Synthesis, Antimicrobial, and Antioxidant Activities of Some Fused Heterocyclic [1,2,4]Triazolo[3,4-b][1,3,4]thiadiazole Derivatives

Article abstract of DOI:10.1002/jhet.2348

In the present work, we synthesized a series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives (6a, 6b, 6c, 6d, 6e, 6f and 7a, 7b, 7c, 7d, 7e, 7f) by using simple starting materials, namely, β-amino acids and different aromatic acid hydrazides. The

Full text of DOI:10.1002/jhet.2348

May 2016
Synthesis, Antimicrobial, and Antioxidant Activities of Some Fused
Heterocyclic [1,2,4]Triazolo[3,4-b][1,3,4]thiadiazole Derivatives
929
*
Gangadhara Seelolla and Venkateswarlu Ponneri
Department of Chemistry, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India
*
E-mail: ponneri.chem@gmail.com
Received July 30, 2014
DOI 10.1002/jhet.2348
Published online 29 September 2015 in Wiley Online Library (wileyonlinelibrary.com).
In the present work, we synthesized a series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives (6a–f and
7a–f) by using simple starting materials, namely, β-amino acids and different aromatic acid hydrazides. The
newly synthesized compounds were characterized by mass, IR, 1H, and13C-NMR spectral data analysis. The
newly synthesized compounds were tested for their antimicrobial activities and antioxidant properties. Compound
6c was a potent microbial agent particularly against Staphylococcus aureus (MIC 3.12 μg/mL) and Candida
albicans (MIC 6.25 μg/mL) when compared with the reference drugs ciprofloxacin and fluconazole, respectively.
The antioxidant activity of the synthesized compounds was also evaluated by 1,1-diphenyl-2-picryl hydrazyl,
nitric oxide, and hydrogen peroxide radical scavenging methods. Compounds 6c, 6f, 7c, and 7f showed good
radical scavenging activity due to the presence of electron-donating group on phenyl ring.
J. Heterocyclic Chem., 53, 929 (2016).
INTRODUCTION
compounds containing nitrogen, sulfur, and bicyclic systems
with anticipated antibacterial activities [9,10,16].
The high resistance acquired by microbes against the anti-
microbial drugs existing in the market is of a great challenge
to the scientific organization, involved in the development of
novel and effective drugs against human diseases. It is, there-
fore, necessary to develop therapeutic agents with improved
potential for treating broad spectrum microbial infections.
For the last few decades, there has been a tremendous growth
of research in the synthesis of nitrogen and sulfur containing
heterocyclic derivatives because of their utility in various
pharmaceuticals applications.
1,2,4-Triazole derivatives are an important group of hetero-
cyclic compounds and have been the subject of extensive
study in the recent years [1]. It has been reported in the liter-
ature that certain compounds bearing 1,2,4-triazole nucleus
possess significant antimicrobial activities [2–4]. Recently,
triazole–thiadiazole-fused compounds have been frequently
found to display a broad spectrum of biological activities
[5,6]. Further, triazolo–hiadiazole system may be viewed
as a cyclic analog of two important components, namely,
thiosemicarbazide and biguanide [7,8], which often displays
diverse biological activities including antimicrobial [9,10],
antifungal [11], antiinflammatory [12], anticancer [13], anal-
gesic [14], and antitumor [15] activities in continuation to
our efforts directed toward the synthesis of heterocyclic
In addition, β-amino acids (BAA) are not as common in na-
ture as their α-analogs. Some of the BAA were found in more
complex structures such as peptides, dipeptides, lactones, alka-
loids, and other natural products, and in free form, they show
interesting pharmacological effects. These are currently of
growing interest not only because of their natural roles but also
because of their use in the synthesis of peptide mimetics and
certain quite new biologically active substances [17]. BAA
is a constituent of some toxins and a special class of pore-
forming lipopeptides, which exhibit antibacterial and antifun-
gal activities [18]. Peptidomimetics containing BAA represent
one of the strategies in developing therapeutic candidates with
increased oral bioavailability and resistance to metabolic deg-
radation [19]. Recently, the chemistry of hybrid heterocyclic
derivatives has received considerable attention owing to their
synthetic and effective biological importance.
By finding the antimicrobial and antioxidant activities of
heterocyclic compounds, we decided to synthesis of some fused
triazol thiadizol derivatives such as 1-(3,4-dimethoxyphenyl)-2-
(3-(para-substitutedphenyl)[1,2,4]triazolo[3,4-b][1,3,4] thiadiazol-
6-yl)ethnamine derivatives and 1-(4-chlorophenyl)-2-(3-
(para-substitutedphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-
6yl)ethanamine derivatives and to evaluate their antimicrobial
activities and antioxidant properties.
© 2015 HeteroCorporation

930
G. Seelolla and V. Ponneri
Vol 53
Scheme 1
Compound
R
R1
H
Cl
6a
6b
6c
6d
6e
6f
OH
CH3
NO2
OCH3
H
3,4 di-OMe
7a
7b
7c
7d
7e
7f
Cl
OH
CH3
NO2
OCH3
4-Cl
RESULTS AND DISCUSSION
The general synthetic pathway for the
vibrations, which were indicating the formation of 1,3,4-
thiadiazole. The other absorptions band appear in the range
between 1551–1651 cmꢀ1 and 3338–3392 cmꢀ1 for C¼N
Chemistry.
preparation of [1,2,4]triazolo[3,4-b][1,3,4] thiadiazole
derivatives is depicted in Scheme 1. Compounds 5a–f
were prepared according to a methodology [20,21]
involving condensation of different para-substituted
benzoic acid hydrazides (3a–f) with carbon disulfide and
potassium hydroxide under reflux condition that gave the
potassium dithiocarbazate salt (4a–f). Potassium dithio
carbazate salt reaction with hydrazine hydrate under reflux
condition to produce the amino thiols (5a–f). Finally,
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles were prepared by
cyclic condensation of the SH and NH2 functions of 5a–f
compounds with aromatic carboxylic acids 1 and 2 in the
presence of phosphorous oxychloride (POCl3), under reflux
condition that afforded compounds 6a–f and 7a–f with
good yield.
1
and N–H bonds, respectively. H-NMR spectra of com-
pounds 6a–f and 7a–f showed the signals in the region of
δ 3.35–4.35 and 4.25–5.85ppm that correspond to chiral
–CH group and –NH2, respectively. 13C-NMR further con-
firmed the structures of the desired products.
Antimicrobial activity. A broad panel of microbes was
used for testing in vitro antibacterial and antifungal
properties of the synthesized molecules. The results
obtained were reproduced in Table 1 as MIC values. The
antimicrobial values showed that most of the synthesized
derivatives exhibited excellent activities against different
bacterial strains and moderate activity against fungal
strains. Ciprofloxacin and fluconazole were used as the
standard drugs for antimicrobial and antifungal testing,
respectively. When ciprofloxacin was used as a standard
drug for testing the MIC values against the Gram-positive
bacterial strains, it exhibited MIC values of 6.25 and
12.5 μg/mL against Staphylococcus aureus and Bacillus
subtilis, respectively. The MIC values of the standard
ciprofloxacin were found to be 6.25 μg/mL against Gram-
negative bacterial strains (Klebsiella pneumoniae and
The structures of the newly synthesized compounds
1
were established on the basis of their mass, IR, H, and
13C-NMR spectral data. The (M + H)+ molecular ion peak
of all compounds was confirmed by their molecular
weights. The IR spectra of 6a–f and 7a–f compounds
which showed characteristic absorption band in the range
between 1062 and 1138 cmꢀ1 attributed to N¼C–S–C¼N
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2016
Synthesis, Antimicrobial and Antioxidant Activities of Some
[1,2,4]Triazolo[3,4-b][1,3,4]thiadiazole Derivatives
931
Table 1
Antimicrobial activity (MIC profiles) of the synthesized compounds (6a–f and 7a–f).
Minimum inhibitory concentration (MIC) (μg/mL)
Antibacterial activity
Gram-positive bacteria
Gram-negative bacteria
Antifungal activity
Candida Aspergillus
Staphylococcus
Bacillus
subtilis
Klebsiella
Escherichia
coli
Compound
aureus
pneumoniae
albicans
flavus
6a
6b
12.5
25
12.5
25
25
25
12.5
50
12.5
–
50
–
6c
6d
6e
6f
7a
7b
3.12
6.25
50
6.25
12.5
50
6.25
6.25
50
6.25
100
50
6.25
12.5
100
3.12
12.5
25
3.12
12.5
–
6.25
6.25
50
6.25
25
>100
12.5
50
50
50
–
12.5
–
–
50
7c
7d
7e
7f
3.12
6.25
>100
6.25
6.25
–
12.5
50
>100
6.25
12.5
–
3.12
12.5
50
6.25
6.25
–
3.12
12.5
–
3.12
6.25
–
6.25
50
–
12.5
–
12.5
6.25
>100
–
25
–
Ciprofloxacin
Fluconazole
12.5
(–), No activity.
Escherichia coli). The standard drug fluconazole displayed
an MIC value of 12.5 μg/mL against fungal strains
Candida albicans and Aspergillus flavus.
activity with the value of MIC 3.12 μg/mL compared
with the standard drug ciprofloxacin. Other derivatives 6c
and 7f also showed good MIC value of 6.25 μg/mL,
equivalent to the standard ciprofloxacin. The remaining
compounds 6a, 6b, 6d, 6e, 7a, 7b, 7d, and 7e were
found to exhibit low activity. When E. coli was
introduced as another Gram-negative strain for
conducting the antibacterial test, it was found that 6c, 7c,
and 7f compounds exhibited better activity (3.12 μg/mL)
compared with the standard. The remaining compounds
6f and 7a showed an MIC value (6.25 μg/mL) equivalent
to that reported by the standard ciprofloxacin. The
remaining compounds 6a, 6b, 6d, and 7b were found to
exhibit low activity, and 6e and 7e did not showed any
activity compared with the standard.
Antibacterial activity.
All the newly synthesized
compounds (6a–f and 7a–f) were screened for their
in vitro antimicrobial activity determined by well plate
method [22,23]. All compounds were screened against
Gram-positive bacterial strains such as S. aureus and B,
subtilis and Gram-negative bacterial strains such as K.
pneumoniae and E. coli. Compounds 6c and 7c exhibited
excellent activity with the value of MIC 3.12 μg/mL
against the Gram-positive bacteria strain S. aureus.
Compounds 6d, 6f, 7d, and 7f showed an MIC value of
6.25μg/mL, which is equivalent to the standard
ciprofloxacin. The remaining compounds 6a, 6b, 6e, 7a,
7b, and 7e showed low activity (≥25 μg/mL) when
compared with the standard. B. subtilis was introduced as
another Gram-positive strain for conducting the
antibacterial test, it was found that 6b, 6e, 7a, 7b, 7d,
and 7e compounds exhibited poor activity compared with
the standard. The derivatives 6c, 6d, 6f, and 7f showed
excellent activity with the value of MIC 6.25 μg/mL
against B. subtilis. The remaining compounds 6a and 7c
showed an MIC value of 12.5 μg/mL, which was
equivalent to the standard ciprofloxacin. All prepared
compounds were tested for their antibacterial property
against Gram-negative bacterial strains such as K.
pneumoniae and E. coli. When compounds were exposed
against Gram-negative bacteria K. pneumoniae, it was
observed that compounds 6f and 7c exhibited excellent
Antifungal activity.
The synthesized molecules were
tested against C. albicans and A. flavus for their antifungal
activities. It was found that compared with the antibacterial
activity, very few derivatives exhibited good activity.
When molecules 6a–f and 7a–f were tested against C.
albicans, derivatives 6c and 7c showed good activity
(6.25μg/mL) compared with the standard fluconazole. A
few molecules 6a, 6f, and 7f exhibited an MIC value
(12.5 μg/mL) equivalent to that of the standard drug
fluconazole. The remaining molecules 6d, 6e, 7a, and 7d
showed less activity when compared with the standard
drug. Further, A. flavus was also used as a fungal strain
for checking the antifungal potency of the derived
bioactive compounds. It was observed that one of the
compounds 7c showed good activity (6.25 μg/mL) and
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

932
G. Seelolla and V. Ponneri
Vol 53
another compound 6f showed good activity equivalent to
the standard (12.5 μg/mL). The remaining synthesized
compounds displayed poor activity.
with the standard (ascorbic acid 15.57 0.01) with the
IC50 values of 16.83 0.02, 17.29 0.03, 17.25 0.02,
and 17.53 0.01, respectively (Table 2). The remaining
compounds 6a, 6b, 6d, 6e, 7a, 7b, 7d, and 7e showed
moderate activity. Compounds 6c and 7c displayed
greater NO radical scavenging activity with the IC50
values of 16.81 0.04 and 16.99 0.08, respectively,
compared with standard ascorbic acid (16.61 0.02).
The remaining compounds 6f and 7f showed moderate
activities of 17.26 0.02 and 17.33 0.06, respectively
(Table 3). All the other derivatives were found to
exhibit less activity. The good scavenging effect by
H2O2 was detected in compounds 6c, 6f, 7c, and 7f
Antioxidant testing. Compounds 6a–f and 7a–f were
tested for their antioxidant activity by 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging assay, nitric
oxide (NO), and hydrogen peroxide (H2O2) [24–28]
methods. The obtained results were summarized in
Tables 2–4. All synthesized compounds were tested
for their antioxidant activities compared with the
standard ascorbic acid by DPPH method. Among the
synthesized compounds, 6f, 6c, 7c, and 7f were found
to have moderate antioxidant activities when compared
Table 2
The in vitro antioxidant activity of 6a–f and 7a–f in DPPH method.
Concentration (μg/mL)
Compound
25
50
75
100
IC50
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
60.19 0.84
59.25 0.27
78.28 0.23
62.16 0.99
58.34 0.64
74.24 0.07
60.46 0.10
61.36 0.08
72.46 0.06
62.40 0.30
57.20 0.85
71.28 0.05
80.28 0.03
–
61.39 0.72
60.09 0.93
73.42 0.19
63.10 0.95
59.08 0.96
75.28 0.07
61.09 0.06
62.29 0.07
73.28 0.03
63.09 0.92
59.26 0.08
72.14 0.03
81.16 0.02
–
62.46 0.63
61.18 0.07
74.16 0.14
64.24 0.96
60.19 1.08
71.86 0.03
62.29 0.89
63.46 0.04
74.24 0.06
64.23 0.12
60.16 0.92
73.46 0.01
82.25 0.01
–
63.09 0.94
62.04 0.97
75.24 0.78
65.28 0.82
61.07 0.98
77.38 0.10
63.07 1.06
64.49 1.26
75.29 0.02
65.10 0.90
61.07 1.04
74.29 0.03
83.20 0.05
–
20.76 0.30
21.09 0.93
17.29 0.03
20.10 1.05
21.42 0.33
16.83 0.02
20.67 0.18
20.37 1.03
17.25 0.02
20.03 0.97
21.85 0.47
17.53 0.01
15.57 0.01
–
Ascorbic acid
Blank
(ꢀ), Showed no scavenging activity. Values were the means of three replicates SD.
Table 3
The in vitro antioxidant activity of 6a–f and 7a–f in NO method.
Concentration (μg/mL)
Compound
25
50
75
100
IC50
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
60.38 0.84
60.27 1.02
74.36 0.10
61.42 0.52
60.24 0.86
72.39 0.52
61.27 1.07
60.46 0.46
73.56 0.05
63.46 0.33
60.24 0.86
72.12 0.08
75.22 0.05
–
62.13 0.88
61.21 0.17
75.21 0.09
63.45 0.35
60.78 0.43
74.25 0.13
62.10 0.95
61.68 0.90
74.52 0.04
63.91 0.41
60.78 0.43
7.164 0.06
78.26 0.05
–
64.36 0.34
63.52 0.47
76.45 0.05
64.72 0.43
62.46 1.11
76.19 0.09
64.27 0.19
63.17 0.93
76.48 0.30
64.68 1.08
62.46 1.11
75.44 0.22
80.44 0.02
–
65.19 0.98
65.91 0.21
78.16 0.09
67.17 0.90
63.59 0.54
77.13 0.03
66.19 0.10
64.76 1.07
78.29 0.06
65.27 1.02
63.59 0.54
78.10 0.04
81.26 0.03
–
20.70 0.70
20.74 0.76
16.81 0.04
20.35 0.61
20.75 0.90
17.26 0.02
20.40 0.35
20.67 0.48
16.99 0.08
19.69 0.96
20.75 0.90
17.33 0.06
16.61 0.02
–
Ascorbic acid
Blank
(ꢀ), Showed no scavenging activity. Values were the means of three replicates SD.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2016
Synthesis, Antimicrobial and Antioxidant Activities of Some
[1,2,4]Triazolo[3,4-b][1,3,4]thiadiazole Derivatives
933
Table 4
The in vitro antioxidant activity of 6a–f and 7a–f in H2O2 method.
Concentration (μg/mL)
Compound
25
50
75
100
IC50
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
59.33 0.09
56.44 0.66
73.29 0.02
60.43 0.18
57.14 0.96
72.16 0.06
60.15 0.87
59.19 0.98
72.42 0.04
61.28 0.23
58.32 0.90
74.31 0.03
76.27 0.07
–
62.25 0.87
57.32 0.85
74.16 0.06
61.29 1.07
60.15 0.87
74.19 0.04
62.48 0.92
62.08 0.96
74.62 0.02
64.12 0.93
59.16 0.84
76.18 0.04
78.44 0.04
–
64.37 0.81
59.14 0.96
75.42 0.07
62.34 0.96
62.46 0.26
77.35 0.01
64.62 0.36
64.16 0.05
76.48 0.08
65.26 1.04
60.29 0.21
78.29 0.06
80.32 0.04
–
66.19 0.94
61.56 1.15
76.33 0.24
66.29 0.18
64.59 0.36
80.25 0.03
65.29 0.90
65.29 0.87
78.21 0.02
67.48 0.20
64.16 0.88
80.33 0.02
83.46 0.03
–
21.06 0.98
22.14 0.91
17.05 0.04
20.68 0.48
21.87 1.10
17.32 0.03
20.78 0.55
21.11 0.26
17.26 0.02
20.39 0.35
21.43 0.55
16.82 0.03
16.38 0.02
–
Ascorbic acid
Blank
(ꢀ), Showed no scavenging activity. Values were the means of three replicates SD.
Table 5
Analytical data of synthesized compounds (6a–f and 7a–f).
Elemental analysis
Calculated (%)
H
Found (%)
S.
No
Molecular
formula
Melting point
C
N
C
H
N
Mass M + 1
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
C
19H19N5SO2
183-185
178-180
193-195
181-183
213-215
198-200
164-166
179-181
161-163
163-165
185-187
163-165
59.82
54.87
57.42
60.74
53.51
58.38
57.38
52.32
54.91
58.45
50.94
56.03
5.02
4.36
4.82
5.35
4.25
5.14
3.97
3.36
3.79
4.36
3.27
4.18
18.36
16.84
17.62
17.71
19.71
17.02
19.68
17.94
18.83
18.93
20.97
18.15
59.73
54.80
57.35
60.68
53.46
58.30
57.32
52.27
54.83
58.39
50.88
55.94
5.00
4.33
4.80
5.31
4.22
5.12
3.94
3.34
3.75
4.34
3.25
4.15
18.30
16.78
17.57
17.65
19.65
17.96
19.60
17.86
18.75
18.88
20.91
18.15
382
416
398
396
427
412
356
390
372
370
401
386
C19H18ClN5SO2
C19H19N5SO3
C20H21N5SO2
C19H18N6SO4
C20H21N5SO3
C17H14ClN5S
C17H13Cl2N5S
C17H14ClN5SO
C18H16ClN5S
C17H13ClN6SO2
C18H16ClN5SO
shown as 17.05 0.04, 17.32 0.03, 17.26 0.02, and
16.82 0.03, respectively, (Table 4). Analytical data of
the target compounds also were displayed in Table 5.
Structure activity relationship study helped to reveal the
effect of different substituents on the microbial strains,
depending upon the different electronic environments
developed on the aromatic ring by substituting both
electron-withdrawing and electron-donating groups. It
was observed that compounds 6c (4–OH), 6f (4–OCH3),
7c (4–OH), and 7f (4–OCH3) with electronic-donating
groups executed good antifungal results, displaying much
lesser MIC values than the standard drugs. The remaining
compounds, namely, 6b (4–Cl), 6e (4–NO2), 7b (4–Cl),
and 7e (4–NO2) showed lesser activity compared with the
standard due to the presence of electron-withdrawing
group on the aromatic ring.
CONCLUSION
In the conclusion of this work, a new series of [1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole was synthesized from
simple starting materials, namely, β-amino acids and
para-substituted aromatic acid hydrazides. The newly syn-
thesized compounds were screened for their antimicrobial
and antioxidant activity studies. The investigation of anti-
oxidant activity screening data reveals that, among all com-
pounds, only few of them exhibited good activity.
Compounds 6c, 6f, 7c, and 7f showed an excellent, almost
equivalent to that of the standard, the rest of the com-
pounds showed moderate to mild inhibition activity. The
presence of electron-donating substituent on the phenyl
ring enhances the activity, whereas electron-withdrawing
group decreases the activity.
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G. Seelolla and V. Ponneri
Vol 53
2986 (C–H), 1580 (C¼N), 1252 (N–N¼C), 1062 (N¼C–S–
C¼N), 710 (C–S–C); 1H–NMR (DMSO-d6) δ: 7.69 (d, 2H,
–ArH, J = 8.0 Hz), 6.95 (d, 2H, –ArH, J = 8.0 Hz), 7.34 (s, 1H,
–ArH, J = 8.0 Hz), 6.73 (d, 1H, –ArH, J = 8.0 Hz), 6.45 (dd,
1H, –ArH, J = 4.0 Hz), 8.75 (s, 1H, –OH), 4.45 (s, 2H, –NH2),
4.35 (t, 1H, –CH), 3.48 (s, 6H, –OCH3), 3.10 (d, 2H, –CH2);
13C–NMR (DMSO-d6) δ (ppm): 164.7, 164.2, 157.3, 152.3,
146.2, 144.5, 134.2, 132.1, 126.2, 124.3, 118.4, 112.2, 109.3,
55.6, 55.5, 42.1; MS: m/z 398 (M + H)+ for C19H19N5O3S.
1-(3,4-Dimethoxyphenyl)-2-(3-(p-tolyl)-[1,2,4]triazolo[3,4-b]
EXPERIMENTAL
Chemistry. Melting points were determined in open capillaries
on a Mel-Temp apparatus and are uncorrected. All reactions
were monitored by TLC on precoated silica gel 60 F254 (mesh); spots
were visualized with UV light. Merck (Merck KGaA, Darmstadt,
Germany) silica gel (60–120 mesh) was used for column
chromatography. The IR spectra were recorded on a Perkin Elmer
(Waltham, MA) BX1 FTIR Spectrophotometer using KBr pellets, and
1
the wave numbers were given in cmꢀ1. H NMR (400 MHz) and
13C NMR (100 MHz) spectra were recorded on a Bruker (Bruker
GmbH, Karlsruhe, Germany) AMX 400 MHz NMR spectrometer
in CDCl3/DMSO-d6 solution using TMS as an internal standard.
All chemical shifts were reported in δ (ppm) using TMS as an
internal standard. The mass spectra were recorded on Agilent
1100 LC/MSD instrument (Agilent, Japan) with method API–ES
at 70eV. The microanalyses were performed on a Perkin Elmer
240C elemental analyzer. The antioxidant property was carried
out by using Shimadzu UV-2450s spectrophotometer.
[1,3,4]thiadiazol-6-yl)ethanamine (6d).
Ash color solid in
68%; mp: 181–183°C; IR (KBr, υ cmꢀ1): 3361 (N–H), 2922
(C–H), 1593 (C¼N), 1264 (N–N¼C), 1083 (N¼C–S–C¼N),
715 (C–S–C); 1H–NMR (DMSO-d6) δ: 8.09 (d, 2H, –ArH,
J = 8.0 Hz), 7.78 (d, 2H, –ArH, J = 8.0 Hz), 6.87 (s, 1H,
–ArH, J = 8.0 Hz), 6.36 (d, 1H, –ArH, J = 8.0 Hz), 6.30 (dd,
1H, –ArH, J = 4.0 Hz), 4.96 (s, 2H, –NH2), 4.35 (t, 1H,
–CH), 3.98 (s, 6H, –OCH3), 3.04 (d, 2H, –CH2), 2.45 (s,
3H, –CH3); 13C–NMR (DMSO-d6) δ (ppm): 164.1, 163.8,
150.4, 148.6, 146.2, 135.5, 130.5, 128.9, 126.7, 124.5, 120.6,
116.2, 107.2, 59.1, 55.7, 41.8, 22.6; MS: m/z 396 (M + H)+
for C20H21N5O2S.
General procedure for the synthesis of 1-(3,4-dimethoxyphenyl)-
2-(3-(para-substituted phenyl)-[1,2,4]triazolo[3,4-b] [1,3,4]
thiadiazol-6-yl)ethanamine (6a–f). An equimolar mixture of
3-amino-3-(3,4-dimethoxyphenyl) propanoic acid with 4-amino-5-
phenyl-4H-[1,2,4]triazole-3-thiol (a)/4-amino-5-(4-chlorophenyl)-
4H[1,2,4] triazole-3-thiol (b)/4-(4-amino-5-mercapto-4H-[1,2,4]
1-(3,4-Dimethoxyphenyl)-2-(3-(4-nitrophenyl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-yl)ethanamine (6e). Yellowish brown solid
in 68%; mp: 213–215°C; IR (KBr, υ cmꢀ1): 3362 (N–H), 2933
(C–H), 1598 (C¼N), 1263(N–N¼C), 1072 (N¼C–S–C¼N),
693 (C–S–C); 1H–NMR (DMSO-d6) δ: 8.22 (d, 2H,
–ArH, J =8.0Hz), 8.16 (d, 2H, –ArH, J = 8.0 Hz), 6.97 (s, 1H,
–ArH, J = 8.0 Hz), 6.73 (d, 1H, –ArH, J = 8.0 Hz), 6.46 (dd, 1H,
–ArH, J = 4.0 Hz), 5.15 (s, 2H, –NH2), 4.15 (t, 1H, –CH), 3.46
(s, 6H, –OCH3), 3.15 (d, 2H, –CH2); 13C–NMR (DMSO-d6) δ
(ppm): 162.1, 161.4, 154.8, 149.1, 146.9, 146.1, 133.9, 137.8,
127.5, 124.5, 122.5, 114.6, 110.1, 54.9, 53.6, 40.4; MS: m/z
427 (M + H)+ for C19H18N6O4S.
triazol-3-yl)-phenol
(c)/4-amino-5-p-tolyl-4H-[1,2,4]triazole-3-
thiol (d)/4-amino-5-(4-nitrophenyl)-4H-[1,2,4]-triazole-3-thiol (e)/
4-amino-5-(4-methoxyphenyl)-4H-[1,2,4]-triazole-3-thiol (f) in
POCl3 (7mL) was refluxed over a steam bath for 10–12h. The
progress of reaction was monitored by TLC. The excess POCl3
was removed under reduced pressure, and the residue was poured
in to crushed ice. The resulting precipitate was filtered, washed
with saturated sodium bicarbonate solution and then with water. It
was dried and recrystallized from ethanol.
1-(3,4-Dimethoxyphenyl)-2-(3-(4-methoxyphenyl)-[1,2,4]triazolo
1-(3,4-Dimethoxyphenyl)-2-(3-phenyl-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-6-yl)ethanamine (6a).
[3,4-b][1,3,4]thiadiazol-6-yl)ethanamine (6f).
Orange solid in
74%; mp: 198–200°C; IR (KBr, υ cmꢀ1): 3370 (N–H), 2943
(C–H), 1605 (C¼N), 1252 (N–N¼C), 1138 (N¼C–S–C¼N), 683
Pale yellow solid in
67%; mp: 183–185°C; IR (KBr, υ cmꢀ1): 3350 (N–H), 2937
(C–H), 1579 (C¼N), 1230 (N–N¼C), 1084 (N¼C–S–C¼N), 695
(C–S–C); 1H–NMR (DMSO-d6) δ: 8.12 (d, 2H, –ArH, J=8.0Hz),
7.60 (d, 2H, –ArH, J= 8.0 Hz ), 7.32 (t, 1H, –ArH, J= 8.0 Hz), 6.87
(s, 1H, –ArH, J= 8.0 Hz), 6.63 (d, 1H, –ArH, J= 8.0 Hz), 6.56 (dd,
1H, –ArH, J= 4.0 Hz), 4.75 (s, 2H, –NH2), 4.05 (t, 1H, –CH), 3.78
(s, 6H, –OCH3), 3.09 (d, 2H, –CH2); 13C–NMR (DMSO-d6) δ
(ppm): 164.2, 163.5, 151, 147.3, 144.2, 141., 132.9, 127.4, 127.1,
126.2, 122.9, 115.4, 111.3, 55.9, 55.6, 43; MS: m/z 382 (M + H)+
for C19H19N5O2S.
1
(C–S–C); H–NMR (DMSO-d6) δ: 7.82 (d, 2H, –ArH, J=8.0Hz),
7.36 (d, 2H, –ArH, J=8.0Hz), 7.22 (s, 1H, –ArH, J=8.0Hz), 6.87
(d, 1H, –ArH, J= 8.0 Hz), 6.56 (dd, 1H, –ArH, J=4.0Hz), 4.65
(s, 2H, –NH2), 4.35 (t, 1H, –CH), 3.79 (s, 9H, –OCH3), 3.10
(d, 2H, –CH2); 13C NMR (DMSO-d6) δ (ppm) : 166.6, 160.7,
152.6, 151, 149.1, 145.3, 141.1, 127.4, 127.2, 122.8, 118, 115.8,
114.4, 111.6, 110.1, 55.6, 55.5, 55.3; MS: m/z 412 (M + H)+ for
C20H21N5O3S.
General procedure for the synthesis of 1-(4-chlorophenyl)-2-(3-
(para-substituted phenyl)-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazol-6-yl)
2-(3-(4-Chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-
6-yl)-1-(3,4dimethoxy-phenyl)ethanamine (6b).
Pale brown
ethanamine (7a–f).
An equimolar mixture of 3-amino-3-
solid in 69%; mp: 178–180°C; IR (KBr, υ cmꢀ1): 3365 (N–H),
2996 (C–H), 1590 (C¼N), 1262 (N–N¼C), 1082 (N¼C–S–
C¼N), 683 (C–S–C); 1H–NMR (DMSO-d6) δ: 8.17 (d, 2H,
–ArH, J = 8.0 Hz), 7.52 (d, 2H, –ArH, J = 8.0 Hz), 6.94 (s, 1H,
–ArH, J = 8.0 Hz), 6.61 (d, 1H, –ArH, J = 8.0 Hz), 6.43 (dd, 1H,
–ArH, J = 4.0 Hz), 5.65 (s, 2H, –NH2), 4.35 (t, 1H, –CH), 3.63
(s, 6H, –OCH3), 3.26 (d, 2H, –CH2);13C–NMR (DMSO-d6) δ
(ppm): 167.2, 153.5, 151, 149.1, 144.5, 141.4, 134.9, 129.2,
127.4, 127.2, 124.4, 122.9, 115.6, 55.6, 55.2, 44.2; MS: m/z
416 (M + H)+ for C19H18ClN5O2S.
(4-chlorophenyl) propanoic acid with 4-amino-5-phenyl-
4H-[1,2,4]triazole-3-thiol (a)/4-Amino-5-(4-chlorophenyl)-4H
[1,2,4] triazole-3-thiol (b)/4-(4-amino-5-mercapto-4H-[1,2,4]
triazol-3-yl)-phenol (c)/4-amino-5-p-tolyl-4H-[1,2,4] triazole-3-
thiol (d)/4-amino-5-(4-nitrophenyl)-4H-[1,2,4]-triazole-3-thiol
(e)/4-amino-5-(4-methoxyphenyl)-4H-[1,2,4]-triazole-3-thiol (f)
in POCl3 (7 mL) was refluxed over a steam bath for 10–12 h.
The progress of reaction was monitored by TLC. The
excess POCl3 was removed under reduced pressure, and the
residue was poured on to crushed ice. The resulting
precipitate was filtered, washed with saturated sodium
bicarbonate solution, and then with water. It was dried and
recrystallized from ethanol.
4-(6-(2-Amino-2-(3,4-dimethoxyphenyl)ethyl)-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-3-yl)phenol (6c).
Yellowish brown
solid in 72%; mp: 193–195°C; IR (KBr, υ cmꢀ1): 3345 (N–H),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2016
Synthesis, Antimicrobial and Antioxidant Activities of Some
[1,2,4]Triazolo[3,4-b][1,3,4]thiadiazole Derivatives
935
1-(4-Chlorophenyl)-2-(3-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazol-6-yl)ethanamine (7a). Pale brownish yellow solid
in 69%; mp: 164–166°C; IR (KBr, υ cmꢀ1): 3370 (N–H), 2930
(C–H), 1569 (C¼N), 1230 (N–N¼C), 1090 (N¼C–S–C¼N),
693 (C–S–C); 1H–NMR (DMSO-d6) δ: 8.45 (d, 2H, –ArH,
J = 8.0 Hz), 7.68 (d, 2H, –ArH, J = 8.0 Hz), 7.46 (d, 2H, –ArH,
J = 8.0 Hz), 7.30 (d, 2H, –ArH, J = 8.0 Hz), 7.19 (t, 1H, –ArH,
J = 8.0 Hz), 5.85 (s, 2H, –NH2), 3.90 (t, 1H, –CH), 3.10 (d,
2H, –CH2); 13C–NMR (DMSO-d6) δ (ppm): 165.1, 162.6,
150.8, 141.9, 133.2, 131.6, 130.2, 128.6, 127.4, 126.7, 126.1,
53.8, 40.5; MS: m/z 356 (M + H)+ for C17H14ClN5S.
7.06 (d, 2H, –ArH, J = 8.0 Hz), 4.25 (s, 2H, –NH2), 3.65 (s, 1H,
–CH), 3.40 (s, 3H, –OCH3), 2.95 (d, 2H, –CH2); 13C–NMR
(DMSO-d6) δ (ppm): 162.3, 161.4, 159.5, 150.7, 141.3, 136.4,
133.9, 126.3, 124.5, 120.3 115.4, 55.3, 54.2, 41.8; MS: m/z 386
(M + H)+ for C18H16ClN5OS.
PHARMACOLOGICAL SCREENING
Antioxidant screening (in vitro). Compounds 6a–f and
7a–f are tested for antioxidant property by DPPH, NO,
and H2O2 methods.
1-(4-Chlorophenyl)-2-(3-(4-chlorophenyl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-yl)ethanamine (7b).
Greenish brown
DPPH radical scavenging activity. The hydrogen atom
or electron donation ability of the compounds was
measured from the bleaching of the purple-colored
methanol solution of 1,1-diphenyl-1-picrylhydrazyl. The
spectrophotometric assay uses the stable radical DPPH as
a reagent. About 1 mL of various concentrations of the test
compounds (25, 50, 75, and 100μg/mL) in methanol was
added to 4mL of 0.004% (w/v) methanol solution of DPPH.
After a 30-min incubation period at room temperature, the
absorbance was read against blank at 517 nm. The
percentage of inhibition (I%) of free radical production from
DPPH was calculated by the following equation
solid in 66%; mp:179–181°C; IR (KBr, υ cmꢀ1): 3340 (N–H),
2885 (C–H), 1588 (C¼N), 1238 (N–N¼C), 1087 (N¼C–S–
C¼N), 714 (C–S–C); 1H–NMR (DMSO-d6) δ: 8.24 (d, 2H,
–ArH, J = 8.0 Hz), 7.85 (d, 2H, –ArH, J = 8.0 Hz), 7.70 ( d, 2H,
–ArH, J = 8.0 Hz), 7.52 (d, 2H, –ArH, J = 8.0 Hz), 5.67 (s, 2H,
–NH2), 3.35 (t, 1H, –CH), 2.50 (d, 2H, –CH2); 13C–NMR
(DMSO-d6) δ (ppm): 164.2, 163.1, 150.5, 140.2, 136.2, 134.2,
128.6, 128.4, 127.1, 125.8, 124.6, 56.4, 41.1; MS: m/z 390 (M
+ H)+ for C17H13Cl2N5S.
4-(6-(2-Amino-2-(4-chlorophenyl)ethyl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-3yl) phenol (7c).
Pale yellow solid in
72%; mp: 161–163°C; IR (KBr, υ cmꢀ1): 3300 (O–H), 3354
(N–H), 2882 (C–H), 1551 (C¼N), 1240 (N–N¼C), 1120
1
(N¼C–S–C¼N), 693 (C–S–C); H–NMR (DMSO-d6) δ: 7.52
% of scavenging ¼ ½ðA control – A sampleÞ =
A blankꢁ x 100 →
(1)
(d, 2H, –ArH, J = 8.0 Hz), 7.42 (d, 2H, –ArH, J = 8.0 Hz), 7.29
(d, 2H, –ArH, J = 8.0 Hz), 6.99 (d, 2H, –ArH, J = 8.0 Hz), 6.98
(s, 1H, –OH); 4.76 (s, 2H, –NH2), 3.91 (t, 1H, –CH), 3.14 (d,
2H, –CH2); 13C–NMR (DMSO-d6) δ (ppm):163.2, 161.4,
155.4, 150.2, 142.7, 134.9, 131.5, 129.4, 128.9, 119.4, 117.4,
51.4, 42.5; MS: m/z 372 (M + H)+ for C17H14ClN5OS.
where A control is the absorbance of the control reaction
(containing all reagents except the test compound), and A
sample is the absorbance of the test compound. Tests were
carried at in triplicate.
1-(4-Chlorophenyl)-2-(3-(p-tolyl)-[1,2,4]triazolo[3,4-b][1,3,4]
Nitric oxide scavenging activity.
Nitric oxide
thiadiazol-6-yl)ethanamine (7d).
Pale brown solid in 68%;
mp: 163–165°C; IR (KBr, υ cmꢀ1): 3340 (N–H), 2882 (C–H),
1651 (C¼N), 1294 (N–N¼C), 1088 (N¼C–S–C¼N), 717 (C–
scavenging activity was measured by slightly modified
methods of Green et al. and Marcocci et al. NO radicals
were generated from sodium nitro prusside. About 1 mL
of sodium nitroprusside (10 mM) and 1.5mL of
phosphate buffer saline (0.2 M, pH 7.4) were added to
different concentrations (25, 50, 75, and 100 μg/mL) of
the test compounds and incubated for 150min at 25°C,
and 1 mL of the reaction mixture was treated with 1 mL
of Griess reagent (1% sulfanilamide, 2% H3PO4, and
0.1% naphthyl ethylenediamine dihydrochloride). The
absorbance of the chromatophore was measured at
546 nm. NO scavenging activity was calculated using
Equation (1).
1
S–C); H–NMR (DMSO-d6) δ: 8.16 (d, 2H, –ArH, J = 8.0 Hz),
7.88 (d, 2H, –ArH, J = 8.0 Hz), 7.64 (d, 2H, –ArH, J = 8.0 Hz),
7.42 (d, 2H, –ArH, J = 8.0 Hz), 5.80 (s, 2H, –NH2), 3.48 (t, 1H,
–CH), 3.23(d, 2H, –CH2), 2.42 (s, 3H,–CH3); 13C–NMR
(DMSO-d6) δ (ppm): 161.8, 160.2, 149.4, 137.4, 134.3, 130.7,
127.9, 126.5, 124.2, 122.3, 121.6, 57.5, 42.4, 20.9; MS: m/z
370 (M + H)+ for C18H16ClN5S.
1-(4-Chlorophenyl)-2-(3-(4-nitrophenyl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-yl)ethanamine (7e).
Yellowish brown
solid in 68%; mp: 185–187°C; IR (KBr, υ cmꢀ1): 3338 (N–H),
2883 (C–H), 1607 (C¼N), 1256 (N–N¼C), 1065 (N¼C–S–
C¼N), 691 (C–S–C); 1H–NMR (DMSO-d6) δ: 7.93 (d, 2H,
–ArH, J = 8.0 Hz), 7.86 (d, 2H, –ArH, J = 8.0 Hz), 7.53 (d, 2H,
–ArH, J = 8.0 Hz), 6.73 (d, 2H, –ArH, J = 8.0 Hz), 5.85
(s, 2H,–NH2), 4.10 (s, 1H, –CH), 3.04 (d, 2H, –CH2); 13C–
NMR (DMSO-d6) δ (ppm): 172.4, 171.1, 154.9, 147.1, 143.3,
139.4, 134.9, 128.3, 126.4, 115.7, 114.8, 54.2, 40.8; MS: m/z
401 (M + H)+ for C17H13ClN6O2S.
Hydrogen peroxide scavenging activity.
The H2O2
scavenging ability of the test compound was determined
according to the method of Ruch et al. A solution of
H2O2 (40 mM) was prepared in phosphate buffer
(pH 7.4).
A total of 25, 50, 75, and 100 μg/mL
1-(4-Chlorophenyl)-2-(3-(4-methoxyphenyl)-[1,2,4]triazolo[3,4-
concentrations of the test compounds in 3.4mL
phosphate buffer were added to H2O2 solution (0.6 mL,
40mM). The absorbance value of the reaction mixture
was recorded at 230nm. The percentage of scavenging of
H2O2 was calculated using Equation 1.
b][1,3,4]thiadiazol-6-yl)ethanamine (7f).
Pale yellow solid in
73%; mp:163–165°C; IR (KBr, υ cmꢀ1): 3392 (N–H), 2894
(C–H), 1612 (C¼N), 1252 (N–N¼C), 1089 (N¼C–S–C¼N), 682
(C–S–C); 1H–NMR (DMSO-d6) δ: 8.19 (d, 2H, –ArH, J= 8.0Hz),
7.87 (d, 2H, –ArH, J= 8.0 Hz), 7.52 (d, 2H, –ArH, J= 8.0Hz),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

936
G. Seelolla and V. Ponneri
Vol 53
[13] Sunil, D.; Isloor, A. M.; Shetty, P.; Satyamoorthy, K.; Bharath-
Prasad, A. S. Arab J Chem 2010, 3, 211.
[14] Tozkoparan, B.; Aytac, S. P.; Aktay, G. Arch Pharm 2009,
342, 291.
[15] Ibrahim, D. A. Eur J Med Chem 2009, 44, 2776.
[16] Dawood, K. M.; Farag, A. M.; Abdel-Aziz, H. A. Heteroat
Chem 2005, 16, 621.
Acknowledgments. The author (S. Gangadhara) was grateful to
the Council of Scientific and Industrial Research (CSIR), New
Delhi, for the financial assistance under the Junior Research
Fellow (JRF).
REFERENCES AND NOTES
[17] Okada, Y.; Zasshi, Y. Overview in 45 years of studies on
peptide chemistry [Article in Japanese] 2009, 129, 1141.
[18] Bonmatin, J. M.; Laprevote, O.; Peypoux, F. Comb Chem
High Throughput Screen 2003, 6, 541.
[19] Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.; Aguilar, M. I.
Curr Med Chem 2002, 9, 811.
[1] Eswaran, S.; Adhikari, A. V.; Shetty, N. S. Eur J Med Chem
2009, 44, 4637.
[2] Sahoo, P. K.; Sharma, R.; Pattanayak, P. Med Chem Res 2010,
19, 127.
[3] El-Ashry, S. H.; Kassem, A. A.; Abdel-Hamid, H. M.; Louis, F.;
Khattab, S. A.; Aouad, M. R. Carbohydr Res 2009, 344, 725.
[4] Feng, C. T.; Wang, L. D.; Yan, Y. G.; Liu, J.; Li, S. H. Med
Chem Res 2012, 21, 315.
[5] Mathew, V.; Keshavayya, J.; Vaidya, V. P.; Giles, D. Eur J
Med Chem 2007, 42, 823.
[6] Badr, S. M. I.; Barwa, R. M. Bioorg Med Chem 2011, 19, 4506.
[7] Gilani, S. J.; Khan, S. A.; Siddiqui, N. Bioorg Med Chem Lett
2010, 20, 4762.
[8] Fan, Z. J.; Yang, Z. K.; Zhang, H. K.; Mi, N.; Wang, H.;
Cai, F.; Zuo, X.; Zheng, Q. X.; Song, H. J Agri Food Chem 2010,
58, 2630.
[20] Mathew, V.; Keshavayya, J.; Vaidya, V. P. Eur J Med Chem
2006, 41, 1048.
[21] Chidananda, N.; Poojary, B.; Sumangala, V.; Kumari, N. S.;
Shetty, P.; Arulmoli, T. Eur J Med Chem 2012, 51, 124.
[22] Koneman, E. W.; Allen, S. D.; Winn, W. C.; Colour Atlas
and Textbook of Diagnostic Microbiology, Lippincott Raven Pub:
Philadelphia, 1997; pp 86–856.
[23] NCCLS, Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeasts Approved Standard (2nd Edn.), 2002.
ISBN: 1-56238-469-4 NCCLS document M27-A2.
[24] Burits, M.; Bucar, F. Phytother Res 2000, 14, 323.
[25] Cuendet, M.; Hostettmann, K.; Potterat, O. Helv Chim Acta
1997, 80, 1144.
[9] Almajan, G. L.; Barbuceanu, S. F.; Saramet, I.; Draghici, C.
Eur J Med Chem 2010, 45, 3191.
[10] Almajan, G. L.; Barbuceanu, S. F.; Bancescu, G.; Saramet, I.;
Saramet, G.; Draghici, C. Eur J Med Chem 2010, 45, 6139.
[11] Sangshetti, J. N.; Lokwani, D. K.; Sarkate, A. P.; Shinde, D. B.
Chem Biol Drug Des 2011, 78, 800.
[12] Karegoudar, P.; Prasad, D. J.; Ashok, M.; Mahalinga, M.;
Poojary, B.; Holla, B. S. Eur J Med Chem 2008, 43, 808.
[26] Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.;
Wishnok, J. K. S. R. Anal Biochem 1982, 126, 131.
[27] Marcocci, L.; Maguire, J. J.; Droy-Lefaix, M. T.; Packer, L.
Biochem Biophys Res Commun 1994, 201, 748.
[28] Ruch, R. J.; Cheng, S. J.; Klaunig, J. E. Carcinogenesis 1989,
10, 1003.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet