Synthesis, Antimicrobial, and Antioxidant Screening of Aryl Acetic Acid Incorporated 1,2,4-Triazolo-1,3,4-Thiadiazole Derivatives

Article abstract of DOI:10.1002/jhet.3513

Some novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were synthesized from aryl acetic acids. All the synthesized derivatives were selected for the screening of antibacterial potential against Gram-positive bacteria [Staphylococcus aureus (MTCC 3160) and Micrococcus luteus (MTCC 1538)] and Gram-negative bacteria [Escherichia coli (MTCC 1652) and Pseudomonas aeruginosa (MTCC 424)] and antifungal potential against Aspergillus niger (MTCC 8652) and Candida albicans (MTCC 227), and free radical scavenging activity through 2,2-diphenyl-2-picrylhydrazyl hydrate method. The compounds TH-4, TH-13, and TH-19 were found to be more potent antimicrobial agents compared to standard drugs. The compounds TH-3, TH-9, and TH-18 also showed significant antimicrobial activity. The compound TH-13 showed antioxidant activity with IC₅₀ value better than the standard compound. The structures of all the synthesized compounds were confirmed by Fourier transform infrared, ¹H-NMR, liquid chromatography–mass spectrometry, and CHN analyzer.

Full text of DOI:10.1002/jhet.3513

Month 2019
Synthesis, Antimicrobial, and Antioxidant Screening of Aryl Acetic Acid
Incorporated 1,2,4-Triazolo-1,3,4-Thiadiazole Derivatives
Vipan Kumar Kamboj,a,b
Archana Kapoor,a and Sandeep Jaina
*
aDepartment of Pharmaceutical Sciences, Guru Jambheshwar University of Science & Technology, Hisar125001, India
bDepartment of Pharmaceutical Chemistry, JCDM College of Pharmacy, Sirsa125055, India
*E-mail: vk.kamboj123@gmail.com
Received September 21, 2018
DOI 10.1002/jhet.3513
Published online 00 Month 2019 in Wiley Online Library (wileyonlinelibrary.com).
Some novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were synthesized from aryl acetic acids.
All the synthesized derivatives were selected for the screening of antibacterial potential against Gram-
positive bacteria [Staphylococcus aureus (MTCC 3160) and Micrococcus luteus (MTCC 1538)] and
Gram-negative bacteria [Escherichia coli (MTCC 1652) and Pseudomonas aeruginosa (MTCC 424)] and
antifungal potential against Aspergillus niger (MTCC 8652) and Candida albicans (MTCC 227), and free
radical scavenging activity through 2,2-diphenyl-2-picrylhydrazyl hydrate method. The compounds TH-4,
TH-13, and TH-19 were found to be more potent antimicrobial agents compared to standard drugs. The
compounds TH-3, TH-9, and TH-18 also showed significant antimicrobial activity. The compound TH-
13 showed antioxidant activity with IC50 value better than the standard compound. The structures of all
the synthesized compounds were confirmed by Fourier transform infrared, 1H-NMR, liquid
chromatography–mass spectrometry, and CHN analyzer.
J. Heterocyclic Chem., 00, 00 (2019).
INTRODUCTION
that
the
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
heterocyclic ring has gained much attention during last
few years due to their high utilization as antimicrobial,
anti-inflammatory, antioxidant, anticancer, antianxiety,
sedatives, and anticonvulsant potential.
Based on this concept, we have synthesized [1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole derivatives from aryl
acetic acids with the aim to combine the pharmacological
activities of 1,2,4-triazole and 1,3,4-thiadiazole as fused
heterocyclic moiety. The substitutions on aryl acetic acid
and aromatic carboxylic acids were changed, and the
effect of substitution on antimicrobial and antioxidant
activities was studied.
1,2,4-Triazole nucleus by virtue of their ambident
nucleophilic centers is good starting materials for the
synthesis of several interesting N-bridged, and sulfur
group containing heterocyclic compounds [1,2]. 1,3,4-
Thiadiazole moiety is connected to a broad spectrum of
biological activities possibly due to the presence of
pharmacophoric (N–C=S) moiety [3]. 1,2,4-Triazole or
1,3,4-thiadiazole moieties has been integrated into a
variety of therapeutically important drug candidates
including antimicrobial, anti-inflammatories, anticancer,
antioxidant, sedatives, antianxiety, and anticonvulsant
compounds
[4–9].
Fluconazole,
itraconazole,
voriconazole, triazolam, alprazolam, and etizolam are
triazole containing drugs available in the market [10–13].
Acetazolamide, methazolamide, and megazol are
thiadiazole containing drugs available in the market [7].
Oxidative stress is one of the major causes of various
RESULTS AND DISCUSSION
The 4-amino-5-aryl-4H-1,2,4-triazole-3-thiol (4) was
prepared by exploiting intermediates, methyl arylacetate
pathological
atherosclerosis,
inflammation, cardiovascular diseases, and myocardial
infarction [9,14]. The extensive literature survey reveals
diseases
like
diabetics,
arthritis,
cancer,
chronic
(1),
2-arylacetohydrazide
(2),
and
potassium
rheumatoid
dithiocarbazinate (3). Intermediate (1) was prepared by an
esterification reaction between four different substituted
phenylacetic acids (R1) and methanol. The needle-like
© 2019 Wiley Periodicals, Inc.

V. K. Kamboj, A. Kapoor, and S. Jain
Vol 000
white crystals of (2) were prepared by reacting (1) with
hydrazine hydrate. Potassium dithiocarbazinate (3) was
produced quantitatively by reacting carbon disulfide with
(2) under stirring conditions. The 4-amino-5-aryl-4H-
1,2,4-triazole-3-thiol (4) was obtained by reflexing
potassium salt (3) with hydrazine hydrate. The unique
Furthermore, the structure of final compounds was
supported by H-NMR. The absence of signals of NH2
1
and SH protons in 1H-NMR spectra of [1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (TH1–20) confirms the
synthesis of final compounds. The characteristic signals
at δ 7.0–8.0 ppm were observed for (ArH) aromatic rings.
The –CH2 of R1 linker showed a signal at δ 4.0–5.0 ppm,
whereas for alkyl chain on R2, –CH2 group showed
signals in the region of δ 1.1–4.1 ppm and –CH3 groups
observed at δ 0.8–0.9 ppm. LC–MS and elemental
analysis also supported the structures of final compounds.
In vitro antimicrobial activity. The synthesized [1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole compounds (TH1–20)
were evaluated for their in vitro antibacterial potential
against Gram-positive (Staphylococcus aureus and
Micrococcus luteus) and Gram-negative (Escherichia coli
and Pseudomonas aeruginosa) and antifungal activity
against Candida albicans and Aspergillus niger by cup
plate and serial dilution method. Ciprofloxacin
(antibacterial) and fluconazole (antifungal) were used as
standards drugs, and results are summarized in Table 2.
The results of antimicrobial activity by serial dilution
followed the same pattern of activity as shown by the cup
plate method. Antimicrobial activity by cup plate method
and pMIC indicated that the compounds TH-4 and TH-
19 were more potent than standard drug against
S. aureus; TH-3 and TH-13 were most active against
M. luteus. TH-13 showed excellent antibacterial activity
than the standard drug against E. coli. The compound
TH-18 exhibited significant antimicrobial activity against
P. aeruginosa, and values were comparable to standards
drug. Among fungal strains, compounds TH-3 and TH-9
were found to be the most potent against A. niger, and
TH-13 showed moderate potency against C. albicans.
series
of
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
derivatives (TH1–20; Scheme 1) were prepared by
reacting (4) with different aliphatic/aromatic carboxylic
acids (R2) in presence of phosphorus oxychloride under
reflux conditions. The progress of reaction and purity of
compounds were confirmed on every step by thin-layer
chromatography (TLC), and all the compounds were
obtained in good yield. The physicochemical
characteristics of synthesized compounds are presented in
Table 1. The structures of all the newly synthesized
compounds were confirmed by Fourier transform
infrared,
1H-NMR,
liquid
chromatography–mass
spectrometry (LC–MS), and CHN analyzer, which were
in full agreement with their structures.
The IR spectra of synthesized compounds (TH1–20)
exhibit the absorption bands for aromatic vibrations in
the regions of 3100–3000 cmꢀ1, C=N 1690–1600 cmꢀ1
,
and C–S–C 704–680 cmꢀ1, respectively. The absence of
strong stretching bands of NH/NH2 between 3291 and
3239 cmꢀ1 and the presence of C–S–C stretching peaks
confirmed
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
formation. Besides the earlier absorption peaks, intense
absorption peaks for C–Cl in the region of 760–
720 cmꢀ1 and for –NO2 functional group, two peaks
observed between 1570–1490 cmꢀ1 and 1355–
1315 cmꢀ1 for respective compounds. An aliphatic chain
containing compounds TH-4, TH-9, TH-14, and TH-19
showed absorption bands between 1450 and 1370 cmꢀ1
for –CH3 and –CH2 peaks between 1470 and 1465 cmꢀ1
,
Free radical scavenging activity.
Free radical
respectively. Fluoro group containing compounds TH-5,
TH-10, TH-15, and TH-20 showed peaks for C–F in the
scavenging activity of synthesized [1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole derivatives was checked by 2,2-
diphenyl-2-picrylhydrazyl hydrate (DPPH) method and
results are summarized in Table 3. Overall, 50%
inhibition concentration activity (IC50) of screened
region of 1100–1000 cmꢀ1
.
compounds ranging between 8.1
239.6 0.361 μg/mL. The compound TH-13 was more
potent with the IC50 value of 8.1 0.325 μg/mL than
0.325 to
Scheme 1. Synthesis of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles.
standard ascorbic acid with IC50 value 36.3 0.21 μg/
mL, respectively. The compound TH-18 showed least
free radical scavenging activity with IC50 value
239.6 0.36 μg/mL, whereas compounds TH-4, TH-6,
and TH-15 showed moderate IC50 value 40.5
0.274,
47.6 0.431, and 43.4 0.338 μg/mL, respectively.
Conclusion. In order to develop new antimicrobial and
antioxidant compounds, we have successfully synthesized
and reported a series of [1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole derivatives from different substituted aryl
acetic acids. The compounds 3-(4-chlorobenzyl)-6-nonyl-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis, Antimicrobial, and Antioxidant Screening of 1,2,4-Triazolo-1,3,4-
Thiadiazole Derivatives
Table 1
Substitution arrangement and physicochemical properties of final derivatives.
Compound
R1
R2
Rf value
% age yield
TH-1
TH-2
TH-3
TH-4
TH-5
TH-6
TH-7
TH-8
C6H5
C6H5
C6H5
C6H5
2-Cl-5-NO2C6H3
(C6H5)2CH
2,4-Cl2C6H3CH2
n-C9H19
4-F-C6H4
2-Cl-5-NO2C6H3
(C6H5)2CH
2,4-Cl2C6H3CH2
n-C9H19
4-F-C6H4
2-Cl-5-NO2C6H3
(C6H5)2CH
2,4-Cl2C6H3CH2
n-C9H19
4-F-C6H4
2-Cl-5-NO2C6H3
(C6H5)2CH
0.89a
0.90a
0.82a
0.89a
0.75a
0.89a
0.90a
0.87a
0.92a
0.94a
0.88a
0.91b
0.83b
0.90b
0.94a
0.89b
0.92a
0.79a
0.78a
0.88a
75.1
81.4
69.3
76.0
71.2
71.4
77.3
83.0
75.0
69.0
80.0
83.0
79.0
72.0
81.0
61.0
64.0
77.0
80.0
68.3
C6H5
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
2,4-Cl2C6H3
2,4-Cl2C6H3
2,4-Cl2C6H3
2,4-Cl2C6H3
2,4-Cl2C6H3
TH-9
TH-10
TH-11
TH-12
TH-13
TH-14
TH-15
TH-16
TH-17
TH-18
TH-19
TH-20
2,4-Cl2C6H3CH2
n-C9H19
4-F-C6H4
TLC solvent system.
aCHCl3: Methanol (7:3).
bToluene: Ethylacetate: Formic acid (5:4:1).
Table 2
pMIC in μmol/mL and zone of inhibition (mm) of [1,2,4]triazolo[3,4-b] [1,3,4]thiadiazole derivatives.
Staphylococcus
aureus
Micrococcus
luteus
Escherichia
coli
Pseudomonas
aeruginosa
Aspergillus
niger
Candida
albicans
Compound
TH-1
TH-2
TH-3
TH-4
TH-5
TH-6
TH-7
TH-8
2.07 (16)
2.08 (17)
2.38 (18)
2.64 (20)
1.39 (12)
1.80 (13)
2.42 (18)
2.11 (17)
2.38 (18)
1.43 (12)
2.41 (19)
1.82 (14)
2.11 (17)
1.78 (13)
1.44 (12)
1.54 (11)
2.46 (15)
2.15 (16)
2.72 (19)
1.18 (9)
1.47 (9)
2.07 (19)
1.18 (11.5)
1.47 (10)
2.03 (18)
-
1.47 (11)
1.47 (10.5)
1.48 (11.5)
2.07 (15.5)
1.73 (12)
1.09 (9)
1.80 (11)
1.21 (9)
1.81 (13)
2.08 (15.5)
1.43 (11)
1.21 (9)
1.52 (11)
1.81 (12)
1.47 (11)
1.74 (12.5)
1.54 (10.5)
1.55 (10)
1.55 (10.5)
1.21 (9)
1.47 (9)
1.48 (9)
1.47 (9)
-
1.69 (10)
-
-
-
-
1.77 (13)
-
-
1.73 (12)
-
1.50 (9)
1.21 (9)
-
1.50 (11)
1.51 (11)
1.51 (12)
2.08 (17)
1.43 (10)
-
2.12 (15)
2.72 (24)
1.47 (10)
1.44 (12)
-
1.55 (12)
-
1.21 (9)
2.08 (15)
2.32 (24)a
-
-
-
-
1.51 (10)
TH-9
1.47 (9)
-
1.17 (9)
1.13 (8.5)
-
TH-10
TH-11
TH-12
TH-13
TH-14
TH-15
TH-16
TH-17
TH-18
TH-19
TH-20
Standard
-
-
-
1.43 (10)
-
-
1.22 (8.5)
1.89 (12)
1.78 (10)
1.13 (8.5)
1.54 (9)
1.25 (8.5)
1.55 (9)
-
1.81 (13.5)
1.17 (9)
-
1.91 (15)
-
-
-
-
-
1.55 (8.5)
1.24 (8)
1.20 (8.5)
1.78 (12)
2.32 (20)a
2.15 (15)
1.51 (11)
1.78 (14)
2.32 (25)a
1.18 (9)
1.18 (8)
2.32 (27)a
2.29 (17)b
1.99 (16)b
aCiprofloxacin.
bFluconazole.
Most active to moderately active compounds in series are highlighted in bold.
standard drug against S. aureus which may be attributed
to the presence of long alkyl chain (n-C9H19) at R2
position. The fact is further supported by the higher
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
dichlorobenzyl)-6-nonyl-[1,2,4]triazolo
thiadiazole exhibited higher antibacterial potential than
and
[3,4-b][1,3,4]
3-(2,4-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

V. K. Kamboj, A. Kapoor, and S. Jain
Vol 000
Table 3
5–6 h at 60–70°C, and completion of the reaction was
checked by silica gel G coated TLC plates using ethyl
acetate and hexane (3:2) as an eluent, observed under
iodine chamber, evaporated the excess of methanol, and
extracted the methyl ester with the help of separating
funnel, and washed with a strong solution of potassium
hydrogen carbonate until all free acid was neutralized and
washed with water, dried, and stored in airtight container.
Results of DPPH free radical scavenging activity of [1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole derivatives.
IC50 SD
IC50 SD
(μg/mL)
Compound
(μg/mL)
Compound
TH-1
TH-2
TH-3
TH-4
TH-5
TH-6
TH-7
TH-8
68.1 0.342
78.1 0.621
53.2 0.812
40.5 0.274
66.2 0.601
47.6 0.431
55.6 0.517
139 0.457
60.4 0.551
60.1 0.363
36.3 0.21
TH-11
TH-12
TH-13
TH-14
TH-15
TH-16
TH-17
TH-18
TH-19
TH-20
Blank
65.3 0.448
60.8 0.387
8.1 0.325
68.2 0.453
43.4 0.338
54.2 0.491
70.2 0.427
239.6 0.361
89.1 0.229
85.1 0.327
--
General
procedure
for
the
synthesis
of
2-arylacetohydrazide (2).
A mixture of methyl aryl
acetate (0.1 mol) and hydrazine hydrate (0.116 mol) was
refluxed with 25 mL of absolute ethanol for 6–7 h, and
reaction completion was checked by TLC plates. The
excess of ethanol was distilled off; the reaction mixture
was cooled and kept overnight. Then, aryl acetohydrazide
was recrystallized with ethanol and stored [15].
TH-9
TH-10
Ascorbic acid
Most active to moderately active compounds in series are highlighted in
bold.
General procedure for the synthesis of potassium
dithiocarbazinate (3).
A
solution of potassium
antifungal activity of the compound 3-(4-methoxybenzyl)-
hydroxide (0.0067 mol) in absolute ethanol (30 mL) was
taken in a beaker; aryl acetohydrazide (0.003 mol) and
carbon disulfide (0.006 mol) were added dropwise along
with stirring, and the mixture was agitated for 16 h. To
the resulting solution, anhydrous ether was added, and
the precipitated potassium dithiocarbazinate was collected
by filtration, washed with diethyl ether, and dried. The
potassium salt was obtained in quantitative yield and was
used in the next step without further purification [16].
6-nonyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
against
A. niger with the pMIC value of 2.08 μmol/mL
evidenced by the presence of the alkyl chain at R2
position (n-C9H19). The presence of Di-substituted chloro
group on R2 aromatic ring and chloro substitution on R1
aromatic ring of compound 6-(2,4-dichlorobenzyl)-3-(4-
chlorobenzyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole showed
excellent antioxidant activity as well as the broad
spectrum of antimicrobial activity and hence can be
selected as lead compound to discover a broad-spectrum
antimicrobial drug candidate with antioxidant properties.
General procedure for the synthesis of 4-amino-5-aryl-4H-
1,2,4-triazole-3-thiol (4).
The earlier potassium salt,
hydrazine hydrate (1.5 mL), and water (1.0 mL) were
heated under reflux for 5 h; hydrogen sulfide gas
evolved, and homogeneous solution was obtained, which
was diluted with 50 mL of water, and subsequent
acidification with dilute acetic acid gives white
precipitate. The precipitates were washed with water and
recrystallized from methanol. The purity was checked by
silica gel G coated TLC plates using toluene: ethyl
acetate: formic acid (5:4:1) as a solvent system [16].
EXPERIMENTAL
Chemicals employed for the synthetic work were
purchased from S.D. Fine Chem Laboratories, Hi-Media
Laboratories, and Sigma Aldrich as “synthesis grade” and
were used without further purification. The reaction
progress was determined by open capillary tubes on
digital auto melting point apparatus. The reaction progress
was monitored by TLC on silica gel-G coated plates and
spot detected under UV light and iodine chamber. Proton
nuclear magnetic resonance (1H-NMR) spectra were
recorded on Bruker Avance II 400 NMR spectrometer
using deuterated CDCl3 as a solvent and were expressed
in parts per million (δ, ppm) downfield from
tetramethylsilane (internal standard). Infrared (IR) spectra
were recorded on Perkin Elmer, BX II spectrophotometer
using KBr pellets. LC–MS were recorded on ABI Sciex
5800 TOF/TOF System with LC-MALDI. Elemental
analysis was performed on Carlo Erba 1106 CHN analyzer.
General procedure for the synthesis of 3-aryl-6-aryl/
alkyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (TH1–20).
An
equimolar mixture of 4-amino-5-aryl-4H-1,2,4-triazole-3-
thiol (0.1 mol) and appropriate aromatic/aliphatic acids in
10 mL of phosphorus oxychloride were refluxed for 5 h.
The reaction mixture was cooled to room temperature
and poured onto crushed ice with continuous stirring. The
mixture was allowed to stand overnight, and the solid
separated out was filtered, treated with dilute sodium
hydroxide solution, and washed thoroughly with cold
water. The compound was dried and recrystallized from
methanol [2].
3-Benzyl-6-(2-chloro-5-nitrophenyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (TH-1). Yield 75.1%; mp: 138–140°C,
General procedure for the synthesis of methyl aryl acetate
(1). A mixture of aryl acetic acid (0.1 mol), methanol
(1 mol), and 1.1 mL of H2SO4 (0.1 mol) was refluxed for
IR (KBr, cmꢀ1): 3060 (C-H str. Ar), 1609 (C=N str),
1578 (C=C str, Ar), 1534, 1345 (C-NO2 str), 739 (C-Cl
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis, Antimicrobial, and Antioxidant Screening of 1,2,4-Triazolo-1,3,4-
Thiadiazole Derivatives
1
1
str), 692 (C-S-C str); H-NMR (CDCl3) δ (ppm): 4.5 (s,
2H, CH2), 7.2–7.4 (m, 5H, ArH), 7.6–7.8 (m, 3H, ArH);
MS (m/z): 371.9933 (M+), 373.9939 (M + 2); Anal.
Calcd for C16H10ClN5O2S (371.80): C, 51.69; H, 2.71;
N, 18.84. Found: C, 51.64; H, 2.68; N, 18.80.
str); H-NMR (CDCl3) δ (ppm): 3.7 (d, 3H, CH3), 4.45
(s, 2H, CH2), 5.0 (s, 1H, CH), 7.2–7.4 (m, 10H, ArH),
7.49–7.56 (m, 4H, ArH); MS (m/z): 412.9383 (M+),
414.8918 (M + 2); Anal. Calcd for C24H20N4OS
(412.50): C, 69.88; H, 4.89; N, 13.58; O, 3.88. Found: C,
69.82; H, 4.85; N, 13.63; O, 3.93.
6-Benzhydryl-3-benzyl-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (TH-2).
Yield 81.4%; mp: 140–142°C; IR
6-(2,4-Dichlorobenzyl)-3-(4-methoxybenzyl)-[1,2,4]
(KBr, cmꢀ1): 3019 (C-H str, Ar), 1629 (C=N str) 1598
triazolo[3,4-b][1,3,4]thiadiazole (TH-8).
Yield 83%; mp:
1
(C=C str, Ar), 1511 (C-N str), 695 (C-S-C str); H-NMR
168–170°C; IR (KBr, cmꢀ1): 3064, 3002 (C-H str, Ar),
(CDCl3) δ (ppm): 4.6 (s, 2H, CH2), 5.8 (s, 1H, CH), 7.2–
7.5 (s, 15H, ArH); MS (m/z): 382.9834 (M+); Anal.
Calcd for C23H18N4S (382.48): C, 72.22; H, 4.74; N,
14.65. Found: C, 72.18, H, 4.76, N, 14.61.
1611 (C=N str), 1585 (C=C str, Ar), 1255, 1036 (C-O-C
str), 739 (C-Cl str), 684 (C-S-C str); H-NMR (CDCl3) δ
1
(ppm): 2.9–3.0 (s, 2H, CH2), 3.7 (s, 3H, CH3), 4.3–4.5
(d, 2H, CH2), 6.8 (m, 3H, ArH), 7.2–7.3 (m, 4H, ArH);
MS (m/z): 404.9604 (M+); Anal. Calcd for
C18H14Cl2N4OS (405.30): C, 53.34; H, 3.48; N, 13.82;
O, 3.95. Found: C, 53.29; H, 3.43; N, 13.87; O, 3.99.
3-(4-Methoxybenzyl)-6-nonyl-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (TH-9). Yield 75%; mp: 56–58°C; IR (KBr,
6-(2,4-Dichlorobenzyl)-3-benzyl-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (TH-3).
Yield 69.3%; mp: 162–164°C; IR
(KBr, cmꢀ1): 3020 (C-H str, Ar), 1618 (C=N str), 1576
1
(C=C str, Ar), 733 (C-Cl str), 704 (C-S-C str); H-NMR
(CDCl3) δ (ppm): 3.6 (s, 2H, CH2), 4.1–4.3 (t, 2H,
CH2), 6.9–7.1 (m, 5H, ArH), 7.2 (m, 3H, ArH); MS
(m/z): 374.9303 (M+). Anal. Calcd for C17H12Cl2N4S
(375.27): C, 54.41; H, 3.22; N, 14. Found: C, 54.45;
cmꢀ1): 3002 (C-H str, Ar), 1611 (C=N str), 1580 (C=C
str, Ar), 1465 (C-H bend, CH2), 1375 (C-H bend,
CH3), 1251, 1031 (C-O-C str), 692 (C-S-C str); 1H-
NMR (CDCl3) δ (ppm): 0.86–0.89 (d, 3H, CH3), 1.2
(s, 2H, CH2), 1.3–1.4 (t, 4H, CH2), 1.6 (t, 2H, CH2),
1.7–1.8 (t, 2H, CH2), 2.1 (s, 2H, CH2), 2.3 (t, 2H,
CH2), 2.9 (t, 2H, CH2), 3.77–3.83 (q, 3H, CH3), 4.3 (s,
2H, CH2), 7.2–7.3 (m, 4H, ArH); MS (m/z): 372.9270
(M+); Anal. Calcd for C20H28N4OS (372.52): C, 64.48;
H, 7.58; N, 15.04; O, 4.29. Found: C, 64.44; H, 7.62;
H, 3.20; N, 14.90.
3-Benzyl-6-nonyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
(TH-4). Yield 76%; mp: 58–60°C; IR (KBr, cmꢀ1): 3025
(C-H str, Ar), 1655 (C=N str), 1646 (C-N str), 1560 (C=C
band, Ar), 1465 (C-H bend, CH2), 1375 (C-H bend, CH3),
1
696 (C-S-C str); H-NMR (CDCl3) δ (ppm): 0.8–0.9 (t,
3H, CH3), 1.27–1.40 (t, 8H, CH2), 1.8 (t, 2H, CH2), 2.17
(s, 2H, CH2), 3.0 (s, 2H, CH2), 4.1 (s, 2H, CH2), 4.5 (s,
2H, CH2), 7.2–7.4 (m, 5H, ArH); MS (m/z): 342.9183 (M
+); Anal. Calcd for C19H26N4S (342.50): C, 66.63; H,
7.65; N, 16.36. Found: C, 66.60; H, 7.61; N, 16.41.
3-Benzyl-6-(4-fluorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]
N, 15.09; O, 4.33.
3-(4-Methoxybenzyl)-6-(4-fluorophenyl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole (TH-10). Yield 69%; mp: 130–132°C,
IR (KBr, cmꢀ1): 3014 (C-H str, Ar), 1604 (C=N str), 1546
(C=C str, Ar), 1515 (C-N str), 1251, 1032 (C-O-C str),
1104 (C-F str), 692 (C-S-C str); 1H-NMR (CDCl3) δ
(ppm): 3.77 (s, 3H, CH3), 4.49 (s, 2H, CH2), 6.8 (m, 4H,
ArH), 7.1–7.2 (m, 4H, ArH); MS (m/z): 340.9981 (M+);
Anal. Calcd for C17H13FN4OS (340.37): C, 59.99; H,
3.85; N, 16.46; O, 4.70. Found: C, 59.94; H, 3.82; N,
thiadiazole (TH-5).
Yield 71.2%; mp: 164–166°C; IR
(KBr, cmꢀ1): 3081 (C-H str, Ar), 1656 (C=N band),
1
1562 (C=C str, Ar), 1528 (C-N str), 693 (C-S-C str); H-
NMR (CDCl3) δ (ppm): 4.4–4.5 (d, 2H, CH2), 7.4–7.3
(m, 5H, ArH), 7.4 (m, 4H, ArH); MS (m/z): 309.9961 (M
+), 311.9922 (M + 2); Anal. Calcd for C16H11FN4S
(310.34): C, 61.92; H, 3.57; N, 18.05. Found: C, 61.87;
16.49; O, 4.73.
3-(4-Chlorobenzyl)-6-(2-chloro-5-nitrophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (TH-11). Yield 80%; mp:
H, 3.55; N, 18.11.
142–144°C; IR (KBr, cmꢀ1): 3067 (C-H str, Ar), 1608
3-(4-Methoxybenzyl)-6-(2-chloro-5-nitrophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (TH-6). Yield 71.4%; mp:
(C=N str), 1568, 1344 (-NO2 str), 1525 (C-N str), 742
164–166°C; IR (KBr, cmꢀ1): 3065 (C-H str, Ar), 1608
(C-Cl str), 689 (C-S-C str); H-NMR (CDCl3) δ (ppm):
1
(C=N band), 1569, 1343 (-NO2 str), 1243, 1031 (C-O-C
str), 744 (C-Cl str), 695 (C-S-C str); H-NMR (CDCl3) δ
(ppm): 3.78 (s, 3H, CH3), 4.5 (s, 2H, CH2), 6.88–6.90
(m, 3H, ArH), 7.2–7.4 (m, 4H, ArH); MS (m/z):
401.9901 (M+); Anal. Calcd for C17H12ClN5O3S
(401.82): C, 50.81; H, 3.01; N, 17.43; O, 11.94. Found:
4.5 (d, 2H, CH2), 7.3 (m, 3H, ArH), 7.4 (m, 4H, ArH);
MS (m/z): 406.9921 (M+); Anal. Calcd for
C16H9Cl2N5O2S (406.24): C, 47.30; H, 2.23; N, 17.24;
1
O, 7.88. Found: C, 47.34; H, 2.21; N, 17.19; O, 7.91.
3-(4-Chlorobenzyl)-6-benzhydryl-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (TH-12). Yield 83%; mp: 64–66°C; IR (KBr,
cmꢀ1): 3058, 3025 (C-H str, Ar), 1615 (C=N str), 1580
(C=C str, Ar), 1516 (C-N str), 747 (C-Cl str), 699 (C-S
C, 50.78; H, 2.97; N, 17.49; O, 11.90.
3-(4-Methoxybenzyl)-6-benzhydryl-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (TH-7). Yield 77.3%; mp: 128–130°C;
1
str); H-NMR (CDCl3) δ (ppm): 4.6 (d, 2H, CH2), 5.7
IR (KBr, cmꢀ1): 3028 (C-H str, Ar), 1610 (C=N str),
1590 (C=C str, Ar), 1249, 1030 (C-O-C str), 698 (C-S-C
(s, 1H, CH), 7.1–7.4 (m, 14H, ArH); MS (m/z):
418.9903 (M + 2); Anal. Calcd for C23H17ClN4S
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

V. K. Kamboj, A. Kapoor, and S. Jain
Vol 000
(416.92): C, 66.26; H, 4.11; N. 13.44. Found: C, 66.21;
MS (m/z): 443.9751 (M+); Anal. Calcd for
H, 4.14; N, 13.41.
C17H10Cl4N4S (444.16): C, 45.97; H, 2.27; N, 12.61.
6-(2,4-Dichlorobenzyl)-3-(4-chlorobenzyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (TH-13). Yield 79%; mp:
Found: C, 45.94; H, 2.30; N, 12.57.
3-(2,4-Dichlorobenzyl)-6-nonyl-[1,2,4]triazolo[3,4-b][1,3,4]
84–86°C; IR (KBr, cmꢀ1): 3070, 3025 (C-H str, Ar),
thiadiazole (TH-19).
Yield 80%; mp: 134–136°C; IR
(KBr, cmꢀ1): 3010 (C-H str, Ar), 1660 (C=N str), 1582
(C=C str, Ar), 1522 (C-N str), 1470 (C-H bend, CH2),
1385 (C-H bend, CH3), 721 (C-Cl str), 695 (C-S-C str);
1H-NMR (CDCl3) δ (ppm): 0.8–0.9 (t, 3H, CH3), 1.2–1.4
(t, 8H, CH2), 1.81 (s, 2H, CH2), 2.17 (s, 2H, CH2), 2.99–
3.0 (s, 2H, CH2), 3.4 (s, 2H, CH2), 4.6 (s, 2H, CH2),
7.23–7.40 (m, 3H, ArH); MS (m/z): 412.9015 (M + 2);
Anal. Calcd for C19H24Cl2N4S (411.39): C, 55.47; H,
1610 (C=N str), 1584 (C=C str, Ar), 759 (C-Cl str), 685
(C-S-C str); H-NMR (CDCl3) δ (ppm): 3.6 (s, 2H, CH2),
4.1–4.3 (t, 2H, CH2), 6.9–7.1 (m, 4H, ArH), 7.2 (m, 3H,
ArH); MS (m/z): 407.9929 (M+); Anal. Calcd for
C17H11Cl3N4S (409.72): C, 49.83; H, 2.71; N, 13.67.
Found: C, 49.80; H, 2.73; N, 13.64.
1
3-(4-Chlorobenzyl)-6-nonyl-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (TH-14).
Yield 72%; mp: 110–112°C; IR
(KBr, cmꢀ1): 3010 (C-H str, Ar), 1610 (C=N str), 1580
(C=C str, Ar), 1517 (C-N str), 1467 (C-H bend, CH2),
5.88; N, 13.62. Found: C, 55.44; H, 5.86; N, 13.65.
3-(2,4-Dichlorobenzyl)-6-(4-fluorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (TH-20). Yield 68.3%; mp:
1
1376 (C-H bend, CH3), 721 (C-Cl str), 697 (C-S str); H-
124–126°C; IR (KBr, cmꢀ1): 3114 (C-H str, Ar), 1600
(C=N str), 1520 (C-N str), 1043 (C-F str), 693 (C-S str);
1H-NMR (CDCl3) δ (ppm): 4.5–4.6 (t, 2H, CH2), 7.3 (m,
4H, ArH), 7.2 (m, 3H, ArH); MS (m/z): 378.9764 (M+);
Anal. Calcd for C16H9Cl2FN4S (379.23): C, 50.67; H,
2.39; N; 14.77. Found: C, 50.62; H, 2.36; N, 14.79.
NMR (CDCl3) δ (ppm): 0.8–0.9 (q, 3H, CH3), 1.2–1.4 (t,
2H, CH2), 1.6 (q, 2H, CH2), 1.8 (t, 2H, CH2), 2.1 (s, 2H,
CH2), 2.3 (s, 2H, CH2), 2.99 (s, 2H, CH2), 3.49 (s, 2H,
CH2), 4.5 (s, 2H, CH2), 7.2–7.3 (m, 4H, ArH); MS (m/z):
376.9735 (M+); Anal. Calcd for C19H25ClN4S (376.94):
C, 60.54; H, 6.68; N, 14.86. Found: C, 60.51; H, 6.71;
N, 14.83.
Biological
1,2,4-triazolo-1,3,4-thiadiazole derivatives: Evaluation of
antimicrobial activity.
performed by cup plate and serial dilution methods
against Gram-positive bacteria [S. aureus (MTCC 3160)
and M. luteus (MTCC 1538)] and Gram-negative bacteria
[E. coli (MTCC 1652) and P. aeruginosa (MTCC 424)]
and antifungal activity against A. niger (MTCC 8652)
and C. albicans (MTCC 227) [15,17].
evaluation
of
synthesized
3-(4-Chlorobenzyl)-6-(4-fluorophenyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (TH-15). Yield 81%; mp: 158–160°C;
The antibacterial activity was
IR (KBr, cmꢀ1): 3042 (C-H str, Ar), 1602 (C=N str),
1523 (C-N str), 1092 (C-F str), 758 (C-Cl str), 686 (C-S
1
str); H-NMR (CDCl3) δ (ppm): 4.4–4.5 (d, 2H, CH2),
7.4–7.3 (m, 4H, ArH), 7.4 (m, 4H, ArH); MS (m/z):
344.9116 (M+); Anal. Calcd for C16H10ClFN4S (344.79):
C, 55.74; H, 2.92; N; 16.25. Found: C, 55.70; H, 2.87;
N, 16.29.
Cup-plate method.
The 10 μg/mL of dilutions of
3-(2,4-Dichlorobenzyl)-6-(2-chloro-5-nitrophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (TH-16). Yield 61%; mp:
standard drugs (ciprofloxacin and fluconazole) and test
compounds were prepared from 100 μg/mL of stock
solutions and used for cup plate method. After the
incubation period, the diameter of the zone of inhibition
in plates was recorded.
142–144°C; IR (KBr, cmꢀ1): 3067 (C-H str, Ar), 1602
(C=N str), 1566, 1350 (-NO2 str), 1525 (C-N str), 743
(C-Cl str), 684 (C-S-C str); H-NMR (CDCl3) δ (ppm):
4.6–4.7 (d, 2H, CH2), 7.2–7.3 (m, 3H, ArH), 7.4 (m, 3H,
ArH); MS (m/z): 438.9981 (M+). Anal. Calcd for
C16H8Cl3N5O2S (440.69): C, 43.61; H, 1.83; N, 15.89.
1
Serial dilution method.
On the confirmation of the
antimicrobial activity of compounds by the cup-plate
method, serial dilution method was performed. Different
seven concentrations (50, 25, 12.5, 6.25, 3.12, 1.56, and
0.78 μg/mL) of each test compound and standard drugs
were prepared by serial dilution method and used for the
antimicrobial activity. The minimum inhibitory
concentration was observed by the turbidity of assay
tubes, and pMIC values were calculated by the following
formula:
Found: C, 43.65; H, 1.86; N, 15.85.
3-(2,4-Dichlorobenzyl)-6-benzhydryl-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (TH-17). Yield 64%; mp: 60–62°C; IR
(KBr, cmꢀ1): 3058, 3028 (C-H str, Ar), 1586 (C=N str),
1
1514 (C-N str), 745 (C-Cl str), 699 (C-S str); H-NMR
(CDCl3) δ (ppm): 4.4–4.6 (t, 2H, CH2), 5.7 (d, 2H, CH2),
6.99–7.5 (m, 13H, ArH); MS (m/z): 452.9912(M + 2);
Anal. Calcd for C23H16Cl2N4S (451.37): C, 61.20; H,
3.57; N, 12.41. Found: C, 61.25; H, 3.53; N; 12.39.
3,6-Bis(2,4-dichlorobenzyl)-[1,2,4]triazolo[3,4-b][1,3,4]
ꢀ
ꢁ
MIC
pMIC ¼ ꢀ log
(1)
Molecular weight
thiadiazole (TH-18).
Yield 77%; mp: 138–142°C; IR
(KBr, cmꢀ1): 3010 (C-H str, Ar), 1653 (C=N str), 1586
(C=C str, Ar), 1512 (C-N str), 758 (C-Cl str), 676 (C-S-C
Free radical scavenging activity.
Free radical
scavenging activity of synthesized derivatives was
screened by using 2,2-diphenyl-2-picrylhydrazyl hydrate
(DPPH). On reduction, the deep violet color of DPPH in
1
str); H-NMR (CDCl3) δ (ppm): 4.3 (d, 2H, CH2), 4.5
(d, 2H, CH2), 7.1 (m, 3H, ArH), 7.2 (m, 3H, ArH);
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis, Antimicrobial, and Antioxidant Screening of 1,2,4-Triazolo-1,3,4-
Thiadiazole Derivatives
[3] Noolvi, M. N.; Patel, H. M.; Kamboj, S.; Cameotra, S. S. Arab
J Chem 2016, 9, S1283.
[4] Zhong, Z.; Zhang, D.; Peng, Z.; Li, Y.; Shan, G.; Zuo, L.; Wu,
L.; Li, S.; Gao, R.; Li, Z. Eur J Med Chem 2013, 69, 32.
[5] Xinyang, L.; Lan, Y.; Zhijiang, F. J. Saudi Chem Soc 2018,
22, 101.
[6] Akhter, M. W.; Hassan, M. Z.; Amir, M. Arab J Chem 2014, 7,
955.
[7] Jain, A. K.; Sharma, S.; Vaidya, A.; Ravichandran, V.;
Agrawal, R. K. Chem Biol Drug Des 2013, 81, 557.
[8] Kamboj, V. K.; Verma, P. K.; Dhanda, A.; Ranjan, S. Cent
Nerv Syst Agents Med Chem 2015, 15, 17.
[9] Karrouchi, K.; Chemlal, L.; Taoufik, J.; Cherrah, Y.; Radi, S.;
Faouzi, M. E. A.; Ansar, M. Ann Pharm Fr 2016, 74, 431.
[10] Karegoudar, P.; Prasad, D. J.; Ashok, M.; Mahalinga, M.;
Poojary, B.; Holla, B. S. Eur J Med Chem 2008, 43, 808.
[11] Ghosh, S.; Verma, A.; Mukerjee, A.; Mandal, M. K. Arab J
Chem 2015. https://doi.org/10.1016/j.arabjc.2015.07.003.
[12] Mallikarjuna, B. P.; Sastry, B. S.; Kumar, G. V. S.;
Rajendraprasad, Y.; Chandrashekar, S. M.; Sathisha, K. Eur J Med Chem
2009, 44, 4739.
[13] Sunil, D.; Isloor, A. M.; Shetty, P.; Satyamoorthy, K.; Prasad,
A. S. B. Arab J Chem 2010, 3, 211.
[14] Prakash, C.; Kamboj, V. K.; Ahlawat, P.; Kumar, V. Toxicol
Environ Chem 2015, 97, 1408.
methanol changes into yellow color, and a decrease in
absorption was observed at 517 nm. Five concentrations
(25, 50, 75, 100, and 125 μg/mL) was prepared in
methanol and 2 mL of each concentration of test was
mixed with equal volume of 3 μg/mL of concentration of
DPPH in methanol solution. The absorption was
determined by UV–Vis spectrophotometer against blank,
and screening was performed in triplicate. Ascorbic acid
was used as positive control. The percentage of free
radical scavenging was calculated by equation 2. IC50
values of the test compounds were calculated from
percentage scavenging activity (Table 3) [18,19].
ꢂ
ꢃ
A ꢀ B
Scavenging activity ð%Þ ¼
ꢁ100
(2)
A
where A is the absorbance of the control (containing meth-
anolic DPPH and ascorbic acid) and B is the absorbance of
the test compound (containing methanolic DPPH and test
compound).
[15] Küçükgüzel, I.; Küçükgüzel, Ş. G.; Rollas, S.; Ötük-Sanış, G.;
Özdemir, O.; Bayrak, I.; Altuğ, T.; Stables, J. P. Farmacoterapia 2004, 59,
893.
[16] Anu; Kamboj, V. K.; Sati, B.; Verma, P. K. RJPBCS 2014, 5,
1491.
Acknowledgment. The authors are thankful to All India Council
for Technical Education (AICTE) for awarding postgraduate
fellowship to the first author.
[17] Sharma, S. K.; Singh, H. Inventi Rapid Planta Activa 2013,
3, 1.
[18] Gunthanakkala, A. K.; Mangali, M.; Gudi, Y.; Tatha, S.;
Venkatapuram, P.; Adivireddy, P. J Heterocyclic Chem 2018, 55, 2142.
https://doi.org/10.1002/jhet.3259.
REFERENCES AND NOTES
[19] Anu; Kamboj, V. K.; Sati, B.; Verma, P. K. RJPBCS 2014, 5,
866.
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Chandrashekar, S. M.; Kistayya, C. Eur J Med Chem 2010, 45, 2063.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet