Facile synthesis, cytotoxic and antimicrobial activity studies of a new group of 6-aryl-3-[4-(methylsulfonyl)benzyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazines

Article abstract of DOI:10.1016/j.ejmech.2012.04.024

The reaction of 4-(methylsulfonyl)phenylacetohydrazide (3) with carbon disulfide and potassium hydroxide followed by hydrazine hydrate gave 4-amino-5-[4-(methylsulfonyl)benzyl]-4H-[1,2,4]triazole-3-thione (4). The resulting triazole was subjected to cyclo

Full text of DOI:10.1016/j.ejmech.2012.04.024

European Journal of Medicinal Chemistry 54 (2012) 59e64
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Facile synthesis, cytotoxic and antimicrobial activity studies of a new group of
6-aryl-3-[4-(methylsulfonyl)benzyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines
,
,
a
b
c
V. Sumangala a b, Boja Poojary a , N. Chidananda , T. Arulmoli , Shalini Shenoy
*
a Department of Chemistry, Mangalore University, Mangalagangothri 575199, Karnataka, India
b SeQuent Scientific Limited, 120 A & B, Industrial area, Baikampady, New Mangalore 575011, Karnataka, India
c Department of Microbiology, KMC, Mangalore 575001, Karnataka, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< A series of novel 6-substituted-3-[4-
(methylsulfonyl)benzyl]-7H-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazines
were synthesized in good yields.
< The structures of synthesized
compounds were established on the
basis of spectral and analytical data.
< Synthesized compounds were eval-
uated for their cytotoxic activity and
antimicrobial activity.
H
N
N
N
N
COOH
S
N
S
N
N
NH2
R1
O
H3C
S
O
S
O
H3C
O
S
4
O
< Most of the compounds exhibited
significant cytotoxicity.
H3C
O
5(a-i)
1
< A few derivatives exhibited antimi-
crobial activity comparable with
that of the standard drugs used for
testing.
a r t i c l e i n f o
a b s t r a c t
Article history:
The reaction of 4-(methylsulfonyl)phenylacetohydrazide (3) with carbon disulfide and potassium
hydroxide followed by hydrazine hydrate gave 4-amino-5-[4-(methylsulfonyl)benzyl]-4H-[1,2,4]triazole-
3-thione (4). The resulting triazole was subjected to cyclocondensation reaction with different phenacyl
bromides to afford 6-substituted-3-[4-(methylsulfonyl)benzyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zines (5aei). All structures of the newly synthesized compounds were confirmed by IR, NMR, mass
spectral studies and elemental analyses. The newly synthesized compounds were screened for their
cytotoxic, antibacterial and antifungal activity. Some of the derivatives have exhibited promising bio-
logical activity.
Received 28 February 2012
Received in revised form
15 April 2012
Accepted 18 April 2012
Available online 28 April 2012
Keywords:
[1,2,4]-Triazoles
[1,3,4]-Thiadiazines
4-(Methylsulfonyl)benzyl
Cytotoxicity
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antimicrobial activity
1. Introduction
[1,2,4]-Triazoles and N-bridged heterocycles derived from them
are found to be associated with diverse pharmacological activities
[1e6]. They are also reported to possess significant anticancer
properties [7]. It has also been reported that derivatives of [1,2,4]-
* Corresponding author. Tel.: þ91 824 2287262; fax: þ91 824 2287367.
E-mail address: bojapoojary@gmail.com (B. Poojary).
triazole and their fused systems exert diverse pharmacological
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.024

60
V. Sumangala et al. / European Journal of Medicinal Chemistry 54 (2012) 59e64
activities such as antibacterial [8], antifungal [9], anti-inflammatory
[10,11], anticancer [12] and antitubercular [13], properties. Certain
[1,2,4]-triazoles find applications in the preparation of photo-
graphic plates, polymers and also as analytical agents [14]. It is
found that the incorporation of various substituents into the [1,2,4]-
triazole ring and the synthesis of condensed heterocycles derived
from [1,2,4]-triazoles led to compounds with enhanced biological
activities. Moreover 4-amino-3-substituted-5-mercapto-[1,2,4]-
triazoles by virtue of their ambident nucleophilic centers are good
starting materials for the synthesis of several interesting N-bridged
heterocycles.
triazole. Aromatic and alkyl stretching vibrations were observed at
3091 and 2944 cmꢀ1 respectively. The peaks observed at 1408 and
1144 cmꢀ1 were assigned to S]O. The C]S stretch was observed at
1290 cmꢀ1. The 400 MHz 1H NMR spectrum of compound (4)
showed two doublets at
phenyl moiety. The singlets at
d
7.55 and 7.87 due to (4-methylsulfony)
5.58 and 13.61 were corresponding
d
to NH2 and NH protons respectively. The mass spectrum of
compound (4) showed its characteristic molecular ion peak at m/z
285.1.
All structures of the title compounds were confirmed by IR, 1H
NMR and mass spectra. IR spectrum of (5a) showed absorption
bands at 3005 cmꢀ1 (AreH), 1598 cmꢀ1 (C]N), 1467 (NeCeS), 690
(CeSeC) respectively. The absence of the absorption bands corre-
sponding to eNH2 and C]S stretching frequencies of the reactants
clearly revealed the formation of triazolothiadiazine (5a). The
400 MHz 1H NMR spectrum of compound (5a) showed two
2. Chemistry
The key starting material 4-methylthiophenylacetic acid (1) on
esterification yielded 2 which on treatment with hydrazine hydrate
gave acid hydrazide (3). The reaction of the acid hydrazide with
carbon disulphide in the presence of potassium hydroxide in
ethanol gave the corresponding intermediate potassium dithio-
carbazinate. This salt underwent ring closure with an excess of
hydrazine hydrate to give 4-amino-5-[4-(methylsulfonyl)benzyl]-
2,4-dihydro-3H-[1,2,4]-triazole-3-thione (4). The compound 4 was
then subjected to cyclocondensation reaction with different phe-
nacyl bromides to afford 6-substituted-3-[4-(methylsulfonyl)
benzyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (5aei).
doublets at
The characteristic eSCH2e protons of the thiadiazine ring reso-
nated as a sharp singlet at 3.97. The multiplet at 7.51e7.60 was
d 7.81 and 7.87 due to (4-methylsulfony)phenyl moiety.
d
corresponding to the phenyl moiety. The mass spectrum of
compound (5a) showed its characteristic molecular ion peak at m/z
385.4. The IR and 1H NMR spectra of other compounds of the series
showed similar characteristic properties.
4. Pharmacological evaluation
3. Results and discussion
4.1. Cytotoxic study
Synthetic strategy of the title compounds is outlined in Scheme 1.
The formation of thiadiazines (5aei) was evidenced by their
elemental analyses and spectral data. Characterization data of all the
newly synthesized compounds are presented in Table 1.
Procedure for MTT assay: RAW 264.7 (murine macrophage cell
line) HT-29 (human colonic epithelial cell lines) cell lines were
seeded at a density of 1 ꢁ104 cells (cell viability was determined by
Trypan blue exclusion dye method) [15]. Per each well in 100 ml of
The formation of the compound (4) was confirmed by IR, 1H
NMR and mass spectra. In the IR spectrum of compound (4) the
absorption due to the NH2 group of triazole ring was observed at
3215 and 3177 cmꢀ1, thus confirming the formation of amino
DMEM (Dulbeccos modified Eagles Medium) supplemented with
10% FBS (Fetal bovine serum). Twelve hours after seeding, above
media was replaced with fresh DMEM supplemented with 10% FBS,
then 20 ml samples from above stock solutions were added to each
Scheme 1. Synthetic route of the title compounds.

V. Sumangala et al. / European Journal of Medicinal Chemistry 54 (2012) 59e64
61
Table 1
Characterization data of compounds (5aei).
Sample
R1
Mol. formula
Mol. wt
m.p (ꢂC)
Yield (%)
Analysis (%) (calculated) found
C
H
N
5a
5b
5c
5d
5e
5f
5g
5h
5i
eC6H5
4-CH3eC6H4
2,4-Cl2eC6H3
4-C6H5eC6H4
4-CONH2e5-OHeC6H3
3-Coumarinyl
2-SO2NH2e5-Cle3-thienyl
2,5-Cl2e3-thienyl
2,7-Cl2-9H-4-fluorenyl
C18H16N4O2S2
C19H18N4O2S2
C18H14Cl2N4O2S2
C24H20N4O2S2
C19H17N5O4S2
C21H16N4O4S2
C16H14ClN5O5S4
C16H19Cl2N4O2S3
C26H18Cl2N4O2S2
384.47
398.50
453.36
460.57
443.49
452.50
504.025
459.39
541.47
182e183
170 (dec)
138e140
119e123
220e222
167e169
219e220
226e228
250e252
82
83
80
79
82
80
80
83
89
(56.23)56.20
(57.27)57.25
(47.69)47.65
(62.59)62.56
(51.46)51.42
(55.74)55.73
(38.13)38.10
(41.83)41.80
(55.45)55.43
(4.19)4.15
(4.55)4.53
(3.11)3.10
(4.38)4.36
(3.86)3.88
(3.56)3.54
(2.56)2.56
(2.63)2.65
(3.35)3.33
(14.57)14.55
(14.06)14.03
(12.36)12.35
(12.16)12.14
(15.79)15.75
(12.38)12.36
(13.89)13.85
(12.20)12.13
(10.35)10.33
well in triplicates which gives final concentration of 100, 50, 25 and
10
g/well. The above cells were incubated for 48 h at 37 ꢂC with 5%
CO2. After 48 h incubation, the above media was replaced with
100 l of fresh DMEM without FBS and to this 10 l of MTT [3-(4,5-
4.2.1. Preparation of antimicrobial suspension (1 mg/mL)
Dissolved 10 mg of each compound in 10 mL of Dimethyl
formamide (DMF) to get 1 mg/mL concentration.
m
m
m
dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (5 mg
dissolved in 1 mL of FBS) was added and incubated for 3 h at 37 ꢂC
with 5% CO2. After 3 h of incubation the above media was removed
4.2.2. Preparation of dilutions
In all, for each of the 9 antimicrobial compounds, standard
antibacterial i.e Penicillin and standard antifungal i.e Flucanazole,
30 tubes of 5 mL capacity were arranged in 5 rows with each row
containing 6 tubes. Then 1.9 mL of trypticase soya broth was added
in the first tube in each row and then 1 mL in the remaining tubes.
with a multi channel pipette, then 200 mL of DMSO was added to
each well and the incubated at 37 ꢂC for 15 min. Finally the plate
was read at 570 nm using spectrometer (Spectra-Max, Molecular
devices). The % cell death is calculated by the formula:
Now, 100
ml of antimicrobial suspension dissolved in DMF was
added to the first tube in each row and then after mixing the
content, 1 mL was serially transferred from these tubes to the
second tube in each of the rows. Then the contents in the second
tube of each of the rows were mixed and transferred to the third
AbsorbanceðDMSOÞ ꢀ Absorbance
% Cell death ¼
ðsampleÞ ꢁ 100
Absorbance
ðDMSOÞ
The IC50 value1 is obtained based on the % cell death value. The
cytotoxicity study revealed that some of the 6-substituted-3-[4-
(methylsulfonyl)benzyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zines, 5a, 5d, 5i containing phenyl, biphenyl and dichloro fluorenyl
groups respectively at C-6 position of thiadiazines showed signifi-
cant activity compared to the standard Doxorubicin. The cytotox-
icity of thiadiazine containing biphenyl derivative is much better
than the phenyl derivative. Other compounds showed moderate to
good activity. The results of cytotoxic bioassay studies are given in
Table 2.
Table 2
Cytotoxic activity data of compounds (5aei).
Compound name
Conc (
m
M)
% Cell death
Standard error
IC50
25
(mM)
Doxorubicin
5a
2.5
12.5
25
78.2
2.5
2.91
2.38
3.61
3.00
4.16
4.35
2.51
6.04
1.15
0.54
1.49
2.60
1.52
0.92
1.89
0.43
1.69
1.73
1.50
2.82
5.12
2.73
1.61
6.11
1.40
2.60
1.47
0.95
11.92
5.50
0.55
6.68
3.74
2.89
7.38
1.02
41.97
50.95
46.73
55.51
Not effective
1.26
50
100
12.5
25
5b
5c
5d
5e
5f
>100
>100
12.5
50
37.91
38.53
27.99
24.19
27.33
30.99
58.20
60.19
67.93
74.77
24.81
24.66
33.06
37.08
22.43
25.58
14.86
10.63
22.80
18.66
27.46
35.22
Not effective
Not effective
25.54
47.27
41.05
44.80
43.12
50.33
4.2. Antimicrobial activity
100
12.5
25
The newly synthesized 1,2,4-triazoles (5aei) were screened for
their in vitro antibacterial and antifungal activity. For antibacterial
studies micro-organisms employed were Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus. For
antifungal screening, Candida albicans strain was used. Both
microbial studies were assessed by Minimum Inhibitory Concen-
tration (MIC) by serial dilution method [16]. The micro-organisms
employed were picked off a fresh isolation plate and incubated in
corresponding tubes containing 5 mL of trypticase soya broth. The
broth was incubated for 6 h at 37 ꢂC until there was visible growth.
Mc Farland No. 5 standard was prepared by adding 0.05 mL of 1% w/
v BaCl2$2H2O in phosphate buffered saline (PBS) to 9.95 mL of 1% v/
v H2SO4 in PBS. The growth of all the five cultures was adjusted to
Mc Farland No. 5 turbidity standard using sterile PBS. This gives
a 108 cfu/mL (colony-forming units per milliliter) suspension. The
working inoculums of aforementioned five different micro-organ-
isms containing 105 cfu/mL suspension was prepared by diluting
the 108 cfu/mL suspension, 103 times in trypticase soya broth.
50
100
12.5
25
50
100
12.5
25
>100
>100
>100
>100
100
50
100
12.5
25
50
100
12.5
25
5g
5h
5i
50
100
12.5
25
50
100
12.5
25
50
100
1
IC50: the concentration of sample which reduced absorbance by 50% relative to
the vehicle-treated control or the half maximal (50%) inhibitory concentration (IC)
of a substance.

62
V. Sumangala et al. / European Journal of Medicinal Chemistry 54 (2012) 59e64
Table 4
tube in each of the rows. This serial dilution was repeated till the
sixth tube in each of the rows. This provided antimicrobial
Antifungal activity data of compounds (5aei).
concentrations of 50, 25, 12.5, 6.25, 3.125, 1.6125 mg/mL in the first
Compound
MIC in mg/mL
to sixth tube respectively in each row. Finally, 1 mL of 105 cfu/mL of
S. aureus, E. coli, P. aeruginosa, B. cereus and C. albicans suspension
were added to the first, second, third, fourth and fifth rows of tubes
respectively. Along with the test samples, standard antibacterial i.e
Penicillin, standard antifungal i.e Flucanazole, the inoculums
control (without antimicrobial compound) and broth control
(without antimicrobial compound and inoculum) were maintained.
All the test sample and control tubes were then incubated for 16 h
at 37 ꢂC.
C. albicans
5a
5b
5c
5d
5e
5f
5g
5h
25.0
1.562
6.25
6.25
3.125
3.125
6.25
3.125
6.25
0.8
***
No growth
5i
Flucanazole (standard)
Inoculum control
Broth control
4.2.3. Interpretation
After incubation, the tubes showing no visible growth were
considered to be representing the MIC. The details of results are
furnished in Table 3 and Table 4. Inoculums control showed visible
growth, where as the broth control showed no growth.
Among the screened samples, compound 5c showed excellent
antibacterial activity against E. coli may be due to the presence of
2,4-dichlorophenyl group at C-6 position of thiadiazine. The
compound 5i was activity against P. aeruginosa, which is due to the
presence of 2,7-Cl2-9H-4-fluorenyl group at C-6 position of thia-
diazine. Also 5e was activity against B. cereus even at low concen-
***Indicates growth in all concentrations.
and 2,5-Cl2-3-thienyl groups at C-6 position of thiadiazine ring
have significant antibacterial and antifungal activity against all the
tested microbial species than the other compounds. The antimicro-
bial screening studies demonstrated that the newly synthesized
compounds have promising antibacterial and antifungal properties.
Therefore, it was concluded that there exists better scope for further
study on this class of compounds.
tration of 3.125
mg/mL may be due to the presence of 2-
hydroxybenzamide group at C-6 position of thiadiazine.
The antifungal activity of the derivatives have much better
results for many of the compounds like 5b, 5e, 5f, 5h. The signifi-
cant activity of these compounds due to the presence of 4-
methylphenyl, 2-hydroxybenzamide, 3-coumarinyl and 2,5-Cl2-3-
thienyl groups at C-6 position of thiadiazine ring.
6. Experimental
Melting points were determined in open capillary tubes and
were uncorrected. IR spectra were recorded in KBr pellets on
a PerkineElmer 157 IR spectrophotometer. 1H NMR and 13C NMR
spectra were recorded on a Varian 400 MHz NMR spectrometer
using TMS as an internal standard. The mass spectra were recorded
on a MDS SCIEX/API4000 spectrophotometer. Elemental analysis
was carried out using Flash EA 1112 Series, CHNSO Analyzer
(Thermo). The progress of the reaction was monitored by thin layer
chromatography (TLC) on pre-coated silica gel G plates using
mixture of ethyl acetate and n-hexane as mobile phase.
5. Conclusions
The research study reports the successful synthesis of 6-aryl-3-
[4-(methylsulfonyl)benzyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zines from 4-amino-5-[4-(methylsulfonyl)benzyl]-2,4-dihydro-3H-
[1,2,4]-triazole-3-thione in good yield. The cytotoxic and antimi-
crobial activity evaluations of these compounds were done. Many of
the synthesized thiadiazines exhibited good cytotoxicity and anti-
microbial activity. The cytotoxicity study revealed that compounds
containing phenyl, biphenyl and dichloro fluorenyl groups respec-
tively at C-6 position of thiadiazines showed significant activity
compared to the standard Doxorubicin. And also from the results of
cytotoxic bioassay study, it was proven that the thiadiazines are
active. The antimicrobial activity study showed that thiadiazines
containing 4-methylphenyl, 2-hydroxybenzamide, 3-coumarinyl
6.1. Procedure for the synthesis of ethyl-4-(methylsulfonyl)
phenylacetate (2)
The ethyl-4-(methylsulfonyl)phenylacetate was prepared by
refluxing 4-(methylsulfonyl)-phenylaceticacid (1) in excess abso-
lute ethanol at 80 ꢂC in the presence of few drops of concentrated
sulfuric acid and excess ethanol was removed and quenched to
water. Aqueous layer was basified to neutral pH using 5% sodium
bicarbonate solution. The product was extracted from aqueous
layer using ethyl acetate. The solvent was removed. The compound
was obtained as low melting (32e34 ꢂC) white solid.
Table 3
Antibacterial activity data of compounds (5aei).
IR (KBr, gmax, cmꢀ1): 3014 (AreH), 1691(C]O),1407 (S]O),1138
(S]O); Anal. Calcd. (%) for C11H14O4S: C, 54.53, H, 5.82; found C,
54.50, H, 5.80.
Compound
MIC in mg/mL
E. coli
S. aureus
P. aeruginosa
B. cereus
5a
5b
5c
5d
5e
5f
5g
5h
6.25
12.5
3.125
6.25
6.25
6.25
6.25
6.25
6.25
0.12
***
6.25
12.5
12.5
6.25
12.5
6.25
12.5
6.25
12.5
0.12
***
6.25
12.5
6.25
6.25
6.25
6.25
6.25
6.25
6.25
12.5
6.25
6.25
3.125
6.25
12.5
6.25
6.25
6.2. Procedure for the synthesis of 4-(methylsulfonyl)
acetohydrazide (3)
A
mixture of ethyl-4-(methylsulfonyl)phenylacetate (2)
(0.1 mol), hydrazine hydrate (0.15 mol) and 20 mL of ethanol was
refluxed at 80 ꢂC on an oil bath for 10 h. The excess solvent was then
removed and the concentrated solution was quenched to ice cold
water. The solid separated was filtered, washed and dried. The
crude product was purified by re-crystallization from ethanol.
5i
3.125
0.12
***
Penicillin
Inoculum control
Broth control
0.12
***
No growth
No growth
No growth
No growth
***Indicates growth in all concentrations.

V. Sumangala et al. / European Journal of Medicinal Chemistry 54 (2012) 59e64
63
Colorless solid (89%); m.p. 118e120 ꢂC; IR (KBr, gmax, cmꢀ1):
3340, 3240 (NH), 3035 (AreH), 2921 (CeH), 1654 (C]O), 1598 (C]
C), 1409 (S]O), 1145 (S]O).
CH2, AreH), 7.32 (d, 2H, 4-methylphenyl, J ¼ 8.0 Hz), 7.58 (d, 2H,
J ¼ 8.0 Hz, 4-methylphenyl), 7.71 (d, 2H, J ¼ 8.0 Hz, AreH), 7.87 (d,
2H, J ¼ 8.0 Hz, AreH); 13C NMR: (100 MHz, CDCl3,
d ppm): 21.57
(CH3), 23.52 (CH2), 30.95 (CH2), 44.51 (SeCH3), 127.08, 127.80,
6.3. Procedure for the synthesis of 4-amino-5-[4-(methylsulfonyl)
129.95, 130.08, 130.55, 139.35,140.78, 141.99, 143.10, 152.08, 153.46;
benzyl]-2,4-dihydro-3H-[1,2,4]-triazole-3-thione (4)
DEPT (100 MHz, CDCl3, d ppm): CH, CH3: 21.58, 44.51, 127.08,
127.81, 129.95, 130.08, CH2: 23.52, ꢀ30.95; MS (m/z): 399.1 (M þ 1);
Anal. calcd. for C19H18N4O2S2: C, 57.27; H, 4.55; N, 14.06; found: C,
57.25; H, 4.53; N, 14.03.
The acid hydrazide (3) (0.01 mol) was dissolved in absolute
alcohol containing KOH (1.6 g) at room temperature followed by
carbon disulphide (0.013 mol) and the resulting mixture was stirred
at room temperature for 10 h. The mixture was diluted with ether
and stirred for further 1 h. The precipitated solid was filtered and
washed with ether. The solid was stirred in 2 volume of ethanol and
treated with hydrazine hydrate (80%) (0.015 mol). Reaction mixture
was refluxed at 80 ꢂC for 3 h during which hydrogen sulphide
evolved and the color of the reaction mixture changed to dark
green. It was then cooled to 5 ꢂC and acidified with concentrated
HCl to pH 2.5. A yellow solid separated out was filtered, washed
with water and crystallized from ethanol to get pure product (4).
Colorless solid (89%); mp 237e239 ꢂC.
6.4.3. 6-(2,4-Dichlorophenyl)-3-[4-(methylsulfonyl)benzyl]-7H-
[1,2,4]triazolo[3,4-b][1,3,4]-thiadiazine (5c)
IR (KBr, gmax, cmꢀ1): 3000 (AreH), 2925 (CeH), 1592 (C]N),
1520 (C]C), 1466 (NeCeS), 1410 (S]O), 1360 (CeN), 1292 (NeN]
C), 1134 (S]O), 850 (CeCl), 632 (CeSeC); 1H NMR: (400 MHz,
CDCl3,
d ppm): 3.00 (s, 3H, SeCH3), 3.90 (s, 2H, CH2), 4.40 (s, 2H,
CH2, AreH), 7.46 (d, 2H, 2,4-dichlorophenyl, J ¼ 8.0 Hz), 7.71 (d, 2H,
J ¼ 8.0 Hz, AreH), 7.87 (d, 2H, J ¼ 8.0 Hz, AreH), 7.84 (d, 2H,
J ¼ 8.0 Hz, 2,4-dichlorophenyl); Anal. calcd. for C18H14Cl2N4O2S2: C,
47.69; H, 3.11; N, 12.36; found: C, 47.65; H, 3.10; N, 12.35.
IR (KBr, gmax, cmꢀ1): 3315, 3177 (NH2), 3091 (AreH), 2944
(CeH),1627 (C]N),1564 (C]C),1408 (S]O),1290 (C]S),1144 (S]
6.4.4. 3-[4-(Methylsulfonyl)benzyl]-6-biphenyl-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine (5d)
O); 1H NMR: (400 MHz, DMSO-d6,
d ppm): 3.20 (s, 3H, SeCH3), 4.17
(s, 2H, CH2), 5.58 (s, 2H, NH2), 7.55 (d, 2H, AreH, J ¼ 8.0 Hz), 7.87 (d,
IR (KBr, gmax, cmꢀ1): 3007 (AreH), 2920 (CeH), 1591 (C]N),
1522 (C]C), 1461 (NeCeS), 1410 (S]O), 1355 (CeN), 1290 (NeN]
2H, AreH, J ¼ 8.0 Hz), 13.61(s, 1H, NH); 13C NMR: (100 MHz, DMSO-
d6,
d
ppm): 30.55 (SeCH3), 44.03 (CH2), 127.63, 130.35, 139.84,
C), 1130 (S]O), 635 (CeSeC); 1H NMR: (400 MHz, CDCl3,
d ppm):
141.94, 151.15, 166.66; DEPT (100 MHz, DMSO-d6,
d
ppm): CH, CH3:
3.00 (s, 3H, SeCH3), 3.90 (s, 2H, CH2), 4.41 (s, 2H, CH2, AreH), 7.21
(m, 5H, biphenyl), 7.35 (m, 4H, biphenyl), 7.75 (d, 2H, J ¼ 8.0 Hz,
AreH), 7.89 (d, 2H, J ¼ 8.0 Hz, AreH), Anal. calcd. for C24H20N4O2S2:
C, 62.59; H, 4.38; N, 12.16; found: C, 62.56; H, 4.36; N, 12.14.
30.54 (ꢀCH3), 127.64, 130.35, CH2: 44.03; MS (m/z): 285.1 (M þ 1);
Anal. calcd. for C10H12N4O2S2: C, 42.24; H, 4.25; N, 19.70; found: C,
42.21; H, 4.23; N, 19.72.
6.4. General procedure for the synthesis of 6-substituted-3-[4-
(methylsulfonyl)benzyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines
(5aei)
6.4.5. 2-Hydroxy-5-{3-[4-(methylsulfonyl)benzyl]-7H-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazin-6-yl}benzamide (5e)
IR (KBr, gmax, cmꢀ1): 3012 (AreH), 2933 (CeH), 1566 (C]N),
1520 (C]C), 1469 (NeCeS), 1418 (S]O), 1360 (CeN), 1290 (NeN]
An equimolar mixture of 4-amino-5-[4-(methylsulfonyl)
benzyl]-2,4-dihydro-3H-[1,2,4]-triazole-3-thione (4) (0.01 mol)
and substituted phenacyl bromides (0.01 mol) were dissolved in
ethanol. The reaction mass was heated to 80 ꢂC on a water bath for
about 5 h. The completion of reaction was monitored by TLC. After
completion of the reaction, the reaction mixture was cooled and the
precipitated solid was filtered, washed with water, dried and re-
crystallized from ethanol or methanol.
C), 1139 (S]O), 639 (CeSeC); 1H NMR: (400 MHz, CDCl3,
d ppm):
3.05 (s, 3H, SeCH3), 3.94 (s, 2H, CH2), 4.42 (s, 2H, CH2, AreH), 7.71
(d, 2H, J ¼ 8.0 Hz, AreH), 7.85 (d, 1H, salicylamide, J ¼ 8.0 Hz), 7.88
(s, 1H, salicylamide), 7.89 (d, 2H, J ¼ 8.0 Hz, AreH), 7.99 (s, NH2, 1H),
8.49 (s, OH, 1H), Anal. calcd. for C19H17N5O4S2: C, 54.46; H, 3.86; N,
15.79; found: C, 51.46; H, 3.88; N, 15.75.
6.4.6. 3-{3-[4-(Methylsulfonyl)benzyl]-7H-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazin-6-yl}-1H-isochromen-1-one (5f)
6.4.1. 3-[4-(Methylsulfonyl)benzyl]-6-phenyl-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine (5a)
IR (KBr, gmax, cmꢀ1): 3015 (AreH), 2935 (CeH), 1715 (C]O),
1599 (C]N), 1532 (C]C), 1469 (NeCeS), 1419 (S]O), 1368 (CeN),
1298 (NeN]C), 1139 (S]O), 637 (CeSeC); 1H NMR: (400 MHz,
IR (KBr, gmax, cmꢀ1): 3005 (AreH), 2905 (CeH), 1598 (C]N),
1527 (C]C), 1417 (S]O), 1467 (NeCeS), 1337 (CeN), 1297 (NeN]
CDCl3,
d ppm): 3.02 (s, 3H, SeCH3), 3.89 (s, 2H, CH2), 4.40 (s, 2H,
C), 1137 (S]O), 690 (CeSeC); 1H NMR: (400 MHz, CDCl3,
d
ppm):
CH2, AreH), 6.98 (m, 5H, coumarin), 7.71 (d, 2H, J ¼ 8.0 Hz, AreH),
7.87 (d, 2H, J ¼ 8.0 Hz, AreH), 8.11 (s, coumarin, 1H), Anal. calcd. for
C21H16N4O4S2: C, 55.74; H, 3.56; N, 12.38; found: C, 55.73; H, 3.54;
N, 12.36.
3.02 (s, 3H, SeCH3), 3.97 (s, 2H, CH2), 4.43 (s, 2H, CH2, AreH),
7.51e7.60 (m, 5H, phenyl), 7.81 (d, 2H, AreH, J ¼ 8.0 Hz), 7.87 (d,
2H, AreH, J ¼ 8.0 Hz); 13C NMR: (100 MHz, CDCl3,
d ppm): 23.62
(CH2), 30.92 (CH2), 44.50 (SeCH3), 127.13, 127.82, 129.25, 130.07,
132.26, 133.41, 139.38, 141.92, 152.13, 153.53; DEPT (100 MHz,
6.4.7. 5-Chloro-4-{3-[4-(methylsulfonyl)benzyl]-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazin-6-yl}-thiophene-2-sulfonamide (5g)
CDCl3,
d
ppm): CH2: ꢀ23.62 (CH2), ꢀ30.92 (CH2), CH, CH3: 44.50
(SeCH3), 127.13, 127.83, 129.25, 130.07, 132.26; CH2: 23.62, 30.92,
MS (m/z): 385.4 (M þ 1); Anal. calcd. for C18H16N4O2S2: C, 56.23; H,
4.19; N, 14.57; found: C, 56.20; H, 4.15; N, 14.55.
IR (KBr, gmax, cmꢀ1): 3000 (AreH), 2920 (CeH), 1590 (C]N),
1530 (C]C), 1465 (NeCeS), 1423 (S]O), 1372 (CeN), 1295 (NeN]
C), 1130 (S]O), 880, 828 (CeCl), 631 (CeSeC); 1H NMR: (400 MHz,
DMSO-d6 þ CDCl3,
d ppm): 3.18 (s, 3H, SeCH3), 4.27 (s, 2H, CH2),
4.38 (s, 2H, CH2, AreH), 7.57 (s, 1H, thienyl), 7.59 (d, 2H, AreH,
6.4.2. 6-(4-Methylphenyl)-3-[4-(methylsulfonyl)benzyl]-7H-[1,2,4]
triazolo[3,4-b][1,3,4] thiadiazine (5b)
J ¼ 8.0 Hz), 7.86 (d, 2H, AreH, J ¼ 8.0 Hz); 13C NMR (100 MHz,
IR (KBr, gmax, cmꢀ1): 3009 (AreH), 2924 (CeH), 1598 (C]N),
1524 (C]C), 1460 (NeCeS), 1413 (S]O), 1366 (CeN), 1293 (NeN]
DMSO-d6 þ CDCl3,
d ppm): 25.69 (CH2), 30.03(CH2), 44.06 (SeCH3),
127.60, 130.22, 130.41, 132.62, 134.82, 139.73, 142.50, 144.40, 150.20,
152.71; DEPT (100 MHz, DMSO-d6 þ CDCl3, ppm): CH2: ꢀ25.68,
ꢀ30.03, CH, CH3: 44.06, 127.60, 130.22, 130.41; Anal. calcd. for
C), 1137 (S]O), 631 (CeSeC); 1H NMR: (400 MHz, CDCl3,
d
ppm):
d
2.45 (s, 3H, CH3), 3.05 (s, 3H, SeCH3), 3.94 (s, 2H, CH2), 4.42 (s, 2H,

64
V. Sumangala et al. / European Journal of Medicinal Chemistry 54 (2012) 59e64
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N, 13.85.
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6.4.8. 6-(2,5-Dichloro-3-thienyl)-3-[4-(methylsulfonyl)benzyl]-7H-
[1,2,4]triazolo[3,4-b][1,3,4]-thiadiazine (5h)
IR (KBr, gmax, cmꢀ1): 3007 (AreH), 2930 (CeH), 1590 (C]N),
1533 (C]C), 1460 (NeCeS), 1410 (S]O), 1362 (CeN), 1290 (NeN]
C), 1132 (S]O), 885, 825 (CeCl), 630 (CeSeC); 1H NMR: (400 MHz,
CDCl3,
d ppm): 3.15 (s, 3H, SeCH3), 4.22 (s, 2H, eCH2), 4.35 (s, 2H,
CH2, AreH), 7.50 (s, 1H, thienyl), 7.60 (d, 2H, AreH, J ¼ 8.0 Hz), 7.88
(d, 2H, AreH, J ¼ 8.0 Hz); 13C NMR (100 MHz, CDCl3,
d ppm): 25.65
(CH2), 30.00(CH2), 44.00 (SeCH3), 127.56, 130.15, 130.35, 132.45,
134.68, 139.70, 142.35, 144.35, 150.12, 152.63; DEPT (100 MHz,
CDCl3,
d ppm): CH2: 25.65, 30.00, CH, CH3: 44.01, 127.56, 130.14,
130.35; Anal. calcd. for C16H19Cl2N4O2S3: C, 41.83; H, 2.63; N, 12.20;
found: C, 41.80; H, 2.65; N, 12.13.
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substituted
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Acknowledgment
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We are grateful to the Mangement, SeQuent Scientific Ltd. New
Mangalore. The authors are also thankful to Prof. A. Srikrishna,
Department of Organic Chemistry, IISc, Bangalore for providing 1H
NMR and 13C NMR spectral facilities. Thanks are also due to
Department of Pharmacology, Manipal College of Pharma science,
Manipal University for the analysis of cytotoxicity.
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