H), 1.48 (d, J=6.8 Hz,
3H). 13C-NMR (75.4 MHz, CDCl3): d 158.6, 156.8, 146.7,
143.9, 132.4, 129.8, 128.9, 128.6, 127.1, 126.7, 121.0, 111.1, 55.4,
46.6, 39.6, 27.7, 27.6, 20.6, 20.1. MS (MM-ES + APCI) 470.2 (M+).
3-[1-(4-Chlorophenyl)-1-methylethyl]-7H-1,2,4-triazolo[3,4-b]-
1,3,4-thiadiazine-6-carboxylic acid-(3-methoxybenzyl)methylamide
amide (13a).
Yield (0.25g, 85%), off white solid, Found: C,
55.88; H, 4.68; N, 15.11. Calcd for C22H21Cl2N5OS: C, 55.70; H,
4.46; N, 14.76. IR (KBr) υ/cmꢂ1: 3389 (amide —NH—), 2975,
2926 (C—H), 1676 (amide C═O), 1460 (C═C). 1H-NMR
(300 MHz, CDCl3): d 7.42 (d, J = 9.9 Hz, 2H), 7.24–7.11 (m, 6H),
6.29 (d, J = 7.7 Hz, 1H), 5.00 (q, J = 7.3 Hz, 1H), 3.89–3.75 (m,
2H), 1.81 (d, J= 7.9 Hz, 6H), 1.47 (d, J = 6.9 Hz, 3H). 13C-NMR
(75.4 MHz, CDCl3): d 159.0, 158.6, 145.9, 144.2, 141.7, 140.0,
133.5, 132.4, 128.9, 128.7, 127.3, 126.8, 48.6, 39.7, 28.1, 27.8,
21.4, 19.9. MS (MM-ES+ APCI) 474.0 (M+).
3-[1-(4-Chlorophenyl)-1-methylethyl]-7H-1,2,4-triazolo[3,4-b]
[-1,3,4-thiadiazine-6-carboxylic
acid-[(S)-1-(4-chlorophenyl)
ethyl]amide (13b). Yield (0.48 g, 92%), off white solid, Found:
C, 55.50; H, 4.31; N, 14.97. Calcd for C22H21Cl2N5OS: C, 55.70;
H, 4.46; N, 14.76. IR (KBr) υ/cmꢂ1: 3389 (amide —NH), 2974,
2926 (C—H), 1677 (amide C═O), 1460 (C═C), 1H-NMR
(300 MHz, CDCl3): d 7.39 (d, J = 8.3 Hz, 2H), 7.21–7.11 (m, 6H),
6.29 (d, J = 8.1 Hz, 1H), 5.03 (q, J = 7.3 Hz, 1H), 3.89–3.75
(m, 2H), 1.81 (d, J = 7.9 Hz, 6H), 1.47 (d, J = 6.9 Hz, 3H). 13C-
NMR (75.4 MHz, CDCl3): d 159.0, 158.6, 145.9, 144.2, 141.7,
140.4, 133.5, 132.4, 128.9, 128.7, 127.3, 126.8, 48.6, 39.7, 28.1,
27.8, 21.4, 19.9. MS (MM-ES + APCI) 474.0 (M+).
(13g).
Yield (0.47 g, 89%), off white solid, Found: C, 59.08;
H, 5.47; N, 15.23. Calcd for C23H24ClN5O2S: C, 58.78; H, 5.15;
N, 14.90. IR (KBr) υ/cmꢂ1: 2973, 2931 (C—H), 1635 (amide
C═O), 1455 (C═C). 1H-NMR (400 MHz, DMSO-d6,): d 7.29
(t, J=7.6 Hz, 1H), 7.22 (d, J=8.6 Hz, 2H), 7.11 (d, J=6.9 Hz,
2H), 6.89 (d, J=8.3 Hz, 1H), 6.73 (br, s, 2H), 4.50 (br, s, 2H),
3.77 (s, 3H), 3.02 (br, s, 3H), 2.55 (br, s, 2H), 1.65 (s, 6H). MS
(MM-ES + APCI) 470.2 (M+).
Pharmacology
Cell lines. Human breast cancer lines, MCF-7, and alveolar
adenocarcinoma cell lines, A549, procured from National Center
for Cell Sciences, Pune, India, were cultured in MEM medium
supplemented with 10% FBS, 1% L-glutamine, and 50mg/mL
gentamicin sulfate in a CO2 incubator in a humidified atmosphere
of 5% CO2 and 95% air. The EAC cells were maintained for
12–14days in the peritoneal cavity of Swiss albino mice. The
tumor cell cultures were started from mouse Ehrlich ascites with at
least one passage in vitro prior to use.
[4-(4-Chlorobenzyl)piperazin-1-yl]-{3-[1-(4-chlorophenyl)-
1-methylethyl])-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine-6-yl}
methanone (13c).
Yield (0.50g, 84%), off white solid, Found:
C, 57.09; H, 5.21; N, 16.10. Calcd for C25H26Cl2N6OS: C, 56.71;
H, 4.95; N, 15.87. IR (KBr) υ/cmꢂ1: 2974, 2912, 2811 (C—H),
1
1633 (amide C═O), 1453 (C═C), H-NMR (300 MHz, CDCl3):
d 7.35–7.26 (m, 4H), 7.19 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.0 Hz,
2H), 3.70 (s, 2H), 3.62 (br, s, 2H), 3.46 (br, s, 2H), 2.92 (br, s,
2H), 2.40 (br, s, 2H), 2.05 (br, s, 2H), 1.79 (s, 6H). 13C-NMR
(75.4 MHz, CDCl3): d 161.4, 158.6, 147.5, 144.6, 141.7, 133.1,
132.1, 130.2, 128.6, 128.5, 126.8, 61.7, 52.7, 52.1, 46.2, 42.4,
40.0, 28.0, 23.4. MS (MM-ES + APCI) 529.0 (M+).
{3-[1-(4-Chlorophenyl)-1-methylethyl])-7H-1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazine-6-yl}-[4-(4-methoxybenzyl)piperazin-1-yl]
methanone (13d). Yield (0.50 g, 85%), off white solid, Found:
C, 59.70; H, 5.81; N, 16.27. Calcd for C26H29ClN6O2S: C, 59.48;
H, 5.57; N, 16.01. IR (KBr) υ/cmꢂ1: 2924, 2804 (C—H), 1633
(amide C═O), 1453 (C═C). 1H-NMR (300MHz, CDCl3): d 7.24
(d, J = 8.3Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.5 Hz,
2H), 6.91 (d, J = 8.4 Hz, 2H), 3.82 (s, 3H), 3.69 (s, 2H), 3.62
(br, s, 2H), 3.45 (s, 2H), 293 (br, s, 2H), 2.40 (br, s, 2H), 2.06
(br, s, 2H), 1.79 (s, 6H). 13C-NMR (75.4MHz, CDCl3): d 161.3,
In vitro cytotoxic activities by MTT assay.
In vitro
cytotoxicity was determined using a standard MTT assay [40] with
protocol appropriate for the individual test system. In brief,
exponentially growing cells were plated in 96-well plates (104 cells/
well in 100mL of medium) and incubated for 24 h for attachment.
Test compounds were prepared prior to the experiment by
dissolving in 0.1% DMSO and diluted with medium. The
cells were then exposed to different concentrations of the drugs
(1–100 mM) in the volume of 100 mL/well. Cells in the control wells
received the same volume of medium containing 0.1% DMSO.
After 24 h, the medium was removed, and cell cultures were
incubated with 100 mL MTT reagent (1 mg/mL) for 4 h at 37ꢀC.
The formazan produced by the viable cells was solubilised
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E66
P. Puthiyapurayil, B. Poojary, and S. K. Buridipad
Vol 51
by addition of 100 mL DMSO. The suspension was placed on micro-
vibrator for 5 min, and absorbance was recorded at 540 nm by the
enzyme linked immunosorbent assay reader. The experiment was
performed in triplicate. The percentage cytotoxicity was calculated
using the formula.
activity experiment, mice under deep ether anesthesia were killed,
and their stomachs were removed. The abdomen of each mouse
was opened through great curvature and, using a dissecting
microscope, was examined for bleeding.
Acute toxicity and statistical analysis data.
Animals
employed in the carrageenan-induced paw edema experiment were
observed for 71h, and the mortality rate was recorded for each
group at the end of observation period. Data obtained from animals
experiment were expressed as meanꢁ SEM. Statistical differences
between the treatment and the control group of animals were
evaluated by two-tailed student’s t-test.
% cytotoxicity= (Control abs–Blank abs)ꢂ (Test abs–Blank
abs) / (Control abs–Blank abs)ꢃ 100
For MTT time course study, MCF-7 cells (5ꢃ 103 cells/well
seeded in 96-well plates) were exposed to different concentrations
of test compounds (1–50mM). The percentage cytotoxicity and
IC50 values were determined at 24, 48, and 72h of drug incubation.
Albino rats. Albino rats (Wistar strain) of either sex, weighing
between 150 and 200g were selected and fed a standard pellet diet
and water in a temperature-controlled room. One day prior to the
experiment, food was withdrawn, but the animals were allowed
free access of water. The allocation of animals to different groups
was randomized, and the experiments were carried out under blind
conditions. Each groups comprising of three or four mice were
employed for preliminary testing using the carrageenan-induced
paw edema model and writhing test, respectively. For preliminary
activity screening, all test drugs were administered to mice at doses
of 100 mg/kg (body weight). Test compounds that possessed more
than a 20% inhibitory effects were selected for further evaluation
of the activity-dose relationship in two different doses (50 and
200 mg/kg) using groups comprising of four to five animals.
Acknowledgment. The author PP thanks Dr Ashis Baran Mandal,
Dr Santosh Kulkarni, and Dr John Kallikat for helpful discussions.
The authors also grateful to Dr Chadrashekar, Department of
Pharmacology, HSK College, Bagalkote, India, for providing the
facilities to determine the anticancer activity.
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