Synthesis, spectral characterization and biological evaluation of a novel series of 6-arylsubstituted-3-[2-(4-substitutedphenyl)propan-2-yl]-7H-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazines

Article abstract of DOI:10.1016/j.ejmech.2012.06.059

On account of the reported anticancer activity of triazolothiadiazines, we have synthesized a novel series of 6-arylsubstituted-3-[2-(4-substitutedphenyl) propan-2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and tested for in-vitro cytotoxicity by trypan blue exclusion and MTT assay. These compounds were also evaluated for their in-vivo anthelmintic activity, as well as in-vitro antimicrobial studies. Amongst the tested compounds, the compound 7j was the most promising cytotoxic agent with IC₅₀ value of 10.54 μM in MCF-7 cells. The compounds 7l and 7q exhibited excellent anthelmintic activity. The compounds 7d, 7f, 7j, 7l, 7o, 7p and 7r showed good antibacterial activity, whereas compounds 7e and 7k exhibited excellent antifungal activity. The structures of newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR and LCMS analysis.

Full text of DOI:10.1016/j.ejmech.2012.06.059

European Journal of Medicinal Chemistry 57 (2012) 407e416
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, spectral characterization and biological evaluation of a novel series
of 6-arylsubstituted-3-[2-(4-substitutedphenyl)propan-2-yl]-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazines
,
,
c
d
Pushpan Puthiyapurayil a b, Boja Poojarya , Chandrashekhar Chikkanna , Sunil Kumar Buridipad
*
a Department of Chemistry, Mangalore University, Mangalagangothri, Konaje Post, Mangalore, Karnataka 574199, India
b Syngene International Ltd, Biocon Park, Jigani Link Road, 560099 Bangalore, India
c Department of Medicinal Chemistry, SDM College Ujire, D.K. 574342, Karnataka, India
d Department of Pharmaceutical Chemistry, NET Pharmacy College, Raichur, 584103 Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
On account of the reported anticancer activity of triazolothiadiazines, we have synthesized a novel series
of 6-arylsubstituted-3-[2-(4-substitutedphenyl)propan-2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines
and tested for in-vitro cytotoxicity by trypan blue exclusion and MTT assay. These compounds were also
evaluated for their in-vivo anthelmintic activity, as well as in-vitro antimicrobial studies. Amongst the
Received 14 October 2011
Received in revised form
25 June 2012
Accepted 28 June 2012
Available online 7 July 2012
tested compounds, the compound 7j was the most promising cytotoxic agent with IC50 value of 10.54 mM
in MCF-7 cells. The compounds 7l and 7q exhibited excellent anthelmintic activity. The compounds 7d,
7f, 7j, 7l, 7o, 7p and 7r showed good antibacterial activity, whereas compounds 7e and 7k exhibited
excellent antifungal activity. The structures of newly synthesized compounds were characterized by IR,
1H NMR, 13C NMR and LCMS analysis.
Keywords:
Triazolothiadiazine
Ehrlich ascites carcinoma
MTT assay
Ó 2012 Elsevier Masson SAS. All rights reserved.
Anthelmintic activity
Antimicrobial activity
1. Introduction
taxol and vinca alkaloids are known for effective antimitotic drugs.
However the complex synthesis, difficult formulations, lack of oral
With the advent of combinatorial chemistry, the number of new
chemical entity (NCEs) can be produced in a short space of time by
drug discovery teams. Although this has produced a wealth of
possible new therapeutic compounds, it has also raised an impor-
tant question about screening of most therapeutically active ones,
from thousands of compounds. One approach to solve this problem
has been the use of in-vitro screens to identify the characteristics of
an NCE, particularly with respect to its drug metabolism. Such an
information is crucial to the decision making process of which
compounds to progress with and which to discard. Such an
approach can also be used to screen smaller set of structurally
related compounds, allowing determination of the chemical
structure that is the strongest possible lead candidate [1,2].
availability makes these drugs suboptimum for clinical treatment of
cancer [3]. Hence there is considerable interest in the design and
development of novel molecules that inhibit cell proliferation.
Helminthiasis is a disease caused by infestation of parasitic
worms living in the alimentary canal or in the other tissues of the
host. Anthelmintics are the drugs, which are used to kill or to
remove the parasitic worms. An anthelmintic drug may act by
causing narcosis or paralysis of worms by either penetrating the
cuticle or by the worm ingesting. The specificity of anthelmintics
reveals that they interfere with metabolism of the worm; each
worm is having its own metabolic requirement. The human and
animal parasitic worms zoologically belong to the class Cestodes
(tapeworm), Schistosomes (Trematode, flukes), Filaria (thread
worm), Nematodes (round worms) and Pheritima posthum (Earth
worms, order e Annelida, Class e oligichaeta). As it was difficult to
procure either roundworm, or tapeworm, the experiment
was performed on earth worms (Pheritima posthuma), which have
similar life cycle. The technique adopted was that described
by Giand et al. [4] with slight modifications. Anthelmintic activity
was evaluated by using earthworm Pheritima posthuma
(Class e Annelida and Oligichaeta).
Cancer is a serious disease with complex pathogenesis, which
threats human life greatly. A number of chemotherapeutic drugs
have been developed to treat cancer that includes DNA-alkylating
agents, and antimitotic agents. Many natural products such as
* Corresponding author. Tel.: þ91 824 2287262; fax: þ91 824 2287367.
E-mail address: bojapoojary@gmail.com (B. Poojary).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.059

408
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
The [1,2,4]-triazole nucleus and their derivatives emerge rapidly
2. Results and discussion
with advance of modern heterocyclic chemistry, promising a variety
of medical applications. [1,2,4]-Triazole nucleus has been incorpo-
rated into a wide variety of therapeutically interesting molecules to
transform them into better drugs [5e7]. The [1,2,4]-triazole and
their derivatives were found to be associated with various biological
activities such as antibacterial [8e14], antifungal [11e15], antitu-
bercular [16e18], analgesic [19,20], anti-inflammatory [20e22],
antiviral [23,24] and antidepressant [25]. Several 1,2,4-triazole
containing compounds are used as drugs (Fig. 1); for instance Flu-
conazole is used as an antimicrobial drug, Loreclezole is used as an
anticonvulsant, Itraconazole is used as an antifungal agent, whereas
Vorozole, Letrozole and Anastrozole are non steroidal drugs used
for the treatment of cancer. Moreover recent literature report [26]
showed that, triazolothiadiazine (I) and triazolopyridazine (II)
(Fig. 2) are good inhibitors of phosphodiesterases (PDE4).
A recent literature reveals that modulation of the phenyl cycle
enhances biological activities of [1,2,4]-triazole derivatives, and
incorporation of a methylene chain between the phenyl and 1,2,4-
triazole cycle increased the antifungal activity by two fold on the
yeast species (Candida albicans and Candida tropicalis) [27]. We
therefore are interested in exploring the biological activities of such
molecules through structural modifications, by incorporating three
carbon spacer between the phenyl and [1,2,4]triazolo[3,4-b][1,3,4]
thiadiazine cycle. In view of these observations, we hereby report
the synthesis, spectral characterization, their in-vitro cytotoxicity,
in-vivo anthelmintic and in-vitro antimicrobial activities.
2.1. Chemistry
Synthetic pathway for the preparation of title compounds is
depicted in Scheme 1, we have synthesized three series of 4-amino-
5-[2-(4-substitutedphenyl)propan-2-yl]-4H-[1,2,4]-triazole-3-
thiols (5aec) through a multi step reaction [28]. Substituted phenyl
acetic acids were converted into their methyl substituted arylace-
tates (1aec), which reacted with sodium hydride and methyl iodide
in THF at ꢀ10 ꢁC under N2 atmosphere to yield methyl-2-
arylsubstituted-2-methylpropanoate (2aec). Aromatic esters were
hydrazinolyzed to give aryl hydrazides (3aec). The compounds
(3aec) on reaction with carbon disulfide in methanolic potassium
hydroxide yielded corresponding dithiocarbazinates (4aec) in
good yields and were directly used for the next step without
purification. 4-Amino-5-[2-(4-substitutedphenyl)propan-2-yl]-4H-
[1,2,4]-triazole-3-thiones (5aec) were synthesized by refluxing
(4aec) with hydrazine hydrate. The key condensation reaction
between appropriately substituted-2-bromo-1-phenylethanone
(ultimately the phenyl ring at the C-6 position of the heterocycle)
and appropriately substituted 4-amino-5-[2-(4-substitutedphenyl)
propan-2-yl]-4H-[1,2,4]-triazole-3-thiones (ultimately the phenyl
ring at the C-3 position of the heterocycle) was accomplished in
ethanol at elevated temperatures to yield 6-arylsubstituted-3-[2-
(4-substitutedphenyl)propan-2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazines (7aer). Assignment of structures of the new
N
N
N
N
N
F
OH
N
N
N
N
F
N
N
N
N
N
Anastrozole
Fluconazole
N
N
Letrozole
N
N
N
O
O
Cl
N
N
N
N
O
N
O
Cl
Itraconazole
N
Cl
Cl
N
N
N
N
N
N
N
Cl
N
Cl
Loreclezole
Vorozole
Fig. 1. Drugs containing [1,2,4]-triazole moiety.

P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
409
O
O
O
O
O
O
O
N
S
N
O
N
N
N
N
N
N
I
II
Fig. 2. Structures of several known PDE4 inhibitors.
compounds was based on their spectroscopic data. Characteriza-
tion data of all new compounds were summarized in Table 1.
obtained by needle aspiration of ascetic fluid from the preinocu-
lated mice under aseptic conditions. The cells were washed
repeatedly with phosphate buffered saline (PBS) to free it from
blood. Tested compounds were prepared with various concentra-
3. Pharmacology
tions of 10, 20, 40, 80 and 160 mg/mL in a mixture of 0.7 mL
3.1. In-vitro cytotoxic activity by Trypan blue exclusion method (cell
viability method)
dimethylsulfoxide (DMSO) and 0.3 mL saline.
In a set of sterile test tubes 0.8 mL saline, 0.1 mL of each of the
tested compound solutions were mixed, then 0.1 mL of tumour
cells suspension was added. The test tubes were incubated at 37 ꢁC
In-vitro cytotoxic activity of the compounds was determined
using Ehrlich Ascites Carcinoma (EAC) cell lines [29]. EAC cells were
for 3 h. The control tube had 10
mL of DMSO solvent. To each tube
OH
(i)
O
O
O
(ii)
O
O
R
R
R
2(a-c)
1(a-c)
(iii)
S
H
N
H
(iv)
N
N
H
S-K+
NH2
O
O
R
R
4(a-c)
3(a-c)
(v)
N
N
N
R
H2N
SH
O
O
5(a-c)
(vi)
Br
(vii)
Ar
Ar
6
N
N
N
R
N
S
Ar
R = H, Cl, Br
7(a-r)
Scheme 1. Reagents and conditions: (i) cat. H2SO4, MeOH, Reflux; (ii) NaH, CH3I, THF, RT; (iii) N2H4$H2O, EtOH, Reflux; (iv) CS2, KOH, MeOH, RT; (v) N2H4$H2O, H2O, reflux; (vi)
anhydrous AlCl3, Br2, diethyl ether, RT; (vii) EtOH, reflux.

410
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
Table 1
Characterization data of 6-arylsubstituted-3-[2-(4-substitutedphenyl)propan-2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (7aer).
Compound
R
Ar
Mol. formula
(Mol. weight)
m.p. (ꢁC)
Yield (%)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
H
H
H
H
H
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4eN<ce:glyph name¼"tbnd"/>CeC6H4
3eBreC6H4
3eCleC6H4
C20H17N5S (359.46)
201.1e201.9
173.2e175.4
166.4e168.0
149.6e151.4
238.2e239.4
165.5e167.2
202.1e204.7
172.4e174.0
160.0e162.0
130.0e132.8
206.4e208.1
225.9e227.2
212.1e214.9
179.8e182.3
148.2e150.5
151.8e153.9
215.2e216.9
229.0e231.8
71
73
70
77
71
69
79
75
81
66
81
60
91
89
67
81
66
84
C19H17BrN4S (413.34)
C19H17 ClN4S (368.89)
C20H17F3N4OS (418.44)
C19H16ClN5O2S (413.89)
C19H15Cl2FN4S (421.33)
C20H16ClN5S (393.90)
C19H16 BrClN4S (447.79)
C19H16 Cl2N4S (403.34)
C20H16ClF3N4OS (452.89)
C19H15Cl2N5O2S (448.33)
C19H14Cl3FN4S (455.77)
C20H16BrN5S (438.35)
C19H16Br2N4S (492.24)
C19H15BrF2N4S (449.32)
C20H16BrF3N4OS (497.34)
C19H15BrClN5O2S (492.78)
C19H14BrCl2FN4S (500.22)
4eCF3OeC6H4
4eCl-3eNO2 C6H3
2,4eCl2-5eFꢀC6H2
4eN<ce:glyph name¼"tbnd"/>CeC6H4
3eBreC6H4
3eCleC6H4
4eCF3OeC6H4
4eCl-3eNO2 C6H3
2,4eCl2e5-FꢀC6H2
4eN<ce:glyph name¼"tbnd"/>CeC6H4
3eBreC6H4
3,4-F2eC6H3
4-CF3OeC6H4
4-Cle3-NO2 C6H3
2,4-Cl2e5-FꢀC6H2
100
m
L of trypan blue solution (0.4%) (Cat. No. T8154) was added.
Human breast cancer lines MCF-7 and alveolar adenocarcinoma cell
lines (A549), procured from National centre for cell sciences
(NCCS), Pune, India, were cultured in MEM medium supplemented
The live (without stain) and dead (with blue stain) cells were
counted using hemocytometer. The percentage inhibition of the
viable cells were calculated using the formula, percentage of inhi-
bition viable cells ¼ number viable cells/total number of cells ꢂ 100.
Doxorubicin (CAS-23214-92-8) was used as standard drug. The
results of the experiments are presented in Table 2.
with 10% FBS, 1% L glutamine and 50 mg/mL gentamicin sulphate in
a CO2 incubator in a humidified atmosphere of 5% CO2 and 95% air.
The EAC cells were maintained for 12e14 days in the peritoneal
cavity of Swiss albino mice. The tumour cell cultures were started
from mouse Ehrlich Ascites with at least one passage in-vitro prior
to use.
3.2. In-vitro cytotoxic evaluation by MTT assay
In-vitro cytotoxicity was determined using a standard MTT assay
[30] with protocol appropriate for the individual test system. In
brief, exponentially growing cells were plated in 96-well plates (104
cells/well in 100 mL of medium) and incubated for 24 h for attach-
ment. Test compounds were prepared prior to the experiment by
dissolving in 0.1% DMSO and diluted with medium. The cells were
In-vitro cytotoxicity of the synthesized compounds was assessed
by standard MTT bioassay in different cancer cells at 24 h of drug
administration. In EAC cells, the compounds 7a, 7d, 7e, 7f, 7g, 7i, 7j,
7k and 7l were found to be active with IC50 values in the range of
12.24e29.42 mM (Table 3). To assess the efficacy and to select the
then exposed to different concentrations of the drugs (1e100
m
M)
promising compounds human cancer cells, breast cancer cell line
(MCF-7) and alveolar adenocarcinoma cell line (A549) were used.
in the volume of 100
mL/well. Cells in the control wells received the
Table 2
Table 3
In-vitro cytotoxic activities of 6-arylsubstituted-3-[2-(4-substitutedphenyl)propan-
2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (7aer) by Trypan blue exclusion
method.
In-vitro cytotoxic activities of 6-arylsubstituted-3-[2-(4-substitutedphenyl)propan-
2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (7aer) by MTT assay.
Compound
IC50
EAC
(m
M)a
Compound
% Inhibitions
MCF-7
A549
Concentration (
m
g/mL)
40
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
29.21 ꢃ 0.002
46.22 ꢃ 0.012
32.28 ꢃ 0.009
28.22 ꢃ 0.023
28.20 ꢃ 0.002
29.42 ꢃ 0.005
16.24 ꢃ 0.022
42.28 ꢃ 0.012
28.24 ꢃ 0.017
12.24 ꢃ 0.030
24.42 ꢃ 0.012
22.60 ꢃ 0.005
34.20 ꢃ 0.011
>100
25.28 ꢃ 0.022
42.20 ꢃ 0.005
27.24 ꢃ 0.022
22.44 ꢃ 0.003
24.40 ꢃ 0.011
28.50 ꢃ 0.022
14.28 ꢃ 0.011
40.18 ꢃ 0.005
16.28 ꢃ 0.011
10.54 ꢃ 0.011
28.30 ꢃ 0.009
24.20 ꢃ 0.011
40.20 ꢃ 0.022
>100
36.14 ꢃ 0.012
48.12 ꢃ 0.017
46.14 ꢃ 0.017
40.18 ꢃ 0.011
30.20 ꢃ 0.017
32.28 ꢃ 0.012
18.56 ꢃ 0.022
54.32 ꢃ 0.044
54.20 ꢃ 0.011
18.20 ꢃ 0.022
54.28 ꢃ 0.016
48.22 ꢃ 0.016
46.18 ꢃ 0.017
NTb
160
80
20
10
Controla
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
39.18
9.60
0
31.88
6.03
0
26.52
2.60
0
22.85
4.31
0
12.38
4.00
1.60
5.47
3.52
2.13
6.00
3.30
3.01
6.71
4.05
2.89
3.12
3.89
4.49
3.87
3.06
8.58
15
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
12.11
e
5.59
13.38
15.00
10.22
6.31
14.28
26.01
9.89
9.45
3.87
5.71
7.54
9.41
6.40
40.82
98
4.94
5.60
9.75
8.33
8.01
7.61
15.73
8.73
12.12
5.33
6.48
5.50
7.44
6.92
28.58
73
10.06
8.15
8.33
10.63
5.12
5.45
14.62
4.50
3.50
5.28
6.40
4.74
8.00
6.80
22.46
52
4.27
4.51
4.12
5.00
4.19
5.21
7.80
6.34
4.11
5.55
4.61
4.85
7.20
4.33
16.75
28
44.28 ꢃ 0.005
46.20 ꢃ 0.022
>100
>100
NTb
60.22 ꢃ 0.016
84.20 ꢃ 0.012
7q
7r
>100
NTb
7p
7q
42.40 ꢃ 0.012
44.60 ꢃ 0.016
3.44 ꢃ 0.012
84.26 ꢃ 0.022
4.52 ꢃ 0.18
Doxorubicinc
6.44 ꢃ 0.03
7r
a
Each experiment was independently performed three times and expressed as
Standardb
means ꢃ SD.
a
b
c
DMSO.
Standard ¼ Doxorubicin.
NT, not tested.
Doxorubicin was used as reference drug.
b

P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
411
same volume of medium containing 0.1% DMSO. After 24 h, the
medium was removed and cell cultures were incubated with 100
MTT reagent (1 mg/mL) for 4 h at 37 ꢁC. The formazan produced by
the viable cells was solubilised by addition of 100 L DMSO. The
suspension was placed on micro-vibrator for 5 min and absorbance
was recorded at 540 nm by the ELISA reader. The experiment was
performed in triplicate. The percentage cytotoxicity was calculated
using the following formula and the results were tabulated in
Table 3.
method [32,33]. Serial dilutions of the drug in MuellereHintonbroth
were taken in tubes and their pH was adjusted to 5.0 using phos-
phate buffer. A standardized suspension of the test bacterium was
inoculated and incubated for 18 h at 37 ꢁC. The minimum inhibitory
concentration (MIC) was noted by seeing the lowest concentration
of the drug at which there was no visible growth. A number of
antimicrobial discs are placed on the agar for the purpose of
producing zones of inhibition in the bacterial lawn. The 20 mL
solution of agar media was poured into each Petri dish. Excess of
suspension was decanted and plates were dried by placing in an
incubator at 37 ꢁC for 1 h. Using an agar punch, wells were made on
these seeded agar plates and minimum inhibitory concentrations of
the tested compounds in dimethylsulfoxide (DMSO) were added
into each labelled well. A control was also prepared for the plates in
the same way using DMSO. The Petri dish were prepared in triplicate
and maintained at 37 ꢁC for 3 days. Antibacterial activity was
determined by measuring the diameter of inhibition zone. Activity
of each compound was compared with Streptomycin as standard.
Zone of inhibition was determined for (7aer) and the results are
summarized in Table 5.
mL
m
% cytotoxicity ¼ ðControl abs ꢀ Blank ꢀ absÞ
ꢀ ðTest abs ꢀ Blank ꢀ absÞ=
ðControl ꢀ abs ꢀ Blank ꢀ absÞ ꢂ 100
3.3. Anthelmintic activity
The worms were washed with water to remove adhering
materials and were sorted out for uniform size and length. They
were then kept at 6% dextrose solution for acclimatisation. Worms
with normal motility were selected for the experiment. Petri dishes
of equal size were selected and in each Petri dish, 2 mL of each
extract (50 mg/mL) in 0.1% Tween-80 suspension were placed and
the volume was made up to 25 mL with an aqueous solution of
dextrose (6%). In another Petri dish Tween-80 (25 mL, 0.1% solution)
prepared in 6% dextrose solution was placed, which served as
control. The Albendazole served as standard. In each Petri dish,
eight worms were placed. The time taken by each worm for paral-
ysis was noted and the paralysis of worms were tested by placing
the worms in water maintained at 50 ꢁC. Non motility at 50 ꢁC was
taken as the death of the worm and correspondingly the time for
death was recorded [31]. The results were tabulated in Table 4.
3.5. Antifungal activity
Newly synthesized compounds were screened for their anti-
fungal activity against Rhizopus oryzae, Asperigillus fumigates (NICM
No 524), Asperigillus niger and Trichophyton mentagrophytes
(recultured) in DMSO by serial plate dilution method [34]. Sabo-
urands agar media was prepared by dissolving peptone (1 g), D-
glucose (4 g), and agar (2 g) in distilled water (100 mL) and
adjusting the pH to 5.0. Normal saline was used to make
a suspension of spore of fungal strains for lawning. A loopful of
particular fungal strains was transferred to 3 mL saline to get
a suspension of corresponding species. The 20 mL solution of agar
media was poured into each Petri dish. Excess of suspension was
decanted and plates were dried by placing in incubator at 37 ꢁC for
1 h. Using a punch, wells were made on these agar plates, and
minimum inhibition concentrations of the test compounds in
DMSO were added into each labelled well. A control was also
prepared in the same way using solvent DMSO. The Petri dishes
3.4. Antibacterial activity
The newly synthesized compounds were screened for their
antibacterial activity against Escherichia coli (ATCC-25922), Staph-
ylococuus aureus (ATCC-25923), Pseudomonas aeruginosa (ATCC-
27853) and Bacillus subtilis bacterial stains by serial plate dilution
Table 4
Table 5
In-vivo evaluation of anthelmintic activity of 6-arylsubstituted-3-[2-(4-
substitutedphenyl)propan-2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (7aer).
In-vitro antibacterial activity of 6-arylsubstituted-3-[2-(4-substitutedphenyl)
propan-2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (7aer).
Compound
Concentration
(wt/vol, mg)
Mean paralysing
Mean death time
Compound
MIC in mg/mL and zone of inhibition in mm
time (in min ꢃ SEa)
(in min ꢃ SEa)
E. Coli
S. aureus
P. aeruginosa
B. subtilis
7a
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
e
90 ꢃ 0.02
85 ꢃ 0.044
100 ꢃ 0.011
80 ꢃ 0.009
90 ꢃ 0.044
60 ꢃ 0.012
90 ꢃ 0.020
60 ꢃ 0.044
60 ꢃ 0.022
80 ꢃ 0.020
40 ꢃ 0.0013
35 ꢃ 0.011
90 ꢃ 0.022
85 ꢃ 0.024
60 ꢃ 0.018
45 ꢃ 0.069
38 ꢃ 0.013
40 ꢃ 0.022
e
98 ꢃ 0.18
100 ꢃ 0.025
110 ꢃ 0.17
96 ꢃ 0.044
110 ꢃ 0.012
70 ꢃ 0.022
95 ꢃ 0.022
69 ꢃ 0.18
65 ꢃ 0.044
89 ꢃ 0.022
50 ꢃ 0.0176
38 ꢃ 0.022
95 ꢃ 0.013
95 ꢃ 0.22
70 ꢃ 0.018
50 ꢃ 0.022
40 ꢃ 0.22
45 ꢃ 0.022
e
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
6.25 (21e25)
50 (<10)
6.25 (14e18)
50 (<10)
6.25 (18e22)
50 (<10)
6.25 (16e20)
50 (<10)
12.5 (16e20)
12.5 (14e18)
12.5 (<10)
6.25 (20e22)
12.5 (12e16)
50 (<10)
12.5 (18e22)
12.5 (16e18)
12.5 (14e16)
6.25 (22e26)
6.25 (18e20)
50 (<10)
12.5 (16e18)
12.5 (18e22)
6.25 (12e16)
6.25 (16e18)
6.25 (20e24)
50 (<10)
12.5 (18e20)
12.5 (20e24)
6.25 (16e20)
6.25 (18e22)
12.5 (16e18)
50 (<10)
12.5 (14e16)
12.5 (16e18)
6.25 (12e16)
6.25 (20e24)
6.25 (22e30)
6.25 (18e20)
12.5 (18e24)
6.25 (16e18)
6.25 (20e24)
6.25 (22e24)
50 (<10)
6.25 (18e20)
12.5 (20e22)
6.25 (16e20)
6.25 (20e22)
6.25 (14e16)
50 (<10)
6.25 (16e20)
12.5 (18e20)
6.25 (16e18)
6.25 (10e12)
6.25 (34e40)
12.5 (10e15)
12.5 (18e24)
12.5 (10e12)
12.5 (22e24)
12.5 (10e15)
50 (<10)
12.5 (14e16)
12.5 (18e22)
12.5 (14e18)
12.5 (20e26)
6.25 (16e18)
50 (<10)
12.5 (10e14)
12.5 (14e16)
12.5 (10e14)
12.5 (16e18)
6.25 (28e36)
12.5 (12e16)
12.5 (12e14)
12.5 (12e14)
6.25 (16e18)
6.25 (25e30)
7p
7q
7r
Controlb
Standardc
7r
20
40 ꢃ 0.03
45 ꢃ 0.024
Standarda
a
Each experiment was independently performed three times and expressed as
The MIC values were evaluated at concentration range of 6.25e50
mg/mL.
means ꢃ SE (SE, standard error).
The figures in the table show the MIC values in mg/mL and the corresponding zone of
b
Control: Tween-80.
inhibition in mm.
c
a
Standard: Albendazole.
Standard, streptomycin.

412
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
were prepared in triplicate and maintained at 37 ꢁC for 3 days.
Antifungal activity was determined by measuring the diameter of
inhibition zone. Activity of each compound was compared with
Amphoterin-B as standard, and results are summarized in Table 6.
mainly attributed to the presence of (2-phenylpropan-2yl) and [2-
(4-chlorophenyl)propan-2-yl] substituent at C-3 position of the
triazolothiadiazine ring system. Results from Table 3 further
revealed that, compounds carrying 4-cyanophenyl (7a, 7g), 3-
chlorophenyl (7c, 7i), 4-trifluoromethoxyphenyl (7d, 7j), 4-
chloro-3-nitrophenyl (7e, 7k) and 2,4-dichloro-5-fluorophenyl
(7f, 7l) substituent’s at C-6 position of the triazolothiadiazine ring
system also showed better activity than other compounds.
Compound 7j carrying 4-trifluormethoxyphenyl group at C-6
position and [2-(4-chlorophenyl)propan-2-yl] substituent at C-3
4. Biological activity results
The results of the short term in-vitro cytotoxic activity against
Ehrlich Ascites Carcinoma (EAC) cell lines by trypan blue exclusion
method showed poor to moderate activity. Various phenacyl
bromides were considered for the synthesis of title compounds to
draw the structure activity (SAR) relationship studies in relation to
cytotoxic activity. Only the compounds 7a and 7r showed moderate
position was the most lead molecules with IC50 value 10.54 mM.
These results clearly indicated that compounds having a biologi-
cally active 4-trifluoromethoxyphenyl group at C-6 position and [2-
(4-chlorophenyl)propan-2-yl] substituent at C-3 position could be
responsible for the increased activity.
The results of anthelmintic activity revealed that compounds
7k, 7l, 7p, 7q and 7r showed good anthlemintic activity almost
comparable with standard drug. Compound 7l and 7q exhibited
excellent activity then the standard drug. The excellent activity of 7l
and 7q was attributed to the presence of 2,4-dichloro-5-
fluorophenyl and 4-chloro-3-nitrophenyl groups on the C-6
phenyl ring and halogen groups (4-chloro and 4-bromo) on the C-3
position of the fused triazolothiadiazne ring system.
activity at 80 and 160 mg/mL concentrations, compound 7j showed
poor activity, whereas compounds 7b, 7c, 7d, 7e, 7f, 7g, 7h, 7i, 7k,
7l, 7m, 7n, 7o, 7p and 7q were found to be inactive against EAC cells
lines. To assess the efficacy and to select the promising molecules
we performed in-vitro cytotoxicity by MTT assay.
In-vitro cytotoxicity by MTT assay showed that, in EAC cells; the
compounds 7a, 7d, 7e, 7f, 7g, 7i, 7j, 7k and 7l were found to be
active with IC50 values in the range of 12.24e29.42
cells, IC50 values for the compounds 7aer were in the range of
10.54e60.22 M. Similarly in A549 cells, the IC50 values for 7aer
were found to be in the range of 18.20e84.26 M. In MCF-7 cells,
the compounds 7n, 7o and 7q did not show 50% inhibition even at
a concentration of 100 M, hence they were not evaluated in A549
mM. In MCF-7
m
The investigation of antibacterial and antifungal studies
revealed that all the tested compounds 7a, 7c, 7d, 7e, 7f, 7g, 7i, 7j,
7k, 7l, 7m, 7o, 7p and 7r showed moderate to good inhibition at
m
m
6.25e12.5 mg/mL in DMSO. The compounds 7b, 7h, 7n showed very
cells. Present study revealed that among the human cancer cell
lines tested, MCF-7 cells are more sensitive to all the tested
compounds than A549 cells. The results of the study revealed that,
compounds 7a, 7c, 7d, 7e, 7f, 7g, 7i, 7j, 7k and 7l showed moderate
to good cytotoxic activity in MCF-7 cells. Compound 7j was the
poor activity against all bacterial strains. The poor activity was due
to the presence of bromine at the C-3 phenyl ring of the tri-
azolothiadiazine ring system. The compounds 7d, 7f, 7j, 7l, 7o, 7p,
and 7r showed comparatively very good activity against all bacte-
rial strains. The good activity was attributed to the presence of
pharmacologically active eOCF3, fluorine, (highly electro negative)
in the molecules, which increases the lipophilicity and affects the
partitioning of molecules into membranes and facilitates hydro-
phobic interactions of the molecules with specific binding sites on
either receptor or enzymes (22e24). The compounds 7a, 7c, 7f, 7g,
7i, 7l, 7m and 7o showed moderate to good activity against all the
fungal strains. The good antifungal activity was attributed to the
presence of fluorine, e0CF3, and chloride molecules in the phenyl
ring of triazolothiadiazine ring system. The compounds 7e and 7k,
comprising the electron withdrawing nitro group showed
increased antifungal inhibition, but moderate antibacterial inhibi-
tion. Although compound 7q having electron withdrawing nitro
group on the phenyl ring, the presence of bromine on the C-3
phenyl ring were found to less active against all antifungal strains.
Results showed that combination of triazolothiadiazine containing
thioether linkage and presence of pharmacologically active fluo-
rine, chloride and eOCF3 groups gave an enhanced biological
activity against all microbial strains.
most promising cytotoxic agent with IC50 value of 10.54 mM. We
have also observed that, compounds carrying (2-phenylpropan-
2yl) substituent (7aef), and [2-(4-chlorophenyl)propan-2-yl]
substituent (7gel) at C-3 position of the triazolothiadiazine ring
were more potent than the third series carrying [2-(4-
bromophenyl)propan-2yl] substituent (7mer). The enhanced
activity of compounds 7a, 7c, 7d, 7e, 7f, 7g, 7i, 7j, 7k and 7l was
Table 6
In-vitro antifungal activity of 6-arylsubstituted-3-[2-(4-substitutedphenyl)propan-
2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (7aer).
Compound MIC in
R. oryzae
6.25 (16e20) 6.25 (16e20) 12.5 (14e18) 6.25 (12e16)
m
g/mL and zone of inhibition in mm
A. fumigates A. niger
T. mentagrophytesa
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
50 (<10) 50 (<10) 50 (<10) 50 (<10)
12.5 (12e16) 12.5 (14e16) 12.5 (16e20) 12.5 (16e22)
12.5 (16e20) 12.5 (18e22) 12.5 (20e24) 12.5 (10e12)
6.25 (20e22) 6.25 (20e24) 6.25 (10e12) 6.25 (22e26)
6.25 (20e22) 6.25 (20e24) 6.25 (18e20) 6.25 (24e26)
6.25 (20e22) 6.25 (18e22) 6.25 (18e22) 6.25 (18e20)
50 (<10)
50 (<10)
50 (<10)
50 (<10)
5. Conclusion
6.25 (14e16) 6.25 (16e20) 6.25 (10e14) 6.25 (10e12)
12.5 (20e24) 12.5 (20e24) 12.5 (18e20) 6.25 (16e18)
12.5 (20e24) 12.5 (18e20) 12.5 (22e24) 12.5 (24e28)
6.25 (20e24) 6.25 (20e26) 6.25 (22e24) 6.25 (20e26)
12.5 (16e18) 12.5 (18e22) 12.5 (20e22) 12.5 (18e20)
In conclusion, this work demonstrates the synthesis of a series
of novel 6-arylsubstituted-3-[2-(4-substitutedphenyl)propan-2-
yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and in-vitro evalua-
tion of cytotoxicity by trypan blue exclusion and MTT assay, in-vivo
anthelmintic activity against Pheritima posthum (Earth worms)
and in-vitro evaluation of antimicrobial (bacterial and fungal)
studies. Anticancer study revealed that the compounds 7a and 7r
demonstrated moderate activity against EAC cell lines by trypan
blue exclusion method. Compound 7j was the most promising
50 (<10)
50 (<10)
50 (<10)
50 (<10)
6.25 (20e24) 6.25 (22e26) 6.25 (18e22) 6.25 (18e22)
12.5 (20e22) 12.5 (18e20) 12.5 (18e22) 12.5 (14e16)
12.5 (10e15) 12.5 (12e16) 12.5 (10e14)
50 (<10)
6.25 (10e16) 6.25 (14e16) 6.25 (14e18) 6.25 (16e20)
Standardb 6.25 (24e32) 6.25 (24e30) 6.25 (25e30) 6.25 (25e36)
The MIC values were evaluated at concentration range of 6.25e50 g/mL.
m
The figures in the table show the MIC values in mg/mL and the corresponding zone of
cytotoxic agent with IC50 value of 10.54 mM in MCF-7 cell lines by
MTT assay. The in-vivo anthelmintic screening of these compounds
showed that, compounds 7k, 7l, 7p, 7q and 7r exhibited excellent
inhibition in mm.
a
T. mentagrophytes, Trichophyton mentagrophyte.
Standard, amphoterin-B.
b

P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
413
activity compared with Albendazole as standard drug. The in-vitro
antimicrobial studies revealed that compounds 7a, 7c, 7d, 7e, 7f, 7g,
7i, 7j, 7k, 7l, 7m, 7o, 7p and 7r were active, in particular 7d, 7f, 7j,
7l, 7o, 7p and 7r exhibited excellent antibacterial activity at MIC
mixture was stirred at 0 ꢁC for 1 h and methyl iodide (0.199 mol)
was added slowly at 0 ꢁC and allowed to stir for 1 h, and the
temperature was gradually raised to room temperature. The stir-
ring was continued for 10 h. Upon completion of the reaction (TLC),
the reaction mass was quenched in crushed ice and acidified with
1.5 N HCl. Extracted in ethyl acetate (2 ꢂ150 mL). Combined extract
was washed with water, brine solution and dried over anhydrous
Na2SO4. Solvent was evaporated in rota-evaporator. Crude product
obtained was purified by column chromatography (60e120 mesh
size silica gel) using 4e4.5% EtOAc in pet ether to afford the title
compounds as colourless oils.
6.25 and 12.5 mg/mL respectively, when compared with standard
drug. The compounds 7a, 7c, 7e, 7f, 7g, 7i, 7k, 7l, 7m and 7o
showed good antifungal activity. In conclusion the promising
cytotoxicity, anthelmintic and antimicrobial activity profile of tri-
azolothiadiazine class of compounds make them certain promising
molecules for further lead optimization in the development of
novel anticancer, anthelmintic and antimicrobial agents.
6. Experimental protocols
6.3.1. Methyl-2-methyl-2-phenylpropanoate (2a)
Colourless oil. Yield: 7.5 g (63%). 1H NMR (400 MHz, CDCl3):
6.1. General
d 1.63 (s, 6H, CH3), 3.67 (s, 3H, OCH3), 7.30e7.36 (m, 5H, AreH).
Anal. Calcd. for C11H14O2: C, 74.13; H, 7.92. Found: C, 73.93; H, 7.72.
Thin layer chromatography was used to reach the completion of
the reaction and purity of the compounds synthesized. Melting
points were recorded (uncorrected) on Buchi melting point B-545
instrument. Infrared spectra were recorded on Thermo Scientific
Nicolet 6700-FT-IR spectrometer. 1H NMR, 13C NMR and spectra
were recorded on 400 MHz, 300 MHz and 100 MHz Bruker spec-
trometer in DMSO-d6/CDCl3/CD3OD using TMS as an internal
6.3.2. Methyl-2-(4-chlorophenyl)-2-methylpropanoate (2b)
Colourless oil. Yield: 24 g (70%). 1H NMR (300 MHz, CDCl3):
d
1.57 (s, 6H, CH3), 3.65 (s, 3H, eOeCH3), 7.28 (d, 2H, J ¼ 8.0 Hz,
AreH), 7.30 (d, 2H, J ¼ 8.0 Hz, AreH). Anal. Calcd. for C11H13ClO2: C,
62.12; H, 6.16. Found: C, 62.34; H, 6.38.
standard. Chemical shift values are expressed in
d
ppm, and
6.3.3. Methyl-2-(4-bromophenyl)-2-methylpropanoate (2c)
coupling constants were reported wherever it is necessary in Hz.
Mass spectra were recorded in Finnigan MAT8230 mass spec-
trometer. Reactions were carried out in an oven dried three necked
RB. Yields in table refer to isolated yields of the compounds.
Colourless oil. Yield: 14 g (73%). 1H NMR (400 MHz, CDCl3):
d
1.56 (s, 6H, CH3), 3.65 (s, 3H, eOeCH3), 7.22 (d, 2H, J ¼ 8.8 Hz,
AreH), 7.46 (d, 2H, J ¼ 8.8 Hz, AreeH). Anal. Calcd. for C11H13BrO2:
C, 51.38; H, 5.10. Found: C, 51.17; H, 4.98.
6.2. General method for the synthesis of methyl arylacetates (1aec)
6.4. General method for the synthesis of aryl acid hydrazide (3aec)
To a stirred solution of substitutedphenyl acetic acid (0.11 mol)
in anhydrous methanol (0.55 mol), conc. sulphuric acid (2.25 mL)
was slowly added at 0e5 ꢁC over a period of 10 min and heated to
reflux for 4 h. After the complete conversion of starting material as
monitored by TLC, solvent was removed in rota-evaporator. Residue
obtained was diluted with ether (200 mL), washed with saturated
NaHCO3 solution, water, brine solution and dried over anhydrous
Na2SO4. Solvent was evaporated in rota-evaporator to afford pure
product as colourless oil.
A mixture of Methyl-2-methyl-2-arylpropanoate (1 mol) and
hydrazine hydrate (2 mol) in ethanol (20 mL) was heated under
reflux condition for 6 h. After the completion of the reaction as
monitored by TLC, solvent was removed in rota-evaporator. Residue
obtained was dissolved in ethyl acetate, washed with water, brine
solution and dried over anhydrous Na2SO4. Solvent was evaporated
in rota-evaporator. Solid obtained was recrystallised from meth-
anol to obtain the pure compound.
6.4.1. 2-Methyl-2-phenylpropanehydrazide (3a)
6.2.1. Methyl-2-phenyl acetate (1a)
Off white solid. Yield: 7.2 g (73%); m.p.: 76.5e77.4 ꢁC. 1H NMR
Colourless oil. Yield: 16 g (96%). 1H NMR (400 MHz, CDCl3):
(400 MHz, CDCl3): d 1.60 (s, 6H, CH3), 3.80 (s, 2H, eNH2), 6.50 (s, 1H,
d
3.65 (s, 2H, eCH2eCO2CH3), 3.71 (s, 3H, eCO2CH3), 7.27e7.37(m,
eNHe), 7.27e7.37 (m, 5H, AreH). UPLC 180.2 (M þ H)þ. Anal. Calcd. for
5H, AreH). Anal. calcd. for C9H10O2: C, 71.98; H, 6.71. Found: C,
C10H14N2O: C, 67.39; H, 7.92; N,15.72. Found: C, 67.79; H, 8.22; N,15.97.
72.18; H, 6.91
6.4.2. 2-(4-Chlorophenyl)-2-methylpropanehydrazide (3b)
6.2.2. Methyl-2-(4-chlorophenyl)acetate (1b)
Off white solid. Yield: 15.5 g (77%), m.p. 85e86.4 ꢁC. 1H NMR
(300 MHz, CDCl3): d 1.56 (s, 6H, CH3), 3.65 (s, 2H, eNH2), 6.54 (s, 1H,
Colourless oil. Yield: 43 g (98%). 1H NMR (300 MHz, CDCl3):
d
3.60 (s, 2H, eCH2eCO2CH3), 3.70 (s, 3H, eCO2CH3), 7.23 (d, 2H,
eNHe), 7.30(d, 2H, J ¼ 10.8 Hz, AreH), 7.36 (d, 2H, J ¼ 10.8 Hz,
AreH). UPLC 215.2 (M þ H)þ. Anal. Calcd. for C10H13ClN2O: C, 56.47;
H, 6.16; N, 13.17. Found: C, 56.62; H, 6.31; N, 13.34.
J ¼ 8.4 Hz, AreH), 7.31 (d, 2H, J ¼ 8.4 Hz, AreH). Anal. Calcd. for
C9H9ClO2: C, 58.55; H, 4.91. Found: C, 58.87; H, 4.98.
6.2.3. Methyl-2-(4-bromophenyl)acetate (1c)
6.4.3. 2-(4-Bromophenyl)-2-methylpropanehydrazide (3c)
Colourless oil. Yield: 17.5 g (96%). 1H NMR (300 MHz, CDCl3):
Pale yellow solid. Yield: 11.0 g (78%), m.p. 79.4e80.4 ꢁC. 1H NMR
d
3.58 (s, 2H, eCH2eCO2CH3), 3.70 (s, 3H, eCO2CH3), 7.17 (d, 2H,
(300 MHz, CDCl3): d 1.55 (s, 6H, CH3), 4.1 (s, 2H, eNH2), 6.56 (s, 1H,
J ¼ 8.2 Hz, AreH), 7.47 (d, 2H, J ¼ 8.3 Hz, AreH). Anal. Calcd. for
eNHe), 7.24 (d, 2H, J ¼ 8.4 Hz, AreH), 7.48 (d, 2H, J ¼ 8.4 Hz, AreH).
MS: 259.1 (M þ H)þ. Anal. Calcd. for C10H13BrN2O: C, 46.71; H, 5.10;
N, 10.89. Found: C, 46.93; H, 5.32; N, 10.66.
C9H9BrO2: C, 47.19; H, 3.96. Found: C, 47.49; H, 3.99.
6.3. General method for the synthesis of methyl-2-methyl-2-
(arylsubstituted)propanoate (2aec)
6.5. General method for the synthesis of 4-amino-5-[2-(4-
substitutedphenyl)propan-2-yl]-4H-[1,2,4]-triazole-3-thioles (5aec)
To a stirred solution of compound (1aec, 0.066 mol) in anhy-
drous THF (120 mL), sodium hydride (0.199 mol) was added in
portions at ꢀ10 ꢁC under nitrogen atmosphere [35]. Reaction
2-Aryl-2-methylpropane hydrazide (0.1 mol) was treated with
solution of potassium hydroxide (0.15 mol) dissolved in
a

414
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
anhydrous methanol (50 mL) at 0e5 ꢁC under N2 atmosphere with
stirring. Carbon disulfide (0.15 mol) was added slowly and reaction
mixture was stirred at RT overnight. The precipitated potassium
aryldithiocarbazate was collected by filtration and washed with
anhydrous ether and dried under vacuo. It was directly used for the
next step without purification. The mixture of above potassium
aryldithiocarbazate (4aec, 0.1 mol) in water (10 mL) and hydrazine
hydrate (0.3 mol) was refluxed for 7 h. The colour of the reaction
mixture changed to green with evolution of hydrogen sulphide gas
and finally it became homogeneous. The reaction mixture was then
cooled to RT and diluted with water (100 mL) and acidified with
conc. hydrochloric acid. It was then extracted in ethyl acetate,
washed with water, brine solution and dried over anhydrous
Na2SO4. Final purification was done by using column chromatog-
raphy (60e120 mesh size silica gels) using 5e6% EtOAc-Pet ether as
eluent to afford the title compounds as yellow solids.
6.7. General procedure for the preparation of 6-arylsubstituted-3-
[2-(4-substitutedphenyl)propan-2-yl]-7H-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazines (7aer)
A mixture of 4-amino-5-[2-(4-substitutedphenyl)propan-2-yl]-
4H-[1,2,4]-triazole-3-thioles (1 mmol) and substituted phenacyl
bromides (1.1 mmol) in ethanol (5 mL) was refluxed for 6 h. The
reaction was monitored by TLC. Upon completion of the staring
material the reaction mixture was concentrated in rota-evaporator
to remove the solvent. The residue obtained was diluted with
water, extracted in EtOAc (2 ꢂ 75 mL) The combined extract was
washed with 10% NaHCO3 solution, water followed by brine solu-
tion and dried over anhydrous Na2SO4. The crude product obtained
was purified by column chromatography (60e120 size mesh silica
gel) using 0.75e1.0% methanol in dichloromethane to afford the
title compounds.
6.5.1. 4-Amino-5-(2-phenylpropan-2-yl)-4H-[1,2,4]-triazole-3-
6.7.1. 4-(3-(2-Phenylpropan-2-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]
thiol (5a)
thiadiazin-6-yl)benzonitrile (7a)
Off white solid. Yield: 7.2 g (51%), m.p.: 141e142.8 ꢁC. IR (KBr)
y
/
Off white solid: IR (KBr) y
/cmꢀ1: 3049 (Ar-H), 2979, 2923, 2855
cmꢀ1: 3304, 1323 (NH2), 3128 (AreH), 2980, 2950 (CeH), 1622
(C<ce:glyph name¼"dbnd"/>N), 1488 (C<ce:glyph name¼"dbnd"/
>C), 1235.3 (C<ce:glyph name¼"dbnd"/>S). 1H NMR (300 MHz,
(CeH), 2228 (eC<ce:glyph name¼"tbnd"/>N), 1602 (C<ce:glyph
name¼"dbnd"/>N), 1460 (C<ce:glyph name¼"dbnd"/>C). 1H NMR
(400 MHz, CDCl3): d 1.87 (s, 6H, CH3), 3.84 (s, 2H, eCH2e), 7.19e7.33
CDCl3):
d
1.79 (s, 6H, CH3), 4.23 (s, 2H, eNH2), 7.18e7.36 (m, 5H,
(m, 5H, Ar-H), 7.49 (d, 2H, J ¼ 7.6 Hz, AreH), 7.68 (d, 2H, J ¼ 7.6 Hz,
AreH), 11.4 (s, 1H, tautomeric eNH). MS: 234.9 (M þ H)þ. Anal.
Calcd. for C11H14N4S: C, 56.38; H, 6.02; N, 23.91. Found: C, 56.60; H,
6.17; N, 24.14.
AreH). 13C NMR (100 MHz, CDCl3):
d
22.63, 27.93, 40.21, 111.98,
117.81, 125.75, 126.46, 127.59, 128.37, 132.56, 137.60, 145.64, 149.54.
LCMS: 360 (M þ H)þ. Anal. Calcd. for C20H17N5S: C, 66.83; H, 4.77;
N, 19.48. Found: C, 67.20; H, 4.99; N, 19.58.
6.5.2. 4-Amino-5-[2-(4-chlorophenyl)propan-2-yl]-4H-[1,2,4]-
triazole-3-thiol (5b)
6.7.2. 6-(3-Bromophenyl)-3-(2-phenylpropan-2-yl)-7H-[1,2,4]
Yellow solid. Yield: 13 g (74%), m.p.: 139.1e140.6 ꢁC. IR (KBr)
y
/
triazolo[3,4-b][1,3,4]thiadiazine (7b)
cmꢀ1: 3317, 1303 (eNH2), 3154 (AreH), 2974, 2931 (CeH), 1634
(C<ce:glyph name¼"dbnd"/>N), 1488 (C<ce:glyph name¼"dbnd"/
>C), 1226 (C<ce:glyph name¼"dbnd"/>S). 1H NMR (400 MHz,
Off white solid. IR (KBr) y
/cmꢀ1: 3053 (AreH), 2978, 2912, 2870
(CeH), 1598 (C<ce:glyph name¼"dbnd"/>N), 1459 (C<ce:glyph
name¼"dbnd"/>C), 673 (CeBr). 1H NMR (400 MHz, CDCl3):
d 1.89
DMSO-d6):
d
1.65 (s, 6H, CH3), 5.07 (s, 2H, eNH2), 7.15 (d, 2H,
(s, 6H, CH3), 3.83 (s, 2H, eCH2e), 7.24e7.30 (m, 5H, AreH), 7.36 (d,
2H, J ¼ 8 Hz, AreH), 7.38 (t, 1H, J ¼ 4.1 Hz, AreH), 7.47 (s, 1H,
AreH), 7.61 (d, 2H, J ¼ 7.9 Hz, AreH). 13C NMR (100 MHz, CDCl3):
J ¼ 8.6 Hz, AreH), 7.35 (d, 2H, J ¼ 8.5 Hz, AreH), 13.65 (s, 1H,
tautomeric eNH). MS: 269.1 (M þ H)þ. Anal. Calcd. for C11H13ClN4S:
C, 49.16; H, 4.88; N, 20.85. Found: C, 49.36; H, 5.09; N, 20.74.
d
22.69, 27.87, 40.10, 123.26, 125.52, 125.79, 126.50, 127.59, 128.47,
128.72, 130.07, 130.25, 130.49, 134.51, 135.49, 140.72, 145.55,
149.92, 159.54. LCMS: 414 (M þ 2H)þ. Anal. Calcd. for
C19H17BrN4S: C, 55.21; H, 4.15; N, 13.55. Found: C, 55.06; H, 3.95;
N, 13.60.
6.5.3. 4-Amino-5-[2-(4-bromophenyl)propan-2-yl]-4H-[1,2,4]-
triazole-3-thiol (5c)
Off white solid. Yield: 7.7 g (58%), m.p.: 186.4e188.1 ꢁC. IR (KBr)
y
/cmꢀ1: 3288, 1295 (eNH2), 3144 (AreH), 2976, 2940 (CeH), 1620
(C<ce:glyph name¼"dbnd"/>N), 1492 (C<ce:glyph name¼"dbnd"/
6.7.3. 6-(3-Chlorophenyl)-3-(2-phenylpropan-2-yl)-[1,2,4]7H-
>C), 1218.5 (C<ce:glyph name¼"dbnd"/>S). 1H NMR (400 MHz,
triazolo[3,4eb][1,3,4]thiadiazine (7c)
DMSO-d6):
d
1.66 (s, 6H, CH3), 5.06 (s, 2H, eNH2), 7.10 (d, 2H,
Pale brown solid: IR (KBr) y
/cmꢀ1: 3058 (AreH), 2976, 2917,
J ¼ 14.0 Hz, AreH), 7.48 (d, 2H, J ¼ 14.0 Hz, AreH), 13.64 (s, 1H,
tautomeric -NH). MS: 315.9 (M þ 2H)þ. Anal. Calcd. for
C11H13BrN4S: C, 42.18; H, 4.18; N, 17.89. Found: C, 42.07; H, 3.96; N,
17.64.
2890 (CeH), 1598 (C<ce:glyph name¼"dbnd"/>N), 1458
(C<ce:glyph name¼"dbnd"/>C), 539 (CeCl). 1H NMR (400 MHz,
CDCl3):
d 1.88 (s, 6H, CH3), 3.76 (s, 2H, eCH2e), 7.21e7.30 (m, 8H,
AreH), 7.45 (d, 1H, J ¼ 7.0 Hz, AreH). 13C NMR (100 MHz, CDCl3):
d
22.70, 27.87, 40.11, 125.08, 125.81, 126.49, 127.20, 128.44, 130.04,
6.6. General procedure for the preparation of substituted 2-bromo
acetophenones (6)
131.60, 135.23, 135.27, 140.75, 145.56, 150.04, 159.53. LCMS: 369
(M þ H)þ. Anal. Calcd. for C19H17 ClN4S: C, 61.86; H, 4.65; N, 15.19.
Found: C, 62.11; H, 4.95; N, 14.94.
To a stirred solution of substituted acetophenone (1 mmol) in
anhydrous diethyl ether (5 mL) cooled at 0 ꢁC was added anhydrous
aluminium chloride (0.05 mmol) under N2 atmosphere followed by
bromine (1 mmol). The reaction mixture was stirred at same
temperature for 30 min. After the completion of the starting
material as monitored by the TLC, reaction mass quenched with
crushed ice. Extracted in diethyl ether (2 ꢂ 75 mL), combined
extract was washed with 10% NaHCO3 solution, water followed by
brine solution and dried over anhydrous Na2SO4. The crude product
was purified by recrystallization using pet ether to afford the pure
phenacyl bromides as low melting solids.
6.7.4. 3-(2-Phenylpropan-2yl)-6-[4-(trifluoromethoxy)phenyl]-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7d)
Off white solid: IR (KBr) y
/cmꢀ1: 3064 (AreH), 2978, 2923, 2830
(CeH), 1601 (C<ce:glyph name¼"dbnd"/>N), 1460 (C<ce:glyph
name¼"dbnd"/>C), 1249 (CeO). 1H NMR (400 MHz, DMSO-d6):
d
1.76 (s, 6H, CH3), 4.26 (s, 2H, eCH2e), 7.16e7.33 (m, 5H, AreH),
7.45 (d, 2H, J ¼ 8.3 Hz, AreH), 7.68 (d, 2H, J ¼ 8.8 Hz, AreH). 13C
NMR (100 MHz, DMSO-d6):
d 22.66, 28.28, 39.80, 119.09, 121.58,
121.64, 124.20, 126.01, 126.66, 127.82, 128.74, 129.01, 129.86, 130.11,
130.18, 132.95, 141.52, 146.11, 150.93, 152.62, 158.72. LCMS (MM-

P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
415
ES þ APCl): 419 (M þ H)þ. Anal. Calcd. for C20H17F3N4OS: C, 57.41; H,
127.12, 127.25, 128.48, 130.09, 131.68, 132.24, 135.09, 135.26, 140.90,
144.08, 150.18, 158.96. LCMS: 403 (Mþ). Anal. Calcd. for C19H16
Cl2N4S: C, 56.58; H, 4.00; N, 17.58. Found: C, 56.48; H, 4.37; N, 14.11.
4.10; N, 13.39. Found: C, 57.60; H, 4.35; N, 13.61.
6.7.5. 6-(4-Chloro-3-nitrophenyl)-3-(2-phenylpropan-2yl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7e)
6.7.10. 3-[2-(4-Chlorophenyl)propan-2-yl]-6-[4-(trifluoromethoxy)
phenyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7j)
Pale brown solid: IR (KBr) y
/cmꢀ1: 3096 (AreH), 2977, 2905, 2885
(CeH),1599 (C<ce:glyph name¼"dbnd"/>N),1536 (NO2, anti),1492
Off white solid: IR (KBr) y
/cmꢀ1: 3054 (AreH), 2982, 2922, 2890
(C<ce:glyph name¼"dbnd"/>C), 1464 (NO2, sym). 1H NMR (400
(CeH), 1600 (C<ce:glyph name¼"dbnd"/>N), 1454 (C<ce:glyph
MHz, DMSO-d6): d 1.76 (s, 6H, CH3), 4.30 (s, 2H, eCH2e), 7.20 (t, 1H,
name¼"dbnd"/>C), 1263 (CeO), 536 (CeCl). 1H NMR (400 MHz,
J ¼ 7.2 Hz, AreH), 7.25 (d, 2H, J ¼ 7.2 Hz, AreH), 7.32 (m, 3H, AreH),
DMSO-d6): d 1.76 (s, 6H, CH3), 4.28 (s, 2H, eCH2e), 7.26 (d, 2H,
7.83 (dd,1H, J ¼ 8.4, 2.0 Hz, AreH), 7.91 (d,1H, J ¼ 8.4 Hz, AreH), 8.03
J ¼ 8.6 Hz, AreH), 7.38 (d, 2H, J ¼ 8.6 Hz, AreH), 7.48 (d, 2H,
(s, 1H, AreH). 13C NMR (100 MHz, CDCl3):
d 22.40, 28.30, 39.32,
J ¼ 8.4 Hz, AreH), 7.72 (d, 2H, J ¼ 7.0 Hz, Ar-H). 13C NMR (100 MHz,
124.19, 126.04, 126.70, 128.33, 128.75, 132.29, 134.12, 145.91, 148.23,
151.14, 158.87. LCMS: 414 (Mþ). Anal. Calcd. for C19H16ClN5O2S: C,
55.14; H, 3.90; N, 16.92. Found: C, 55.27; H, 4.12; N, 17.12.
DMSO-d6): d 22.22, 27.67, 39.71, 118.62, 121.19, 127.66, 128.22,
129.41, 130.91, 132.40, 141.31, 144.65, 150.50, 150.52, 152.52, 157.80.
LCMS (MM-ESþAPCI) 453 (Mþ). Anal. Calcd. for C20H16ClF3N4OS: C,
53.04; H, 3.56; N, 12.37. Found: C, 53.26; H, 3.77; N, 12.60.
6.7.6. 6-(2,4-Dichloro-5-fluorophenyl)-3-(2-phenylpropan-2-yl)-
7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7f)
6.7.11. 6-(4-Chloro-3-nitrophenyl)-3-[2-(4-chlorophenyl)propan-
Off colour solid. IR (KBr)
y
/cmꢀ1: 3042 (AreH), 2971, 2922, 2884
2-yl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7k)
(CeH), 1598 (C<ce:glyph name¼"dbnd"/>N), 1462 (C<ce:glyph
Off white solid: IR (KBr) y
/cmꢀ1: 3067 (AreH), 2976, 2921, 2887
name¼"dbnd"/>C), 536 (CeCl). 1H NMR (400 MHz, DMSO-d6):
(CeH),1599 (C<ce:glyph name¼"dbnd"/>N),1537 (NO2, anti),1460
d
1.72 (s, 6H, CH3), 4.12 (s, 2H, eCH2e), 6.91 (d, 1H, J ¼ 9.3 Hz,
(C<ce:glyph name¼"dbnd"/>C), 1346 (NO2, sym), 534 (CeCl). 1H
AreH), 7.14 (d, 2H, J ¼ 8.2 Hz, AreH), 7.31e7.17 (m, 3H, AreH), 7.97
NMR (400 MHz, DMSO-d6): d 1.74 (s, 6H, CH3), 4.32 (s, 2H, -CH2-),
(d, 1H, J ¼ 6.6 Hz, AreH). 13C NMR (100 MHz, CDCl3):
d
22.68, 25.74,
7.27 (d, 2H, J ¼ 6.8 Hz, AreH), 7.35 (d, 2H, J ¼ 8.6 Hz, AreH), 7.89
40.23, 111.81, 119.05, 124.82, 125.94, 125.98, 126.31, 126.49, 127.48,
127.52, 127.62, 128.44, 128.70, 131.80, 134.02, 134.09, 141.18, 146.01,
151.56,155.53, 158.04, 159.41. LCMS (MM-ES þ APCI): 423 (M þ H)þ.
Anal. Calcd. for C19H15Cl2FN4S: C, 54.17; H, 3.59; N, 13.30. Found: C,
53.97; H, 3.87; N, 13.65.
(dd, 1H, J ¼ 8.5, 2.0 Hz, AreH), 7.93 (d, 1H, J ¼ 8.5 Hz, AreH), 8.09 (s,
1H, AreH). 13C NMR (100 MHz, DMSO-d6):
d 22.68, 27.76, 39.77,
123.91, 127.70, 128.08, 128.30, 131.04, 131.93, 132.43, 133.65, 141.25,
144.51, 147.81, 151.14, 157.99. LCMS (MM-ES þ APCI) 450 (M þ H)þ.
Anal. Calcd. for C19H15Cl2N5O2S: C, 50.90; H, 3.37; N, 15.62. Found:
C, 51.07; H, 3.52; N, 15.82.
6.7.7. 4-{3-[2-(4-Chlorophenyl)propan-2-yl]-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazin-6-yl} benzonitrile (7g)
6.7.12. 3-[2-(4-Chlorophenyl)propan-2-yl]-6-(2,4-dichloro-5-
Off white solid: IR (KBr)
y
/cmꢀ1: 3049 (AreH), 2976, 2918, 2854
fluorophenyl)-7H-[1,2,4]triazolo [3,4-b][1,3,4]thiadiazine (7l)
(CeH), 2226 (eC<ce:glyph name¼"tbnd"/>N), 1595 (C<ce:glyph
Off white solid: IR (KBr) y
/cmꢀ1: 3090 (AreH), 2976, 2924, 2893
name¼"dbnd"/>N), 1458 (C<ce:glyph name¼"dbnd"/>C), 534
(CeH), 1595 (C<ce:glyph name¼"dbnd"/>N), 1460 (C<ce:glyph
(CeCl). 1H NMR (400 MHz, CDCl3):
d 1.86 (s, 6H, CH3), 3.82 (s, 2H,
name¼"dbnd"/>C), 533 (CeCl). 1H NMR (400 MHz, DMSO-d6):
eCH2e), 7.21 (d, 2H, J ¼ 6.6 Hz, AreH), 7.30 (d, 2H, J ¼ 6.5 Hz, AreH),
d
1.72 (s, 6H, CH3), 4.13 (s, 2H, eCH2e), 7.03 (d,1H, J ¼ 9.3 Hz, AreH),
7.53 (d, 2H, J ¼ 7.9 Hz, AreH), 7.73 (d, 2H, J ¼ 6.7 Hz, AreH). 13C NMR
7.16 (d, 2H, J ¼ 8.6 Hz, AreH), 7.33 (d, 2H, J ¼ 8.6 Hz, AreH), 7.99 (d,
1H, J ¼ 6.7 Hz, AreH). LCMS (MM-ES þ APCI): 455 (Mþ). Anal. Calcd.
for C19H14Cl3FN4S: C, 50.07; H, 3.10, N, 12.29. Found: C, 50.28; H,
3.32, N, 12.43.
(100 MHz, CDCl3): d 22.70, 27.88, 39.90,115.16,117.74,127.27,127.56,
128.47, 132.34, 132.66, 137.47, 140.96, 144.19, 149.87, 159.09. LCMS
(MM-ES þ APCI) 394.2 (M þ H)þ. Anal. Calcd. for C20H16ClN5S: C,
60.99; H, 4.09; N, 17.78. Found: C, 61.17; H, 4.20; N, 18.03.
6.7.13. 4-{3-[2-(4-Bromophenyl)propan-2-yl]-7H-[1,2,4]triazolo
6.7.8. 6-(3-Bromophenyl)-3-[2-(4-chlorophenyl)propan-2-yl]-7H-
[3,4-b][1,3,4]thiadiazin-6-yl} benzonitrile (7m)
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7h)
Off white solid: IR (KBr) y
/cmꢀ1: 3061 (AreH), 2974, 2920, 2853
Off white solid: IR (KBr)
y
/cmꢀ1: 3059 (AreH), 2980, 2931, 2897
(CeH), 2229.4 (eC<ce:glyph name¼"tbnd"/>N), 1592 (C<ce:glyph
(CeH), 1555 (C<ce:glyph name¼"dbnd"/>N), 1456 (C<ce:glyph
name¼"dbnd"/>N), 1460 (C<ce:glyph name¼"dbnd"/>C), 672
name¼"dbnd"/>C), 538 (CeCl). 1H NMR (400 MHz, DMSO-d6):
(CeBr). 1H NMR (400 MHz, DMSO-d6):
d 1.76 (s, 6H, CH3), 4.32 (s,
d
1.75 (s, 6H, CH3), 4.28 (s, 2H, eCH2e), 7.26 (d, 2H, J ¼ 8.4 Hz,
2H, eCH2e), 7.21 (d, 2H, J ¼ 8.8 Hz, AreH), 7.51 (d, 2H, J ¼ 8.8 Hz,
AreH), 7.39 (d, 2H, J ¼ 8.8 Hz, AreH), 7.45 (t, 1H, J ¼ 8.0 Hz, AreH),
7.55 (s, 1H, AreH), 7.63 (d, 1H, J ¼ 8.0 Hz, AreH), 7.74 (d, 1H,
AreH), 7.75 (d, 2H, J ¼ 8.8 Hz, AreH), 7.97 (d, 2H, J ¼ 8.4 Hz, AreH).
13C NMR (100 MHz, CDCl3):
d 22.70, 27.84, 39.98, 115.18, 117.76,
J ¼ 8.0 Hz, AreH). 13C NMR (100 MHz, DMSO-d6):
d
22.17, 27.82,
120.41, 125.75, 127.57, 127.68, 128.37, 131.45, 132.57, 137.46, 140.98,
144.74, 149.85, 159.04. LCMS (MM-ES þ APCI) 438.0 (Mþ)þ. Anal.
Calcd. for C20H16BrN5S: C, 54.80; H, 3.68; N, 15.98. Found: C, 55.00;
H, 3.83; N, 16.23.
40.01, 122.32, 126.38, 127.62, 128.37, 129.57, 130.98, 131.02, 134.39,
135.57, 141.35, 144.72, 152.27, 157.98. LCMS (MM-ES þ APCI) 449.0
(M þ 2H)þ. Anal. Calcd. for C19H16 BrClN4S: C, 50.96; H, 3.60; N,
12.51. Found: C, 51.24; H, 3.85; N, 12.81.
6.7.14. 6-(3-Bromophenyl)-3-[2-(4-bromophenyl)propan-2-yl]-7H-
6.7.9. 6-(3-Chlorophenyl)-3-[2-(4-chlorophenyl)propan-2-yl]-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7n)
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7i)
Off white solid: IR (KBr) y
/cmꢀ1: 3062 (AreH), 2979, 2897, 2853
Off white solid: IR (KBr)
y
/cmꢀ1: 3068 (AreH), 2980, 2932, 2898
(CeH), 1556 (C<ce:glyph name¼"dbnd"/>N), 1455 (C<ce:glyph
(CeH), 1562 (C<ce:glyph name¼"dbnd"/>N), 1456 (C<ce:glyph
name¼"dbnd"/>C), 673 (CeBr). 1H NMR (400 MHz, DMSO-d6):
name¼"dbnd"/>C), 539 (CeCl). 1H NMR (400 MHz, CDCl3):
d
1.86 (s,
d
1.73 (s, 6H, CH3), 4.27 (s, 2H, eCH2e), 7.19 (d, 2H, J ¼ 8.5 Hz, AreH),
6H, CH3), 3.80 (s, 2H, eCH2e), 7.23 (d, 2H, J ¼ 6.7 Hz, AreH), 7.32 (d,
2H, J ¼ 8.7 Hz, AreH), 7.36e7.34 (m, 3H, AreH), 7.47 (d,1H, J ¼ 6.9 Hz,
7.44 (t, 1H, J ¼ 7.9 Hz, AreH), 7.52 (d, 2H, J ¼ 8.4 Hz, AreH), 7.54 (s,
1H), 7.62 (d, 1H, J ¼ 8.0 Hz, AreH), 7.73 (d, 1H, J ¼ 7.9 Hz, AreH). 13C
AreH). 13C NMR (100 MHz, CDCl3):
d
22.66, 27.76, 39.73, 124.96,
NMR (100 MHz, DMSO-d6): d 22.10, 27.69, 39.69, 119.40, 122.24,

416
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 57 (2012) 407e416
126.32, 127.92, 129.49, 130.93, 131.22, 134.31, 135.49, 141.27, 145.08,
152.20, 157.87. LCMS (MM-ES þ APCI) 492.9 (Mþ). Anal. Calcd. for
C19H16Br2N4S: C, 46.36; H, 3.28; N, 11.38. Found: C, 46.58; H, 3.53;
N, 11.66.
Acknowledgements
The author PP thanks Dr. Ashis Baran Mandal, Dr. Santosh Kul-
karni and Dr. John Kallikat Syngene International ltd. for helpful
discussions. The author PP also thanks Dr. Yashovarma B. SDM
College, Ujire, India, for providing the facilities to determine the
anthelmintic and antimicrobial activities.
6.7.15. 3-[2-(4-Bromophenyl)propan-2-yl]-6-(3,4-difluorophenyl)-
7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7o)
Pale brown solid: IR (KBr) y
/cmꢀ1: 3070.9 (AreH), 2973, 2921,
2852 (CeH), 1577 (C<ce:glyph name¼"dbnd"/>N), 1491
Appendix A. Supplementary information
(C<ce:glyph name¼"dbnd"/>C), 622 (CeF). 1H NMR (400 MHz,
DMSO-d6):
d 1.74 (s, 6H, CH3), 4.28 (s, 2H, eCH2e), 7.20 (d, 2H,
Supplementary data associated with this article can be found, in
theonlineversion, at http://dx.doi.org/10.1016/j.ejmech.2012.06.059.
J ¼ 8.8 Hz, AreH), 7.47e7.44 (m, 2H, AreH), 7.51 (d, 2H, J ¼ 8.8 Hz,
AreH), 7.59e7.55 (m, 1H, AreH), 13C NMR (100 MHz, DMSO-d6):
d
22.55, 28.17, 39.76, 116.60, 116.79, 118.58, 118.75, 119.87, 125.19,
References
125.22, 125.26, 125.29, 125.99, 126.68, 128.48, 128.77, 131.22, 131.26,
131.28, 131.62, 141.72, 145.62, 148.67, 148.80, 150.66, 150.79, 151.13,
152.21, 153.18, 153.30, 158.26. LCMS (MM-ES þ APCI) 451
(M þ 2H)þ. Anal. Calcd. for C19H15BrF2N4S: C, 50.79; H, 3.58; N,
12.47. Found: C, 50.97; H, 3.58; N, 12.71.
[1] N. Plant, Drug Discovery Today 9 (2004) 328.
[2] H.M. Geysen, Nat. Rev. Drug Discov. 2 (2003) 222.
[3] M. Kavallaris, N.M. Verrills, B.T. Hill, Drug. Resist. Update 4 (2001) 392.
[4] K.N. Gaind, B.N. Dhar, R.N. Chopra, K.N. Kaul, Indian J. Pharm. Sci. 27 (1963) 198.
[5] S.J. Sun, H.X. Lou, Y.H. Gao, P.H. Fan, B. Ma, W.Y. Ge, X.N. Wang, J. Pharm.
Biomed. Anal. 34 (2004) 1117.
[6] M. Clemons, R.E. Coleman, S. Verma, Cancer Treat. Rev. 30 (2004) 325.
[7] G.A.R. Johnston, Curr. Top. Med. Chem. 2 (2002) 903.
[8] N. Demirbas, A. Demirbas, S. Alpay Karaoglu, E. Celik, Arkivoc 1 (2005) 75.
[9] B.S. Holla, R. Gonsalves, S. Shenoy, Eur. J. Med. Chem. 35 (2000) 267.
[10] A. Shafiee, A. Sayadi, M.H. Roozbahni, A. Foroumadi, F. Kamal, Arch. Pharm.
Med. Chem. 10 (2002) 495.
[11] S. Sharma, S. Gangal, A. Rauf, M. Zahin, Arch. Pharm. Chem. Life. Sci. 341
(2008) 714.
[12] N.N. Gulerman, H.N. Dogan, S. Rollas, C. Johnanssom, C. Celik, II Farmaco 56
(2001) 953.
[13] S. Papakonstantinou-Garoufalisa, N. Pouli, P. Marakos, A. Chytyroglou-Ladas, II
Farmaco 57 (2002) 973.
[14] G. Turan-Zitouni, Z.A. Kaplacikli, M.T. Yildiz, P. Chevallet, D. Kaya, Eur. J. Med.
Chem. 40 (2005) 607.
[15] A.R. Jalilian, S. Sattari, M. Bineshmarevasti, A. Shafiee, M. Daneshtalab, Arch.
Pharm. Med. Chem. 333 (2000) 347.
[16] I. Kucukguzel, S.G. Kucukguzel, S. Rollas, M. Kiraz, Bioorg. Med. Chem. Lett. 11
(2001) 1703.
6.7.16. 3-[2-(4-Bromophenyl)propan-2-yl]-6-[4-(trifluoromethoxy)
phenyl]-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7p)
Off white solid: IR (KBr) y
/cmꢀ1: 3057 (AreH), 2974, 2924, 2856
(CeH), 1603 (C<ce:glyph name¼"dbnd"/>N), 1460 (C<ce:glyph
name¼"dbnd"/>C), 672 (CeBr). 1H NMR (400 MHz, DMSO-d6):
d
1.75 (s, 6H, CH3), 4.28 (s, 2H, eCH2e), 7.20 (d, 2H, J ¼ 8.6 Hz,
AreH), 7.48 (d, 2H, J ¼ 8.1 Hz, AreH), 7.51 (d, 2H, J ¼ 8.6 Hz, AreH),
7.71 (d, 2H, J ¼ 8.9 Hz, AreH). 13C NMR (100 MHz, DMSO-d6):
d
22.21, 27.62, 39.70, 118.61, 119.39, 121.17, 128.04, 128.28, 129.41,
131.14, 132.40, 141.28, 145.10, 150.50, 152.50, 157.74. LCMS (MM-
ES þ APCI) 499 (M þ 2H)þ. Anal. Calcd. for C20H16BrF3N4OS: C,
48.30; H, 3.24; N, 11.27. Found: C, 48.18; H, 3.09; N, 11.17.
6.7.17. 3-[2-(4-Chlorophenyl)propan-2-yl]-6-(4-chloro-3-
[17] L. Zahajska, V. Klimesova, J. Koci, K. Waisser, J. Kaustova, Arch. Pharm. Med.
Chem. 337 (2004) 549.
nitrophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7q)
Off white solid. IR (KBr) y
/cmꢀ1: 3065 (AreH), 2977, 2922, 2887
[18] A. Foroumadi, Z. Kiani, F. Soltani, II Farmaco 58 (2003) 1073.
[19] G. Turan-Zitouni, M. Sivaci, F.S. Kilic, K. Erol, Eur. Med. Chem. 36 (2001) 685.
[20] B. Tozkoparan, E. Kupeli, E. Yesilada, M. Ertan, Bioorg. Med. Chem. 15(2007) 1808.
[21] S.M. Rabea, N.A. El-Koussi, H.Y. Hassan, T. Aboul-Fadi, Arch. Pharm. Chem. Life.
Sci. 339 (2006) 32.
[22] L. Labanuskas, E. Udrenaite, P. Gaidelis, A. Brukstus, II Farmaco 59 (2004) 255.
[23] M. Kritsanida, A. Mouroutsou, P. Marakos, N. Pouli, S. Papakonstantinou-Gar-
oufalias, C. Pannecouque, M. Witvrouw, E. DeClerc, II Farmaco 57 (2002) 253.
[24] M.T. Abdel-Aal, W.A. El-Sayad, S.M. El-Kosy, E.S.H. El-Ashry, Arch. Pharm.
Chem. Life. Sci. 341 (2008) 307.
[25] A. Varvaresou, T. Siatra-Papastaikoudi, A. Tsotinis, A. Tsantili-Kakouulidou,
A. Vamvakides, II Farmaco 53 (1998) 320.
[26] A.P. Skoumbourdis, C.A. LeClair, E. Stefan, A.G. Turjanski, W. Maguire,
S.A. Titus, R. Huang, D. S Auld, J. Inglese, C.P. Austin, S.W. Michnick, M. Xia,
C.J. Thomas, Bioorg. Med. Chem. Lett. 19 (2009) 3686.
(CeH),1598 (C<ce:glyph name¼"dbnd"/>N),1537 (NO2, anti),1460
(C<ce:glyph name¼"dbnd"/>C), 1346 (NO2, sym), 666 (CeBr). 1H
NMR (400 MHz, DMSO-d6): d 1.75 (s, 6H, CH3), 4.33 (s, 2H, eCH2e),
7.22 (d, 2H, J ¼ 8.4 Hz, AreH), 7.49 (d, 2H, J ¼ 8.4 Hz, AreH), 7.89
(dd, 1H, J ¼ 8.4, 2.0 Hz, AreH), 7.94 (d, 1H, J ¼ 8.4 Hz, AreH), 8.1 0 (d,
1H, J ¼ 2.0 Hz, AreH). 13C NMR (100 MHz, DMSO-d6):
d 22.51, 28.12,
39.77, 119.95, 124.33, 128.49, 131.64, 132.34, 132.83, 134.07, 141.65,
145.37, 148.23, 151.56, 158.35. LCMS (MM-ES þ APCI) 494
(M þ 2H)þ. Anal. Calcd. for C19H15BrClN5O2S: C, 46.31; H, 3.07; N,
14.21. Found: C, 46.50; H, 3.35; N, 14.45.
[27] Xavier Collin, Sauleau Armelle, Joel . Coulon, Bioorg. Med. Chem. Lett. 13
(2003) 2601.
[28] P. Puthiyapurayil, B. Poojary, S. Kumar, R. Hunnur, J. Heterocycl. Chem. 48
(2011) 998.
6.7.18. 3-[2-(4-Bromophenyl)propan-2-yl]-6-(2,4-dichloro-5-
fluorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7r)
Pale brown solid: IR (KBr) y
/cmꢀ1: 3085 (AreH), 2975, 2922,
[29] D.J. Brusick, Clifton, New Jersey (1984), p. 265.
[30] T. Mossman, J. Immunol. Methods 65 (1983) 55.
2893, 2853 (CeH), 1594 (C<ce:glyph name¼"dbnd"/>N), 1460
(C<ce:glyph name¼"dbnd"/>C), 531 (CeCl). 1H NMR (400 MHz,
[31] B. Kalluraya, S. Sreenivas, IL Farmaco 53 (1998) 399.
[32] A.L. Bary, Procedure for testing antimicrobial agents in agar media, in:
V.L. Corian (Ed.), Antibiotics in Laboratory Medicine, Williams and wilins,
Baltimore, MD, 1980, pp. 1e23.
[33] D. James, Mac . Lowry, J.M. Jaqua, T.S. Sally, Appl. Microbiol. 20 (1970) 46.
[34] B.A. Arthington-Skaggs, M. Moltely, D.W. Warnock, C.J. Morrison, J. Clin.
Microbiol. 38 (2000) 2254.
DMSO-d6):
d 1.71 (s, 6H, CH3), 4.14 (s, 2H, eCH2e), 7.00 (d, 1H,
J ¼ 9.3 Hz, AreH), 7.10 (d, 2H, J ¼ 8.4 Hz, AreH), 7.46 (d, 2H,
J ¼ 8.4 Hz, AreH), 7.99 (d, 1H, J ¼ 6.7 Hz, AreH). LCMS (MM-
ES þ APCI): 502 (M þ 2H)þ. Anal. Calcd. for C19H14BrCl2FN4S: C,
45.62; H, 2.82, N, 11.20. Found: C, 45.95; H, 3.12, N, 11.50
[35] T. Hama, J.F. Hartwig, Org. Lett. 10 (2008) 1549.