Synthesis of novel thiadiazolotriazin-4-ones and study of their mosquito-larvicidal and antibacterial properties

Article abstract of DOI:10.1016/j.ejmech.2014.06.072

A series of novel 3-tert-butyl-7-(aryloxymethyl)-4H-[1,3,4]thiadiazolo[2,3- c][1,2,4]triazin-4-ones (5a-5n) were synthesized by refluxing 3,3-dimethyl-2-oxobutanoic acid (trimethyl pyruvic acid) (1) and thiocarbohydrazide (2) in ethanol as solvent for 12 h, to yield 3-mercapto-4-amino-6-tert-butyl-1,2,4-triazine-5(4H)-one (3) (Scheme 1), then the compound (3) was condensed with different substituted aryloxyacetic acids (4) in POCl₃ at 90 °C for 8 h (Scheme 2). The structures of the newly synthesized compounds were confirmed by IR, ¹H NMR, ¹³C NMR, elemental analyses and mass spectroscopic studies. Few of the synthesized compounds exhibited moderate mosquito-larvicidal and antibacterial activities. Among the novel derivatives, the compound (5f) showed relatively high larvicidal activity against a malaria vector. Compounds (5i) and (5m) exhibited a broad spectrum antibacterial activity against Gram positive and Gram negative species and hence they may be considered as drug candidates for bacterial pathogens.

Full text of DOI:10.1016/j.ejmech.2014.06.072

European Journal of Medicinal Chemistry 84 (2014) 194e199
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel thiadiazolotriazin-4-ones and study of their
mosquito-larvicidal and antibacterial properties
,
Prakash Anil Castelino a, Prashantha Naik b, Jagadeesh Prasad Dasappa a *, R.S. Sujayraj b,
K. Sharath Chandra b, Kumara Chaluvaiah a, Ramya Nair c, M.V. Sandya Kumari c,
Guruprasad Kalthur c, Satish Kumar Adiga c
a Department of Chemistry, Mangalore University, Mangalagangothri 574 199, Karnataka State, India
b Department of Biosciences, Mangalore University, Mangalagangothri 574 199, Karnataka State, India
c Division of Clinical Embryology, Kasturba Medical College, Manipal University, Manipal 576 104, Karnataka State, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3-tert-butyl-7-(aryloxymethyl)-4H-[1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-ones (5a
e5n) were synthesized by refluxing 3,3-dimethyl-2-oxobutanoic acid (trimethyl pyruvic acid) (1) and
thiocarbohydrazide (2) in ethanol as solvent for 12 h, to yield 3-mercapto-4-amino-6-tert-butyl-1,2,4-
triazine-5(4H)-one (3) (Scheme 1), then the compound (3) was condensed with different substituted
aryloxyacetic acids (4) in POCl3 at 90 ꢀC for 8 h (Scheme 2). The structures of the newly synthesized
compounds were confirmed by IR, 1H NMR, 13C NMR, elemental analyses and mass spectroscopic studies.
Few of the synthesized compounds exhibited moderate mosquito-larvicidal and antibacterial activities.
Among the novel derivatives, the compound (5f) showed relatively high larvicidal activity against a
malaria vector. Compounds (5i) and (5m) exhibited a broad spectrum antibacterial activity against Gram
positive and Gram negative species and hence they may be considered as drug candidates for bacterial
pathogens.
Received 17 December 2013
Received in revised form
17 June 2014
Accepted 29 June 2014
Available online 30 June 2014
Keywords:
Thiadiazolotriazin-4-one
Mosquito-larvicidal studies
Antibacterial studies
1,3,4-Thiadiazole moiety
Triazin-4-one moiety
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
antidepressant [40] and pesticidal agents [41,42]. Enthused by the
enormous pharmacological importance of triazinones and 1,3,4-
The recent decades have experienced the developments in the
field of science; particularly a huge upsurge in the drug synthesis
due to the advances in technology and the demands. With certainty
and clarity one can pronounce that the novel inventions and the
availability of voluminous literature have boosted the progress of
research. Marching in the same direction it was observed that tri-
azinone derivatives drew considerable interest because of enor-
mous importance in the field of medicine and agriculture [1e6].
Studies have shown that they have potential herbicidal [7e13],
insecticidal [14], antimicrobial [15e18], antifungal [19,20], anti-
cancer [21e25], antibacterial [26], anti-HIV [27], antioxidant [27],
antifish parasites [28,29], anticonvulsant [30,31], antitumour [32]
and antimetastatic properties [33] and so on. In addition to the
activities of triazinones, thiadiazoles are useful as antitumour
[34e36], antimicrobial [37], cytotoxic [38] antituberculosis [39],
thiadiazoles, it was decided to synthesize some novel thiadiazolo-
triazinones (5ae5n) and screen them for mosquito-larvicidal and
antibacterial activities.
2. Pharmacology
The novel 3-tert-butyl-[1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-
ones (5ae5n) were synthesized and evaluated for mosquito-
larvicidal and antibacterial activity studies.
2.1. Mosquito-larvicidal activity
All the synthesized compounds were tested for their larvicidal
activity in mosquito using Malaria Vector Anopheles stephensi as per
the standard WHO guidelines [43]. In 500 mL beakers containing
250 mL of water and 25 numbers of late III or early IV instar mos-
quito larvae for various concentrations of the extracts. A negative
control was kept with each set of experiment and mortality was
recorded after 24 h of exposure. Malathion, the commercial
insecticide (Hindustan Insecticides Ltd, New Delhi, India) was used
* Corresponding author.
E-mail
addresses:
jprasad2003@gmail.com,
jprasad_2003@yahoo.co.in
(J.P. Dasappa).
http://dx.doi.org/10.1016/j.ejmech.2014.06.072
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

P.A. Castelino et al. / European Journal of Medicinal Chemistry 84 (2014) 194e199
195
as the reference standard. Experiments were performed in tripli-
cates for each sample. Median lethal concentration (LC50) with 95%
confidence limit (CI) was calculated using Abbott's formula and Log
5(4H)-one (3) was condensed with different aryloxyacetic acids (4)
in POCl3 at 80 ꢀC for 8 h to yield the resultant series of novel 3-tert-
butyl-[1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-ones
(5ae5n)
probit analysis [44] and results are expressed as
potency was determined for comparison with the reference stan-
dard using the formula.
m
g/mL. Relative
(Scheme 2). The structures of the novel compounds (5ae5n) were
confirmed by spectral and elemental analyses. The data are incor-
porated in experimental section.
3.2. Pharmacological screening
2.2. Antibacterial activity
3.2.1. Mosquito-larvicidal activity
All the synthesized compounds were evaluated for their anti-
bacterial property by Disc diffusion method [45]. The microorgan-
isms were collected from Institute of Microbial Technology
(IMTECH), Chandigarh, India. Two Gram positive bacteria namely
Staphylococcus aureus MTCC-7443 and Bacillus subtilis MTCC-441;
two Gram negative bacteria namely Escherichia coli MTCC-725,
Aeromonas hydrophila MTCC-1739 were used for the screening.
The bacterial strains were inoculated on Nutrient Agar (NA) and
incubated for 24 h at 37 ꢀC. Sterile empty discs (6 mm diameter)
were purchased from Himedia Laboratories Pvt. Ltd. Mumbai. The
test compounds were dissolved in 5 mL of DMSO taken as the
A. stephensi is a malarial vector, breeds commonly stagnant
water known to be the root cause of urban and domestic malaria.
Insects have the tendency of developing resistance to insecticidal/
larvicidal agents due to continual exposure. Therefore, there is al-
ways demand for new agents possessing the larvicidal property to
control the disease vectors. In the present study, 3-tert-butyl-[1,3,4]
thiadiazolo[2,3-c][1,2,4]triazin-4-ones (5ae5n) were subjected to
larvicidal study and all exhibited larvicidal activity but with
different potentiality, the median lethal dose or LC50 values ranging
from 21.7 to 102.8
compounds, (5f) was found to be highly effective followed by (5g)
and (5c) with LC50 values 21.7, 25.7 and 39.8 g/mL respectively.
Compounds with moderate larvicidal activity include (5b) and (5n)
with LC50 values 48.7 and 49.3 g/mL. Among all the synthesized
mg/mL (Fig. 1) (Table 1). Among the tested
solvent; from the stock solution 100
mL of respective compound in
m
the selected concentration (500 g/disc) was loaded on the disc
m
individually and aseptically, dried and were used for screening the
antibacterial assay.
m
compounds (5ae5n), the compound (5f) exhibited highest larvi-
cidal activity against A. stephensi mosquito. The presence of hy-
droxyl moiety may be the plausible reason for the toxicity of (5f)
against mosquito larvae. Similar to hydroxyl moiety, the moiety
with electron releasing groups have been reported to possess po-
tential larvicidal activity [47], which in fact supports the rationale
for the larvicidal activity of compound (5f). Relative potency in-
dicates that novel compounds (5ae5n) tested in the present study
are not as effective as that of Malathion. However (5f) may be
considered as mosquito-larvicidal agent, the further studies on its
half-life, persistence in the nature and non health hazards to non-
target organisms may give insight into the usefulness of (5f) as a
larvicidal agent for mosquito.
Sterile discs were saturated with 100
mL of the test solution,
dried under laminar air flow and placed on the Nutrient Agar (NA)
plate for bacteria, which was inoculated with a lawn of the test
microorganisms. Plates were incubated at 37 ꢀC, for 18e24 h for
bacteria. The compounds that produced distinct circular zones of
inhibition around the discs and the diameters of clear zones were
determined and used as an indication of antibacterial activity.
Streptomycin, an antibiotic drug at a dose of 10
the reference standard.
mg/disc was used as
3. Results and discussion
3.1. Chemistry
3.2.2. Antibacterial activity
4-Amino-6-tert-butyl-3-mercapto-1,2,4-triazine-5(4H)-one (3)
was synthesized according to the well established procedure of
Dornow and co-workers [46]. The equimolar mixture of 3,3-
dimethyl-2-oxobutanoic acid(trimethyl pyruvic acid) (1) and thio-
carbohydrazide (2) were refluxed in ethanol for 12 h (Scheme 1).
The resultant 4-amino-6-tert-butyl-3-mercapto-1,2,4-triazine-
All the tested compounds (5ae5n) possess antibacterial activity
in in vitro test system but with different sensitivity (Fig. 2) (Table 2).
Compounds which gave the values >12 mm are considered as
effective and following interpretations are drawn.
Among the tested compounds (5g) and (5k) are effective against
only Gram positive bacteria and (5h) only against Gram negative
bacterial species; (5i) and (5m) against Gram positive and Gram
negative species. Among the tested compounds, highly effective
include (zone inhibition > 18 mm), (5g) and (5k) (against S. aureus)
(5m) (against B. subtilis and P. aeruginosa), (5i) against (P. aeruginosa
and K. pneumoniae). Among the tested compounds, (5i) (18.4e22.4)
and (5m) (18.7e25.7) exhibited a broad spectrum antibacterial
activity against Gram positive and Gram negative species.
The reference standard, Streptomycin, a well known antibiotic
drug was highly sensitive against Gram positive and Gram negative
bacterial species. However, tested compounds cannot directly be
compared with the reference standard for their efficacy since it
depends on therapeutic index, which varies for different bioactive
compounds depending upon the ratio between lethal dose and
therapeutic dose.
3.2.3. Toxicity studies
Among the novel derivatives, (5f), (5i) and (5m) which showed
comparatively high larvicidal and antibacterial activities were
analyzed for oral acute toxicity considering their future potential
utilities and safer mode of exposures. Median lethal dose (MLD)
Scheme 1. The synthesis of 4-amino-6-tert-butyl-3-mercapto-1,2,4-triazine-5(4H)-
one (3).

196
P.A. Castelino et al. / European Journal of Medicinal Chemistry 84 (2014) 194e199
Scheme 2. Syntheses of 3-tert-butyl-7-(aryloxymethyl)-4H-[1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-ones (5ae5n).
was determined for each of these derivatives using Swiss albino
mice (Mus musculus) as per the OECD guidelines [47]. Handling and
animal experimentation were done according to the guidelines of
CPCSEA (Committee for the Purpose of Control and Supervision on
Experiments on Animals), India [48] with the ethical clearance
certificate IAEC/KMC/69/2012. The oral MLD for (5f), (5i) and (5m)
were found to be 1.36, 2.26 and 1.82 g/kg b.wt. respectively at 72 h
duration. It is evident that the oral LD50 values of these compounds
were within the range between 500 and 5000 mg/kg b.wt. Hence,
as per the Hodge and Sterner Scale toxicity [49], these compounds
can be classified as Slightly Toxic as far as mammalian oral route of
exposure is concerned. The LD50 values obtained for (5i) and (5m)
will be useful for determination of therapeutic index in view of
their relatively good antibacterial activity.
The three compounds with larvicidal and antibacterial activities
exhibiting oral toxicity (LD50 values) which come under the cate-
gory ‘slightly toxic’ stimulate further studies for the potential use of
these lead compounds.
5. Experimental section
Melting points of the compounds (5ae5n) were determined in
open capillary tubes and are uncorrected. The purity of synthesized
compounds was checked by TLC observing single spot on Merck
silica gel 60 F254 coated alumina plates. The structures of thiadia-
zolotriazin-4-ones (5ae5n) were confirmed by spectral studies.
The IR spectra (cmꢁ1) were recorded on a Shimadzu-FTIR 577
Infrared spectrophotometer in KBr pellets. The 1H NMR and 13C
NMR spectra were recorded on a Brucker AMX-400 (400 MHz)
spectrometer using CdCl3-d as solvent and TMS as the internal
standard. The Mass spectra were recorded on PerkineElmer
018444-Y, Triple Quadrupole LC/MS Spectrometer. The novel
synthesized compounds showed the molecular ion peak (m/z)
equivalent to their molecular weight. The elemental analysis was
carried out on an Elementar Vario EL III analyzer.
4. Conclusion
The present study shows that among the thiadiazolotriazin-4-
one derivatives (5ae5n), one with hydroxyl moiety at 4th posi-
tion of phenyl ring (5f) possesses larvicidal activity against a ma-
laria vector. This compound may further be screened against other
insect larvae, which may open up the possibility of using the
compound as a pesticide. Thiadiazolotriazin-4-one derivatives with
chloro and acetyl groups at 4th position of the phenyl ring (5i and
5m) exhibiting broad spectrum antibacterial activity against both
Gram positive and Gram negative species may contribute to the
development of potential antibiotics against bacterial pathogens.
5.1. General procedure for the synthesis of 4-amino-6-tert-buty-3-
mercapto-1,2,4-triazine-5(4H)-one (3) [50]
The synthesis of 4-amino-6-tert-butyl-3-mercapto-1,2,4-
triazine-5(4H)-one (3) was done according to the procedure well
described in the literature. An equimolar mixture of 3,3-dimethyl-
2-oxobutanoic acid (trimethyl pyruvic acid) [CAS no: 815-17-8] (1)
and thiocarbohydrazide (2) were refluxed in ethanol as solvent for
12 h. The progress of the reaction and the purity of the compound
were checked by TLC. The reaction mixture was poured into
crushed ice, the precipitate obtained was dried; yield and melting
points were noted and recrystallized from ethanol (Scheme 1).
5.2. General procedure for the syntheses of 3-tert-butyl-7-
(aryloxymethyl)-4H-[1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-ones
(5ae5n)
An equimolar mixtures of 4-amino-6-tert-butyl-3-mercapto-
1,2,4-triazine-5(H)-one (0.01 mol) (3) and substituted aryloxyacetic
acids (0.01 mol) (4) were condensed in presence of POCl3 at 90 ꢀC,
for 8 h. The reactions were carried out in dry condition. The reaction
mixtures were cooled and poured into crushed ice drop wise with
Fig. 1. Mosquito-larvicidal activity of [1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-ones
(5ae5n) against Anopheles stephensi mosquito and Malathion (Ref. standard).

P.A. Castelino et al. / European Journal of Medicinal Chemistry 84 (2014) 194e199
197
Table 1
Mosquito-larvicidal activity of [1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-ones (5ae5n) against Anopheles stephensi e a malaria vector and Malathion (Ref. standard).
Tested compounds
LC50
(
mg/mL)
95% CI
LC90
(
mg/mL)
95% CI
bRelative potency
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
61.3
48.7
39.8
102.8
70.5
21.7
25.7
79.2
55.2
72.8
81.6
77.5
86.6
49.3
8.7
46.07e76.53
35.81e60.13
26.52e38.92
60.54e92.81
56.22e84.76
14.72e28.69
19.16e32.23
62.23e96.04
45.62e71.08
55.44e89.58
63.58e99.66
61.46e93.56
71.37e101.23
38.28e60.32
6.66e10.79
90.45
73.2
75.24e105.66
56.63e85.92
32.36e57.69
97.14e133.27
88.7e121.32
20.72e43.45
24.17e51.39
103.36e138.58
70.48e104.52
96.08e131.04
96.01e134.12
106.13e143.39
118.98e152.83
57.66e85.24
8.39e15.69
0.14
0.18
0.29
0.11
0.12
0.40
0.34
0.11
0.15
0.12
0.11
0.11
0.10
0.17
1
45.05
115.2
105.04
32.11
37.78
120.98
87.49
113.57
115.05
124.77
135.9
71.45
12.01
5m
5n
aMalathion
Bold represents relatively more significant in terms of zone of inhibition.
CI e Confidence Interval.
a
Malathion e Reference standard.
Relative potency e LC50 standard/LC50 tested substance.
b
vigorous shaking, yielded solid product, filtered and recrystallized
from ethanol to afford analytical samples (5ae5n) (Scheme 2).
and 1483 (C]C), 943 and 875 (AreCl). 1H NMR (CdCl3-d,
d ppm):
1.50 (9H, s, 3CH3), 5.41 (2H, s, eCH2), 6.97 (1H, d, J ¼ 8.8 Hz, 2,4-
dichlorophenoxy), 7.45 (1H, d, J ¼ 2.4 Hz, 2,4-dichlorophenoxy)
5.2.1. The analyses of 3-tert-butyl-7-[(aryloxy)methyl]-4H-[1,3,4]
thiadiazolo[2,3-c]-1,2,4-triazin-4-one (5ae5n)
and 7.25 (dd, H, J ¼ 2 Hz, 2,4-dichlorophenoxy). 13C NMR (
d ppm):
27.67 (3 CH3 groups of t-butyl), 38.26 (C-atom of t-butyl), 66.40
(eCH2), 115.45, 124.75, 128.06, 128.81, 130.78 and 146.90 (2,4-
dichlorophenoxy), 151.29, 158.96 and 161.28 (3C-atoms of thiadia-
zolotriazin-4-one) and 162.01 (eC]O of thiadiazolotriazin-4-one).
LCeMS, [Mþ], (m/z): 384.8/386.8/387.9/389.9. Anal. Cald for
5.2.1.1. 3-tert-Butyl-7-[(4-chlorophenoxy)methyl]-4H-[1,3,4]thiadia-
zolo[2,3-c][1,2,4]triazin-4-one (5a). IR (KBr, cmꢁ1): 3075 (AreH),
2982 (eCH3 of t-butyl), 1709 (eC]O), 1571 (eC]N stretch), 1524
and 1487 (C]C) and 945 (AreCl). 1H NMR (CdCl3-d,
d ppm): 1.50
C
15H14Cl2N4O2S: C, 46.76; H, 3.66; N,14.54. Found: C, 46.75; H, 3.67;
(9H, s, 3CH3), 5.36 (2H, s, eCH2), 6.94 (2H, d, J ¼ 8.8 Hz, 4-
chlorophenoxy), 7.30 (2H, d, J ¼ 8.8 Hz, chlorophenoxy). 13C NMR
N, 14.52. m.p. 180e182 ꢀC, yield: 74%.
(d ppm): 27.67 (3CH3 groups of tert butyl), 38.26 (C-atom of t-
butyl), 65.28 (eCH2), 116.23, 128.22, 129.97 and 146.90 (4-
chlorophenoxy), 155.31, 158.93 and 161.78 (3C-atoms of thiadia-
zolotriazin-4-one) and 162.01 (eC]O of thiadiazolotriazin-4-one).
LCeMS, [Mþ], (m/z): 350.9/351.9. Anal. Cald for C15H15ClN4O2S: C,
55.35; H, 4.31; N, 15.97. Found: C, 55.33; H, 4.32; N, 15.98. m.p.
166e168 ꢀC, yield: 72%.
5.2.1.3. 3-tert-Butyl-7-[(2,3,5-trimethylphenoxy)methyl]-4H-[1,3,4]
thiadiazolo[2,3-c][1,2,4]triazin-4-one (5c). IR (KBr, cmꢁ1): 3021
(AreH), 2925 (eCH3 of t-butyl), 2918 (eCH3 of 2,3,5-
trimethylphenoxy group), 1706 (eC]O), 1561 (eC]N stretch),
1518 and 1462 (C]C); 1H NMR (CdCl3-d,
d ppm): 1.51 (9H, s, 3CH3),
2.17 (3H, s, eCH3, 2,3,5-trimethylphenoxy), 2.21 (3H, s, eCH3 of
5.2.1.2. 3-tert-Butyl-7-[(2,4-chlorophenoxy)methyl]-4H-[1,3,4]thia-
diazolo[2,3-c][1,2,4]triazin-4-one (5b). IR (KBr, cmꢁ1): 3098 (AreH),
2995 (eCH3 of t-butyl), 1705 (eC]O), 1575 (eC]N stretch), 1521
Table 2
Antibacterial activity (zone of inhibition) of [1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-
ones (5ae5n) against Gram positive and Gram negative bacteria and Streptomycin
(Ref. standard).
Tested compounds Diameter of zone of inhibition (mm SD)a
(1 mg/mL) bacterial
species
Gram positive bacteria
Gram negative bacteria
S. aureus
B. subtilis
P. aeruginosa K. pneumoniae
5a
e
11.2
13.9 0.59 12.5 0.42
5b
5c
5d
5e
5f
5g
5h
5i
10.8 0.76 12.9 0.54
17.6 0.83 15.6 0.67
e
9.7 0.23
18.9 0.67 15.6 0.49
11.4 0.39 10.4 0.37
11.7 0.45
14.3 0.47 10.5 0.38
15.6 0.59 13.9 0.58
10.9 0.43
e
9.8 0.59
19.8 0.74 16.8 0.67
11.7 0.48
21.7 0.97 19.2 0.61
12.3 0.54
19.6 0.63 18.4 0.62 21.06 0.48 22.4 0.65
9.6 0.59
e
e
e
5j
5k
16.6 0.74 18.9 0.64
22.4 0.69 19.3 0.42
19.5 0.59 16.5 0.57
16.5 0.44 17.5 0.51
5l
5m
5n
e
11.9 0.53
18.7 0.74 25.7 0.65
12.6 0.56
31.5 0.84 33.6 0.78
e
e
22.7 0.68 19.3 0.49
18.9 0.75 19.6 0.63
26.3 1.08 22.4 0.79
e
bStreptomycin
Bold represents relatively more significant in terms of zone of inhibition.
‘0’ Zone of inhibition <7 mm indicating no sensitivity.
Fig. 2. Antibacterial activity (zone of inhibition) of [1,3,4]thiadiazolo[2,3-c][1,2,4]tri-
azin-4-ones (5ae5n) against Gram positive and Gram negative bacteria and Strepto-
mycin (Ref. standard).
a
Mean values (n ¼ 3).
b
Reference standard (10 mg/disc) used as reference standard.

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P.A. Castelino et al. / European Journal of Medicinal Chemistry 84 (2014) 194e199
2,3,5-trimethylphenoxy), 2.29 (3H, s, eCH3 of 2,3,5-
trimethylphenoxy), 5.34 (2H, s, eCH2), 6.54 (1H, s, 2,3,5-
trimethylphenoxy), 6.73 (1H, s, 2,3,5-trimethylphenoxy). 13C NMR
for C16H18N4O3S: C, 55.48; H, 5.24; N,16.17. Found: C, 55.46; H, 5.25;
N, 16.16. m.p. 172e174 ꢀC, yield: 74%.
(
d
ppm): 11.51, 19.57 and 21.25 (3CH3 groups on 2,3,5-
5.2.1.8. 3-tert-Butyl-7-(phenoxymethyl)-4H-[1,3,4]thiadiazolo[2,3-c]
[1,2,4]triazin-4-one (5h). IR (KBr, cmꢁ1): 3065 (AreH), 2974 (CH3 of
t-butyl), 1701 (eC]O), 1593 (eC]N stretch), 1564 and 1516 (C]C);
trimethylphenoxy), 27.69 (3CH3 groups of t-butyl), 38.23 (C-atom
of t-butyl), 65.25 (eCH2), 110.11, 122.34, 125.29, 136.09, 138.62 and
147.00 (2,3,5-trimethylphenoxy), 154.76, 159.11 and 161.85 (3C-
atoms of thiadiazolotriazin-4-one) and 163.13 (eC]O of thiadia-
zolotriazin-4-one). LCeMS, [Mþ], (m/z): 358.9. Anal. Cald for
1H NMR (CdCl3-d,
d
ppm): 1.50 (9H, s, 3CH3), 5.38 (2H, s, eCH2),
6.98e7.37 (5H, m, phenoxy); 13C NMR (
d
ppm): 27.68 (3CH3 groups
of t-butyl), 38.24 (C-atom of t-butyl), 64.99 (eCH2), 114.88, 123.05,
130.03, 146.95 (6C atoms of phenoxy), 156.76, 159.08 and 161.90
(3C-atoms of thiadiazolotriazin-4-one) and 162.55 (eC]O of
thiadiazolotriazin-4-one). LCeMS, [Mþ], (m/z): 317.08. Anal. Cald
for C15H16N4O2S: C, 56.94; H, 5.10; N, 17.71. Found: C, 56.93; H, 5.11;
N, 17.70. m.p. 150e152 ꢀC, yield: 76%.
C
18H22N4O2S: C, 60.31; H, 6.19; N, 15.63. Found: C, 60.30; H, 6.20; N,
15.62. m.p. 178e180 ꢀC, yield: 72%.
5.2.1.4. 3-tert-Butyl-7-[(2,6-dimethylphenoxy)methyl]-4H-[1,3,4]
thiadiazolo[2,3-c][1,2,4]triazin -4-one (5d). IR (KBr, cmꢁ1): 3035
(AreH), 2924 (eCH3 of t-butyl), 1703 (eC]O), 1560 (eC]N
stretch), 1516 and 1460 (C]C); 1H NMR (CdCl3-d,
d
ppm): 1.51 (9H,
s, 3CH3), 2.32 (6H, s, 2CH3 groups on 2,6-dimethylphenoxy), 5.12
(2H, s, eCH2), 7.04 (3H, m, 2,6-dimethylphenoxy); 13C NMR (
5.2.1.9. 3-tert-Butyl-7-[2-(4-chlorophenoxy)propan-2yl]-4H-[1,3,4]
thiadiazolo[2,3-c][1,2,4]triazin-4-one (5g). IR (KBr, cmꢁ1): 3011
(AreH), 2937 (CH3 of t-butyl), 2922 (CH3 groups), 1704 (eC]O),
1562 (eC]N stretch), 1521 and 1468 (C]C), 941 (AreCl); 1H NMR
d
ppm): 16.35 (2CH3 groups on 2,6-dimethylphenoxy), 27.67 (3CH3
groups of t-butyl), 38.23 (C-atom of t-butyl), 67.95 (eCH2), 125.51,
129.41, 130.37 and 147.04 (2,6-dimethylphenoxy), 154.30, 159.14
and 161.84 (3C-atoms of thiadiazolotriazin-4-one) and 162.54
(eC]O of thiadiazolotriazin-4-one). LCeMS, [Mþ], (m/z): 344.9/
346.1. Anal. Cald for C17H20N4O2S: C, 59.28; H, 5.85; N, 16.27. Found:
C, 59.26; H, 5.87; N, 16.28. m.p. 112e114 ꢀC, yield: 76%.
(CdCl3-d,
(2H, d, J ¼ 8.8 Hz, 4-chlorophenoxy), 7.28 (2H, d, J ¼ 8.8 Hz, 4-
chlorophenoxy); 13C NMR (
ppm): 23. 15 (2C atoms of CH3),
d ppm): 1.50 (9H, s, 3CH3), 1.80 (6H, s, 2 CH3 groups), 6.90
d
27.69 (3CH3 groups of t-butyl), 38.27 (C-atom of t-butyl), 67.98
(eCH2), 125.52, 129.43, 130.39 and 147.07 (4-chlorophenoxy),
154.31, 159.16 and 161.87 (3 C-atoms of thiadiazolotriazin-4-one)
and 162.59 (eC]O of thiadiazolotriazin-4-one). LCeMS, [Mþ], (m/
z): 379.8/381.8. Anal. Cald for C17H19ClN4O2S: C, 53.89; H, 5.05; N,
14.79. Found: C, 53.88; H, 5.06; N, 14.78. m.p. 66e68 ꢀC, yield: 70%.
5.2.1.5. 3-tert-Butyl-7-[(2-nitrophenoxy)methyl]-4H-[1,3,4]thiadia-
zolo[2,3-c][1,2,4]triazin-4-one (5e). IR (KBr, cmꢁ1): 3007 (AreH),
2928 (eCH3 of t-butyl), 1707 (eC]O), 1562 (eC]N stretch), 1542
and 1412 (sym and asym stretch of eNO2), 1524 and 1468 (C]C). 1H
NMR (CdCl3-d,
7.18e7.66 (3H, m, 2-nitrophenoxy), 7.97 (1H, dd, 2-nitrophenoxy);
13C NMR (
ppm): 27.67 (3CH3 groups of t-butyl), 38.27 (C-atom of
5.2.1.10. 3-tert-Butyl-7-[(2-chlorophenoxy)methyl]-4H-[1,3,4]thia-
diazolo[2,3-c][1,2,4]triazin-4-one (5j). IR (KBr, cmꢁ1): 3015 (AreH),
2926 (CH3 of t-butyl), 1705 (eC]O), 1564 (eC]N stretch), 1522
d ppm): 1.50 (9H, s, 3CH3), 5.51 (2H, s, eCH2),
d
and 1464 (C]C) and 932 (AreCl); 1H NMR (CdCl3-d,
d ppm): 1.50
tert butyl), 66.57 (eCH2), 115.60, 119.21, 123.31, 126.42, 134.73 and
146.93 (2-nitrophenoxy), 150.02, 159.05 and 160.79 (3 C-atoms of
thiadiazolotriazin-4-one) and 161.97 (eC]O of thiadiazolotriazin-
4-one). LCeMS, [Mþ], (m/z): 362.3. Anal. Cald for C15H15N5O4S: C,
49.85; H, 4.18; N, 19.38. Found: C, 49.86; H, 4.19; N, 19.37. m.p.
142e144 ꢀC, yield: 70%.
(9H, s, 3CH3), 5.08 (2H, s, eCH2), 7.58 (1H, d, 2-chlorophenoxy), 7.73
(1H, d, 2-chlorophenoxy), 7.31 and 7.54 (2H, 2t, 2-chlorophenoxy);
13C NMR (
d ppm): 27.69 (3CH3 groups of t-butyl), 38.27 (C-atom of
t-butyl), 67.98 (eCH2), 125.52, 129.43, 130.39, 132.25, 134.26 and
147.07 (2-chlorophenoxy), 154.31, 159.16 and 161.87 (3C-atoms of
thiadiazolotriazin-4-one) and 162.59 (eC]O of thiadiazolotriazin-
4-one). LCeMS, [Mþ], (m/z): 351.82/353.9. Anal. Cald for
5.2.1.6. 3-tert-Butyl-7-[(4-hydroxyphenoxy)methyl]-4H-[1,3,4]thia-
diazolo[2,3-c][1,2,4]triazin-4-one (5f). IR (KBr, cmꢁ1): 3351 (-OH),
3098 (AreH), 2982 (CH3 of -butyl), 1709 (eC]O),1571 (eC]N
C15H15ClN4O2S: C, 51.35; H, 4.31; N, 15.96. Found: C, 51.35; H, 4.32;
N, 15.96. m.p. 116e118 ꢀC, yield: 74%.
stretch), 1524 and 1487 (C]C); 1H NMR (CdCl3-d,
s, 3CH3), 5.41 (2H, s, eCH2), 6.98 (d, 2H,
d
ppm): 1.51 (9H,
8.8 Hz, 4-
5.2.1.11. 3-tert-Butyl-7-[(2, 4, 5-trichlorophenoxy)methyl]-4H-[1,3,4]
thiadiazolo[2,3-c][1,2,4]triazin-4-one (5k). IR (KBr, cmꢁ1): 3025
(AreH), 2982 (CH3 of t-butyl), 1710 (eC]O), 1571 (eC]N stretch),
1532 and 1491 (C]C). 945, 927 and 906 (AreCl); 1H NMR (CDCl3-d,
J
¼
hydroxyphenoxy), 7.26 (d, 2H, J ¼ 8.8 Hz, 4-hydroxyphenoxy), 8.5
(1H, s, eOH). 13C NMR (
d
ppm): 27.61 (3CH3 groups of t-butyl),
38.25 (C-atom of t-butyl), 66.34 (eCH2), 124.45, 128.79, 130.19 and
136.90 (4-hydrophenoxy), 151.31, 158.85 and 161.21 (3C-atoms of
thiadiazolotriazin-4-one) and 163.12 (eC]O of thiadiazolotriazin-
4-one). LCeMS, [Mþ], (m/z): 333.4. Anal. Cald for C15H16N4O3S: C,
54.20; H, 4.85; N, 16.86. Found: C, 54.19; H, 4.86; N, 16.85. m.p.
114e116 ꢀC, yield: 68%.
d
, ppm): 1.50 (9H, s, 3CH3), 5.42 (2H, s, eCH2), 7.14 (1H, s, 2,4,5-
trichlorophenoxy), 7.54 (1H, s, 2,4,5-trichlorophenoxy); 13C NMR
ppm): 27.66 (3CH3 groups of t-butyl), 38.28 (C-atom of t-butyl),
(d
66.55 (eCH2), 116.22, 122.95, 127.33, 131.66, 131.81 and 146.85
(2,4,5-trichlorophenoxy), 151.42, 158.88 and 160.62 (3C-atoms of
thiadiazolotriazin-4-one) and 162.09 (eC]O of thiadiazolotriazin-
4-one). LCeMS, [Mþ], (m/z): 420.8/422.7/424.8/426.8; Anal. Cald
for C15H13Cl3N4O2S: C, 42.92; H, 3.12; N, 13.35. Found: C, 42.91; H,
3.14; N, 13.34. m.p. 120e122 ꢀC, yield: 78%.
5.2.1.7. 3-tert-Butyl-7-[(2-methoxyphenoxy)methyl]-4H-[1,3,4]thia-
diazolo[2,3-c][1,2,4]triazin-4-one (5g). IR (KBr, cmꢁ1): 3015 (AreH),
2938 (CH3 of t-butyl), 1708 (eC]O), 1564 (eC]N stretch), 1526
and 1466 (C]C); 1H NMR (CdCl3-d,
d
ppm): 1.51 (9H, s, 3CH3), 3.52
(3H, s, eOCH3), 5.52 (2H, s, eCH2), 7.16e7.62 (3H, m, 2-
methoxyphenoxy), 7.98 (1H, dd, 2-methoxyphenoxy); 13C NMR (
5.2.1.12. 3-tert-Butyl-7-[(4-iodophenoxy)methyl]-4H-[1,3,4]thiadia-
zolo[2,3-c][1,2,4]triazin-4-one (5l). IR (KBr, cmꢁ1): 3011 (AreH),
2988 (CH3 of t-butyl),1707 (eC]O),1574 (eC]N stretch), 1534 and
d
ppm): 27.63 (3CH3 groups of t-butyl), 31.23 (C atom of eOCH3),
38.27 (C-atom of t-butyl), 66.55 (eCH2), 115.62, 119.23, 123.35,
127.44, 135.71 and 145.92 (2-methoxyphenoxy), 151.02, 158.15 and
161.89 (3 C-atoms of thiadiazolotriazin-4-one) and 162.97 (eC]O
of thiadiazolotriazin-4-one). LCeMS, [Mþ], (m/z): 362.3. Anal. Cald
1497 (C]C); 1H NMR (CdCl3-d,
d ppm): 1.51 (9H, s, 3CH3), 5.42 (2H,
s, eCH2), 6.97 (2H, d, J ¼ 8.8 Hz, 4-iodophenoxy), 7.24 (2H, d,
J ¼ 8.8 Hz, 4-iodophenoxy); 13C NMR (
d ppm): 27.62 (3CH3 groups
of t-butyl), 38.27 (C-atom of t-butyl), 66.37 (eCH2 group), 125.45,
128.85, 131.22 and 137.90 (4-iodophenoxy), 152.34, 159.87 and

P.A. Castelino et al. / European Journal of Medicinal Chemistry 84 (2014) 194e199
199
[11] B. Gerhard, B.H. Ulrich, K. Hans (Bayer A-G.), Ger. Offen. DE 3, 009, 043 (1980),
Chem. Abstr. 96 (1982) 6764.
[12] Sumitono Chemical Co. Lyd. Jpn. Kokai Tokkyo Koho JP 5838, 270 [8338, 270]
(1981), Chem. Abstr. 99 (1983) 22505.
[13] W. Roy, H. Santle, R.R. Schmidt. Ger Offen, DE 3, 510, 785, Chem. Abstr. 106
(1987), 33129.
162.44 (3C-atoms of thiadiazolotriazin-4-one) and 162.52 (eC]O
of thiadiazolotriazin-4-one). LCeMS, [Mþ], (m/z): 443.3. Anal. Cald
for C15H15IN4O2S: C, 56.97; H, 5.06; N, 15.03. Found: C, 56.96; H,
5.07; N, 15.62. m.p. 156e158 ꢀC, yield: 76%.
[14] B. Beat, T. Hans (Ciba-Geigy A-G), Eur. Pat. Appl., Ep 150, 677, Chem. Abstr. 104
(1986) 19607.
5.2.1.13. 7-[(4-acetylphenoxy)methyl]3-tert-butyl-4H-[1,3,4]thiadia-
zolo[2,3-c][1,2,4]triazin-4-one (5m). IR (KBr, cmꢁ1): 3021 (AreH),
2970 (CH3 of t-butyl), 2978 (CH3), 1708 (eC]O), 1690 (eC]O, of
acetyl), 1572 (eC]N stretch), 1525 and 1402 (C]C); 1H NMR
[15] K. Singh, M.S. Barwa, P. Tyag, Eur. J. Med. Chem. 42 (2007) 394.
[16] T.E. Ali, Phosphorus Sulfur Silicon Relat. Elem. 182 (2007) 1717.
[17] D.J. Prasad, M.S. Karthikeyan, P.B. Karegoudar, B. Poojary, B.S. Holla,
N.S. Kumari, Phosphorus Sulfur Silicon Relat. Elem. 182 (2007) 1083.
[18] Sraa Abu-Melha, Spectrochim. Acta A 96 (2012) 898.
[19] B.S. Holla, R. Gonsalves, M.K. Shivananda, Bull. Chim. Farm. 137 (1998) 93.
[20] M.A. Ibrahim, K.M. El-Mahdy, Phosphorous Sulfur Silicon Relat. Elem. 184
(2009) 2945.
[21] T.E. Ali, S. Abdel-Ghfaar, H.M. El-Shaaer, F.I. Hanafy, A.Z. El-Fauomy, Phos-
phorous Sulfur Silicon Relat. Elem. 183 (2009) 2143.
[22] Z. El Gendy, J.M. Morsy, H.A. Allimony, W.R. Abdel-Monem, R.M. Abdel-Rah-
man, Phosphorous Sulfur Silicon Relat. Elem. 178 (2003) 2005.
[23] Z. El Gendy, J.M. Morsy, H.A. Allimony, W.R. Abdel-Monem, R.M. Abdel-Rah-
man, Pharmazie 56 (2001) 1.
(CdCl3-d,
d ppm): 1.51 (9H, s, 3CH3), 2.29 (3H, s, CH3), 5.40 (2H, s,
eCH2), 6.81 (2H, d, J ¼ 8.8 Hz, 4-acetylphenoxy), 7.16 (2H, d,
J ¼ 8.8 Hz, 4-acetylphenoxy); 13C NMR (
d ppm): 21.10 (CH3 group on
4-acetyl), 27.69 (3CH3 groups of t-butyl), 38.29 (C-atom of t-butyl),
66.27 (eCH2), 112.65, 126.96, 128.69, 130.04, 131.34 and 138.34 (4-
acetylphenoxy), 147.22, 152.74 and 157.81 (3C-atoms of thiadiazo-
lotriazin-4-one) and 162.25 (eC]O of thiadiazolotriazin-4-one).
LCeMS, [Mþ], (m/z): 359.4; Anal. Cald for C17H18N4 O3S: C, 56.97; H,
5.06; N, 15.63. Found: C, 56.96; H, 5.07; N, 15.62. m.p. 176e178 ꢀC,
yield: 72%.
[24] Z. El-Gendy, J.M. Morsy, H.A. Allimony, W.R. Abdel-Monem, R.M. Abdel-Rah-
man, Pharmazie 56 (2001) 376.
[25] Malgorzata Sztanke, Jolanta Rzymowska, Krzysztof Sztanke, Bioorg. Med.
Chem. 21 (23) (2013) 7465.
[26] Tarik El-Sayed Ali, Eur. J. Med. Chem. 44 (11) (2009) 4539.
[27] A.A. El-Barbary, A.M. Sakran, A.M. El-Madani, N. Claus, J. Heterocycl. Chem. 42
(2005) 935.
5.2.1.14. 3-tert-Butyl-7-[(4-chloro-2-methylphenoxy)methyl]-4H-
[1,3,4]thiadiazolo[2,3-c][1,2,4]triazin-4-one (5n). IR (KBr, cmꢁ1):
3028 (AreH), 297 (CH3 of t-butyl), 1710 (eC]O), 1571 (eC]N
stretch), 1523 and 1398 (C]C), 937 (AreCl); 1H NMR (CdCl3-d,
[28] L.S. Pustoshnaya, V.P. Pustoshnyi, A.K. Mazitova, V.K. Khamaev, E. Ya Davydov,
G.E. Zaikov, K.S. Minsker, Polym. Degrad. Stab. 70 (1) (2000) 39.
[29] a) Günter Schmahl, Heinz Mehlhorn, Eur. J. Protistol. 24 (3) (1989) 252;
ꢀ
d
ppm): 1.50 (9H, s, 3CH3), 2.27 (3H, s, CH3), 5.36 (2H, s, eCH2), 6.79
b) G. Schmahl, J. Senaud, H. Mehlhorn, Arch. Protistenkd. 140 (1) (1991) 83.
[30] Darpan Kaushik, Suroor Ahmad Khan, Gita Chawla, Eur. J. Med. Chem. 45 (9)
(2010) 3960.
[31] Xian-Yu Sun, Lei Zhang, Cheng-Xi Wei, Hu-Ri Piao, Zhe-Shan Quan, Eur. J.
Med. Chem. 44 (3) (2009) 1265.
[32] Krzysztof Sztanke, Tomasz Tuzimski, Małgorzata Sztanke, Jolanta Rzymowska,
Kazimierz Pasternak, Bioorg. Med. Chem. 19 (17) (2011) 5103.
(H, s, J ¼ 8.8 Hz, 4-chloro-2-methylphenoxy), 7.14 (H, d, J ¼ 2.4 Hz,
4-chloro-2-methylphenoxy), 7.16 (2H, dd, J ¼ 2 Hz, 4-chloro-2-
methylphenoxy), 7.19 (H, d,
J
¼
2.4 Hz, 4-chloro-2-
methylphenoxy); 13C NMR (
d ppm): 16.10 (CH3 group on 4-
chloro-2-methylphenoxy), 27.68 (3CH3 groups of t-butyl), 38.27
(C-atom of t-butyl), 65.23 (eCH2), 112.45,126.86, 127.69,129.04 and
131.34 (4-chloro-2-methylphenoxy), 146.92, 153.54 and 158.91 (3C-
atoms of thiadiazolotriazin-4-one) and 162.05 (eC]O of thiadia-
zolotriazin-4-one). LCeMS, [Mþ], (m/z): 364.8/365.9. Anal. Cald for
[33] Krzysztof
Sztanke,
Kazimierz
Pasternak,
Małgorzata
Sztanke,
Martyna Kandefer-Szerszen, Anna E. Koziol, Izabela Dybala, Bioorg. Med.
Chem. Lett. 19 (17) (2009) 5095.
[34] a) Hai-Chuan Zhao, Yan-Ping Shi, Yu-Ming Liu, Cai-Wen Li, Li-Na Xuan,
Peng Wang, Kai Zhang, Bao-Quan Chen, Bioorg. Med. Chem. Lett. 23 (24)
(2013) 6577;
b) B.S. Holla, B.S. Rao, B.K. Sarojini, K. Sridara, Farmaco 57 (2002) 693.
[35] Zhenhua Luo, Baoquan Chen, Shuangyan He, Yanping Shi, Yuming Liu,
Caiwen Li, Bioorg. Med. Chem. Lett. 22 (9) (2012) 3191.
[36] Juan Sun, Yu-Shun Yang, Wei Li, Yan-Bin Zhang, Xiao-Liang Wang, Jian-
Feng Tang, Hai-Liang Zhu, Bioorg. Med. Chem. Lett. 21 (20) (2011) 6116.
[37] a) B. Chandrakantha, Arun M. Isloor, Prakash Shetty, Hoong Kun Fun,
Gurumurthy Hegde, Eur. J. Med. Chem. (2013);
b) Yin Luo, Shuai Zhang, Zhi-Jun Liu, Wu Chen, Jie Fu, Qing-Fu Zeng, Hai-
Liang Zhu, Eur. J. Med. Chem. 64 (2013) 54.
[38] Li-Hong Shen, Hong-Yu Li, Hui-Xia Shang, Shu-Ting Tian, Yi-Sheng Lai, Li-
Jie Liu, Chin. Chem. Lett. 24 (4) (2013) 299.
[39] Shankar G. Alegaon, Kallanagouda R. Alagawadi, Pranali V. Sonkusare, Sagar
M. Chaudhary, Dilip H. Dadwe, Amol S. Shah, Bioorg. Med. Chem. Lett. 22 (5)
(2012) 1917.
[40] Mohammad Yusuf, Riaz A. Khan, Bahar Ahmed, Bioorg. Med. Chem. 16 (17)
(2008) 8029.
C
16H17ClN4O2S: C, 56.67; H, 4.70; N, 15.36. Found: C, 56.66; H, 4.72;
N, 15.35. m.p. 142e144 ꢀC, yield: 74%.
Acknowledgements
The authors are grateful to Rt Rev. Dr Aloysius Paul D’ Souza, the
Bishop of Mangalore Diocese for the financial assistance. Dr Maha-
gundappa R. Maddani for his help in the manuscript. The Head, NMR
Research Centre, SAIF-Cochin-22 for 1H NMR,13C NMR spectroscopy
and CHN analyses, USIC-Mangalore University for FT-IR analyses,
Acquity Labs, Bengaluru and Syngenta, Goa for Mass spectroscopy.
[41] B.S. Holla, R. Gonsalves, B.K. Sarojini, S. Shenoy, Ind. J. Chem. 40B (2001) 475.
[42] Z.Y. Zhang, S.X. Wen, Heterocycles 48 (1998) 561.
[43] World Health Organization, WHO/VBC/81, 807, 1981.
[44] M. Raymond, G. Prato, D. Ratsira, PROBIT Analysis of Mortality Assays Dis-
playing Quantal Response, 1993.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.072.
[45] a) A.W. Bauer, W.M.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45
(1966) 493;
References
€
€
b) G. Vardar-Unlü, F. Candan, A. Sokmen, D. Daferera, M. Polissiou, M. Sokmen,
€
E. Donmez, B. Tepe, J. Agric. Food Chem. 51 (1) (2003) 63.
[46] D. Dornow, H. Mazel, P. Marxd, Ber. 97 (1964) 2173.
[1] H. Neunhoeffer, in: A.R. Katrizky, C.W. Rees (Eds.), Comprehensive Hetero-
cyclic Chemistry, vol. 3, Pergamon, 1984.
[47] OECD Guideline for Testing of Chemicals e Acute Oral Toxicity e Acute Toxic
Class http://ntp.niehs.nih.gov/iccvam/SuppDocs/FedDocs/OECD/OECD_GL423.
pdf (retrieved on 19.02.14.).
[48] EPA 815-R-08-003, Contaminant Candidate List 3 Chemicals: Screeningto a PCCL
e United States e Environment Protection Agency, Retrieved on 18.02.2014:
http://www.epa.gov/ogwdw/ccl/pdfs/repot ccl3 chemicals screening.pdf.
[49] CPCSEA (Committee for the Purpose of Control and Supervision on Experiments
on Animals), India, http://envfor.nic.in/divisions/awd/cpcsea laboratory.pdf.
[50] a) Selvaraj Mohana Roopan, Fazlur-Rahman Nawaz Khan1, Jong Sung Jin, Pak.
J. Pharm. Sci. 26 (4) (2013) 747;
[2] B.S. Holla, B.K. Sarojini, R. Gonsalves, Farmaco 53 (1998) 395.
[3] B.S. Holla, B.K. Sarojini, K. Shridhara, G. Antong, Farmaco 54 (1999) 149.
[4] A. Deeb, F. El-Mariah, M. Hosny, Bioorg. Med. Chem. Lett. 14 (2004) 5013.
[5] M.A. El-Badawi, A.A. El-Barbary, Y.M. Loksha, M. El-Daly, Phosphorus Sulfur
Silicon Relat. Elem. 177 (2002) 587.
[6] A.S. Oganisyan, G.O. Grigoryan, A.S. Noravyan, I.A. Dzhagatspanyan,
G.G. Melikyan, Pharm. Chem. J. 35 (2001) 124.
[7] B. Boehner, A.G. Ciba-Geigy, Eur. Pat., Chem. Abstr. 105 (1986) 6522, 1986.
[8] M. Timer, S. Sauvard, C.M. Georgescu, Biochem. Pharmacol. 15 (1996) 408.
[9] E. Krauz, K. Findeisen, H. Fiege, L. Ene, R.R. Schmidt (Bayer A-G.), Ger. Offen. DE
3, 323, 934 (1983), Chem. Abstr. 102 (1985) 18506.
b) N.N. Su, L.X. Xiong, S.J. Yu, X. Zhang, C. Cui, Z.M. Li, W.G. Zhao, Comb. Chem.
High Throughput Screen. 16 (6) (2013) 484;
c) Yuaho Li, Hongjun Zhu, Kai Chen, Rui Liu, Abdulla Khallaf, Xiangning Zhang,
Jueping Ni, Org. Biomol. Chem. 11 (2013) 3979.
[10] E. Krauz, M. Jautelat, L. Ene, R.R. Schmidt (Bayer A-G.), Ger. Offen. DE 3, 323,
935 (1983), Chem. Abstr.102 (1985) 18505.