Synthesis and biological evaluation of 3,6-dialkylsubstituted-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazoles

Article abstract of DOI:10.1007/s12039-018-1423-6

Abstract: A series of 3,6-dialkyl-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazole (10) analogues were prepared through multistep synthesis and evaluated them for their antimicrobial and cytotoxic activities. Synthesis of target compounds was carried out using undecenoic acid as starting material, which is the renewable product of castor oil. The key step in the synthesis was formation of triazolo [3,4-b][1,3,4]thiadiazole using various free fatty acids in presence of POCl ₃. It was observed that the undecenyl based triazolothiadiazole with butyl (6a), hexyl (6b) and lauryl (6f) derivatives exhibited promising antimicrobial activity against the tested strains. Particularly, Compound 6a exhibited the most promising activity with MIC value 3.9 μ g / mL against most of the tested strains. It also showed potent minimum bactericidal concentration activity with MIC value 7.8 μ g / mL against the tested strains. Cytotoxicity data revealed that most of the tested compounds revealed cytotoxic activity, Compounds 6b, 6d, 6f, 6g, 6h and 6i against SKOV3, 6d, 6e, 6f, 6g, 6h, 6i and 6j against MCF-7 and 6c, 6d, 6e,6g, 6h, 6i and6j against B16–F10 cell lines exhibited significant activities with IC ₅₀ values ranged between 13.67 and 18.62 μ M. Interestingly, all the compounds were non toxic against Chinese hamster ovary cell (CHO-K1) normal cell. Graphical Abstract : A series of 3, 6-dialkyl triazolothiadiazole analogues were prepared using undecenoic acid, which is the renewable product of castor oil and evaluated them for their antimicrobial and cytotoxic activities. Few compounds showed good antimicrobial and cytotoxic activities. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s12039-018-1423-6

J. Chem. Sci. (2018) 130:23
© Indian Academy of Sciences
https://doi.org/10.1007/s12039-018-1423-6
REGULAR ARTICLE
Synthesis and biological evaluation of 3,6-dialkylsubstituted-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazoles
VIJAYENDAR VENEPALLYa,d, K SIRISHAb,d, C GANESH KUMARb,d,
E VAMSHI KRISHNAc,d, SUNIL MISRAc,d and RAM CHANDRA REDDY JALAa,d,∗
aCentre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Tarnaka, Uppal Road, Hyderabad,
Telangana 500 007, India
bMedicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad, Telangana 500 007, India
cPharmacology and Toxicology Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad,
Telangana 500 007, India
dAcademy of Scientific and Innovative Research, New Delhi, India
E-mail: jrcreddy10@gmail.com; ramchandra@iict.res.in
MS received 7 November 2017; revised 29 December 2017; accepted 7 January 2018
Abstract. A series of 3,6-dialkyl-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazole (10) analogues were prepared
through multistep synthesis and evaluated them for their antimicrobial and cytotoxic activities. Synthesis of
target compounds was carried out using undecenoic acid as starting material, which is the renewable product
of castor oil. The key step in the synthesis was formation of triazolo [3,4-b][1,3,4]thiadiazole using various
free fatty acids in presence of POCl3. It was observed that the undecenyl based triazolothiadiazole with butyl
(6a), hexyl (6b) and lauryl (6f) derivatives exhibited promising antimicrobial activity against the tested strains.
Particularly, Compound 6a exhibited the most promising activity with MIC value 3.9 μg/mL against most of the
tested strains. It also showed potent minimum bactericidal concentration activity with MIC value 7.8 μg/mL
against the tested strains. Cytotoxicity data revealed that most of the tested compounds revealed cytotoxic
activity, Compounds 6b, 6d, 6f, 6g, 6h and 6i against SKOV3, 6d, 6e, 6f, 6g, 6h, 6i and 6j against MCF-7 and
6c, 6d, 6e,6g, 6h, 6i and6j against B16–F10 cell lines exhibited significant activities with IC50 values ranged
between 13.67 and 18.62 μM. Interestingly, all the compounds were non toxic against Chinese hamster ovary
cell (CHO-K1) normal cell.
Keywords. Di alkyltriazolothiadiazole; cytotoxicity; antimicrobial; butyl; hexyl; lauryl.
1. Introduction
new compounds with improved selectivity and activ-
ity by chemical modifications. The development of new
antimicrobial and anticancer therapeutic agents is one of
the fundamental goals in medicinal chemistry. There-
fore, continuous efforts are needed to develop novel
compounds with anticancer and antimicrobial activities.
Fatty acids are ubiquitous molecules typically found
bound to glycerol, sugars and phosphate and other
head groups to form lipids. Fatty acids are the impor-
tant components of all of these lipids. Fatty acids and
their derivatives are known for their biological activi-
ties such as antimicrobial,1,2 antifungal,3 and pesticidal4
ones. These fatty acid analogs have been found to be
The prevalence of microbial infections has increased
considerably in recent years. A bacterial and fun-
gal infection remains a serious threat to human life
because of its emerging resistance to existing antibiotic
drugs, which is an increasing public health problem.
Consequently, there is an urge to discover novel antimi-
crobial agents with potent activity against drug resistant
microorganisms. In addition, cancer is a major public
health problem that can affect almost every tissue in
the human body and poses a great challenge to medi-
cal science. Thus, intense efforts are needed to discover
*
For correspondence
Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12039-018-1423-6) contains
supplementary material, which is available to authorized users.

23 Page 2 of 8
J. Chem. Sci. (2018) 130:23
associated with diverse biological activities such as with ethyl acetate, dried over anhydrous sodium sulphate and
anti-inflammatory,5 antioxidant,6 antifeedant,7 antipar-
concentrated under vacuum to afford the title compound.
asitic,8 antimicrobial9 and neuroprotective.10 Literature
studies reported that a variety of modified fatty acids
1H NMR (300 MHz, CDCl3): (δ, ppm) = 5.75−5.85 (m,
-CH = CH2-, 1H), 4.91–5.01 (m, -CH = CH2-, 2H), 3.66(s,
are significant molecules in the treatment of can-
-OCH3, 2H), 2.28–2.31(t, -CH2-, J = 7.4 Hz, 2H), 2.01–
cers.11–13 Undecenoic acid derivatives were found to
2.06(m, -CH2-, 2H), 1.59–1.65(m, -CH2-, 2H), 1.26–1.39(m,
exhibit promising biological activities such as antifun- -(CH2)5-, 10H).
ESI-mass: [M + H]+m/z = 199.
gal, antibacterial, antiviral and anticancer ones.11,14
In addition, 1,2,4-triazoles are a group of hetero-
2.1b Synthesis of undec-10-enehydrazide (3): To a
stirred solution of methyl undec-10-enoate (2) (59.93 mmol)
in ethanol (90 mL), hydrazine hydrate (269.68 mmol) was
added. The reaction mixture was refluxed for about 10 h.
Progress of the reaction was monitored by micro TLC. After
completion of the reaction, the solvent was evaporated under
reduced pressure, ice water (50 mL) was added and the mix-
ture was stirred for 15 min. The solid obtained was filtered
and dried under vacuum to yield undec-10-enehydrazide as a
white solid.
cycliccompoundsexhibitingawidevarietyofbiological
activities such as antibacterial, antifungal,15 antituber-
cular,16 anticancer,17 anticonvulsant,18 antiinflamma-
tory,19 analgesic20 and molluscicidal properties.21–24
1,2,4-triazoles are good starting materials for the syn-
thesis of some interesting N-bridged heterocycles due
to their ambident nucleophilic centers. Triazolothia-
diazoles and triazolothiadiazines are a class of fused
heterocyclic compounds, which attracted great inter-
est in medicinal chemistry owing to their wide range
of pharmacological activities such as antifungal,25,26
antibacterial,27,28 antiviral,29 antihelmentic,30,31 antitu-
mour,32 analgesic33 and anti-inflammatory ones.34,35
Based on the above facts, in the present study
a series of 3,6-dialkylsubstituted-[1,2,4] triazolo[3,4-
b][1,3,4]thiadiazoles were synthesized using various
fatty acids and evaluated for biological activities.
1H NMR (300 MHz, CDCl3): δ(ppm)
=
5.75−5.85
(m, -CH = CH2-, 1H), 4.91–5.01 (m, -CH = CH2, 2H),
2.67–3.01(broad-s, -NH2, 2H), 2.12–2.16(t, -CH2-, J = 7.3
Hz, 2H), 2.00–2.06(m, -CH2-, 2H), 1.59–1.66(m, -CH2-, 2H),
1.25–1.38(m, -(CH2)5-, 10H).
ESI-mass: [M + H]+m/z = 199.
2.1c Synthesis of potassium 2-(undec-10-enoyl) hydr-
azine-1-carbodithioate (4): Potassium hydroxide pellets
(106.54 mmol) were dissolved in ethanol (40 mL). To this
solution, undec-10-enehydrazide (53.27 mmol), carbon disul-
fide (117.19 mmol) were added successively and the contents
were stirred at room temperature for 8 h. Progress of the reac-
tion was monitored by micro TLC. After completion of the
reaction, diethyl ether (100 mL) was added to the reaction
mixture and stirred for 10 min. After filtration, potassium 2-
(undec-10-enoyl) hydrazine-1-carbodithioate was obtained as
an off-white solid.
2. Experimental
2.1 Materials and general synthetic procedures
All the chemicals used in these schemes were of analyti-
cal grade and they were obtained from different commercial
sources and used without any further purification. Reactions
were monitored on micro TLC plates (coated with TLC grade
silica gel, obtained from Merck). Column chromatography
was performed by using silica gel (100–200 mesh) procured
from Qualigens (India) using freshly distilled solvents. All the
1H-NMR and 13C-NMR spectra were recorded with a Bruker
Avance (for 1H-NMR at 300 MHz, 400 MHz, 500 MHz and
for 13C-NMR at 75 MHz, 100 MHz, 125 MHz) spectrometer,
using TMS δ = 0 ppm and δ 77.00 ppm as internal standard
for chemical shifts (δ) in CDCl3 at 25 ◦C. The chemical shift
values are presented in ppm (parts per million) units. Mass
spectra were recorded with HRMS. IR spectra were recorded
in chloroform on a Perkin-Elmer FT-IR spectrum BX.
2.1d Synthesis of 4-amino-5-(dec-9-en-1-yl)-4H-1, 2,
4-triazole-3-thiol (5): Hydrazine hydrate (45.38 mmol)
was added to potassium 2-(undec-10-enoyl) hydrazine-1-
carbodithioate (45.38 mmol) and the contents were refluxed
for 5 h. Progress of the reaction was monitored by micro TLC.
After completion of the reaction, the reaction mixture was
acidified with concentrated hydrochloric acid. The obtained
precipitate was filtered and dried under vacuum to obtain the
crude compound which was subjected to silica gel column
chromatography and the required product was eluted in a sol-
vent mixture (Hexane: EtOAc, 85: 15, v/v) as an off white
solid.
2.1a Synthesis of methyl undec-10-enoate (2): To
a stirred solution of undec-10-enoic acid (73.45 mmol) in
methanol (100 mL), a few drops of concentrated H2SO4 were
ESI-mass: [M + H]+m/z = 255.
added. The reaction mixture was refluxed for 10 h. Progress of 2.1e General procedure for the synthesis of bridged
the reaction was monitored by micro TLC. After completion compounds (6a-j): A mixture of 4-amino-5-(dec-9-en-1-
the reaction, methanol was removed under reduced pressure yl)-4H-1, 2, 4-triazole-3-thiol (0.1 mol), various fatty acids
and water was added and the title compound was extracted (0.1 mol) and phosphorus oxychloride (10 L) was refluxed for

J. Chem. Sci. (2018) 130:23
Page 3 of 8 23
6 h. Progress of the reaction was monitored by micro TLC. 2.1i 3-(Dec-9-en-1-yl)-6-nonyl-[1,2,4]triazolo[3,4-b]
After completion of the reaction, reaction mixture was cooled [1,3,4]thiadiazole (6d): The crude compound was sub-
to room temperature and poured onto crushed ice and the title jected to silica gel column chromatography and the required
compound was extracted with ethyl acetate, dried over anhy- product was eluted in a solvent mixture (Hexane: EtOAc,
drous sodium sulphate. The crude compound was subjected to 20: 80, v/v) as an off-white semi solid with 70% yield.
silica gel column chromatography to get the required product. 1H NMR (500 MHz, CDCl3): δ(ppm) = 5.76−5.84(m, 1H,
CH2-CH = CH2), 4.91–5.01(m, 2H, CH2-CH = CH2),
3.02–3.05(t, J = 7.78Hz, 2H, CH2), 2.94–2.97(t, J =
2.1f 3-(Dec-9-en-1-yl)-6-propyl-[1,2,4]triazolo[3,4-b]
7.62Hz, 2H, CH2), 1.99–2.05(m, 2H, CH2), 1.78–1.89(m,
4H, CH2), 1.25–1.43(m, 22H, (CH2)11), 0.91–0.94(t, J =
7.01Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ(ppm) =
169.9, 153.3, 148.1, 139.0, 114.1, 34.0, 33.7, 31.8, 29.6, 29.3,
3 νmax
3418, 2924, 2853, 1719, 1628, 1467, 1377, 1217, 1091, 771;
HR-MS (ESI) m/z [M+H+]: calc for C22H38N4S is 390.2817,
found 391.28885 (C22H39N4S).
[1,3,4]thiadiazole (6a): The crude compound was sub-
jected to silica gel column chromatography and the required
product was eluted in a solvent mixture (Hexane: EtOAc,
20: 80, v/v) as an off-white semi solid with 67% yield.
1H NMR (400 MHz, CDCl3): δ(ppm) = 5.75−5.85(m,
1H, CH2-CH = C H2), 4.91–5.01(m, 2H, CH2-CH =
CH2), 3.02–3.06(t, J = 7.58 Hz, 2H, CH2), 2.92–2.96(t,
J = 7.45 Hz, 2H, CH2), 2.00–2.06(m, 2H, CH2), 1.81–
1.90(m, 2H, CH2), 1.63–1.66(m, 2H, CH2), 1.25–1.41(m,
29.0, 28.8, 28.4, 26.6, 22.6, 14.0; IR (CHCl
cm−1):
10H, (CH2)5), 1.05-1.09(t, J = 7.33Hz, 3H, CH3);13C NMR 2.1j 3,6-Di(dec-9-en-1-yl)-[1,2,4]triazolo[3,4-b][1,3,
(100 MHz, CDCl3): δ(ppm) = 169.3, 153.3, 148.0, 139.0, 4]thiadiazole (6e): The crude compound was subjected to
114.0, 34.1, 33.6, 29.0, 28.9, 28.8, 26.6, 24.9, 21.8, 13.3; IR silica gel column chromatography and the required product
3 νmax
(CHCl
cm−1): 3422, 2925, 2854, 1468, 1216, 758; HR- was eluted in a solvent mixture (Hexane: EtOAc, 20: 80, v/v)
MS (ESI) m/z [M+H+]: calc for C16H26N4S is 306.1878, as an off white semi solid with 65% yield. H NMR (400
1
found 307.19478 (C16H27N4S).
MHz, CDCl3): 5.75–5.85(m, 2H, CH2-CH = CH2), 4.91–
5.01(m, 4H, CH2-CH = CH2), 3.02–3.06(t, J = 7.70 Hz,
4H, CH2), 2.93–2.97(t, J = 7.58 Hz, 4H, CH2), 1.95–
2.04(m, 4H, CH2), 1.76–1.88(m, 4H, CH2), 1.25–1.42(m,
16H, (CH2)8); 13C NMR (125 MHz, CDCl3): δ(ppm) =
169.5, 153.3, 148.0, 139.0, 138.9, 114.1, 114.0, 33.6, 32.4,
32.2, 29.3, 29.0, 28.9, 28.7, 28.4, 26.6, 24.9, 17.8; IR
3 νmax
2.1g 3-(Dec-9-en-1-yl)-6-pentyl-[1,2,4]triazolo[3,4
-b][1,3,4]thiadiazole (6b): The crude compound was
subjected to silica gel column chromatography and the
required product was eluted in a solvent mixture (Hex-
ane: EtOAc, 20: 80, v/v) as an off white semi solid with
63% yield. 1H NMR (500 MHz, CDCl3): δ(ppm) =5.76–
5.84(m, 1H, CH2-CH = CH2), 4.91–5.01(m, 2H, CH2-CH =
CH2), 3.02–3.05(t, J = 7.62Hz, 2H, CH2), 2.95–2.97(t,
J = 7.62Hz, 2H, CH2), 1.99–2.05(m, 2H, CH2), 1.78-
(CHCl
cm−1): 3423, 3016, 2926, 2854, 1473, 1215,
757, 667; HR-MS (ESI) m/z [M+H+]: calc for C23H38N4S
is 402.2817, found 403.28827 (C23H39N4S).
1.89(m, 4H, CH2), 1.25–1.43(m, 14H, (CH2)7), 0.91–0.94(t, 2.1k 3-(Dec-9-en-1-yl)-6-undecyl-[1,2,4]triazolo[3,4
J = 7.01 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): -b][1,3,4]thiadiazole (6f): The crude compound was sub-
δ(ppm) = 168.9, 152.9, 147.7, 138.7, 113.7, 33.7, 33.3, 28.7, jected to silica gel column chromatography and the required
3 νmax
28.6, 28.4, 26.3, 24.6, 21.5, 13.0; IR (CHCl
cm−1): product was eluted in a solvent mixture (Hexane: EtOAc, 20:
3417, 2925, 2854, 1630, 1468, 1217, 769; HR-MS (ESI) m/z 80, v/v) as an off white semi solid with 71% yield. 1H NMR
[M+H+]: calc for C18H30N4S is 334.2191, found 335.22604 (500 MHz, CDCl3): δ(ppm) = 5.76−5.84(m, 1H, CH2-
(C18H31N4S).
CH = CH2), 4.91–5.00(m, 2H, CH2-CH = CH2), 3.02–
3.05(t, J = 7.62Hz, 2H, CH2), 2.94–2.97(t, J = 7.62Hz,
2H, CH2), 2.01–2.05(m, 2H, CH2), 1.77–1.89(m, 4H, CH2),
1.26–1.44(m, 26H, (-CH2-)13) 0.86–0.89(t, J = 6.71Hz,
3H, CH3); 13C NMR (100 MHz, CDCl3): δ(ppm) = 169.6,
153.0, 147.8, 138.7, 113.7, 33.7, 33.4, 31.5, 29.3, 29.0, 28.7,
3 νmax
2.1h 3-(Dec-9-en-1-yl)-6-heptyl-[1,2,4]triazolo[3,4
-b][1,3,4]thiadiazole (6c): The crude compound was
subjected to silica gel column chromatography and the
required product was eluted in a solvent mixture (Hexane:
EtOAc, 20: 80, v/v) as an off-white semi solid with 66%
yield. 1H NMR(400MHz, CDCl3):δ(ppm) = 5.75−5.85(m,
1H, CH2-CH = CH2), 4.91–5.01(m, 2H, CH2-CH = CH2),
3.02–3.06(t, J = 7.70Hz, 2H, CH2), 2.93–2.97(t, J =
7.58Hz, 2H, CH2), 1.98–2.06(m, 2H, CH2), 1.77–1.90(m, 4H,
28.4, 28.1, 26.3, 22.3, 13.7; IR (CHCl
cm−1): 3419,
3015, 2925, 2854, 1472, 1215, 759, 667; HR-MS (ESI) m/z
[M+H+]: calc for C24H42N4S is 418.3130, found 419.31976
(C24H43N4S).
CH2), 1.25–1.44(m, 18H, (CH2)9), 0.87–0.91(t, J = 6.84Hz, 2.1l 3-Decyl-6-tridecyl-[1,2,4]triazolo[3,4-b][1,3,4]
3H, CH3); 13C NMR (100 MHz, CDCl3): δ(ppm) = 169.6, thiadiazole(6g): The crude compound was subjected to sil-
153.6, 148.4, 139.3, 114.3, 34.4, 34.0, 29.3, 29.2, 29.1, 26.9, ica gel column chromatography and the required product was
3 νmax
25.2, 22.2, 13.6; IR (CHCl
cm−1): 3418, 2924, 2853, eluted in a solvent mixture (Hexane: EtOAc, 20: 80, v/v) as an
1958, 1711, 1629, 1468, 1217, 771; HR-MS (ESI) m/z off white solid with 69% yield. 1H NMR (500 MHz, CDCl3):
[M+H+]: calc for C20H34N4S is 362.2504, found 363.25755 δ(ppm) = 5.76−5.84(m, 1H, CH2-CH = CH2), 4.91–
(C20H35N4S).
5.01(m, 2H, CH2-CH = CH2), 3.23–3.26 (t, J = 7.62 Hz,

23 Page 4 of 8
J. Chem. Sci. (2018) 130:23
2H, CH2), 3.05–3.08(t, J = 7.62Hz, 2H, CH2), 2.01–2.05(m, 130.1, 129.9, 114.1, 33.7, 32.3, 31.8, 29.6, 29.2, 29.0, 28.7,
cm−1): 3423,
3 νmax
2H, CH2), 1.82–1.95 (m, 4H, CH2), 1.25–1.44 (m, 30H, (- 28.4, 26.7, 25.0, 22.6, 14.0; IR (CHCl
CH2-)15) 0.86–0.89(t, J = 6.71 Hz, 3H, CH3); 13C NMR 2925, 2854, 1958, 1734, 1639, 1518, 1471, 1215, 993, 757,
(100 MHz, CDCl3): δ(ppm) = 170.5, 153.3, 148.1, 139.0, 666; HR-MS (ESI) m/z [M+H+]: calc for C30H53N4S is
114.1, 33.7, 32.3, 31.8, 33.9, 29.6, 29.3, 29.0, 28.8, 28.4, 26.6, 501.39854, found 501.39838 (C30H53N4S).
3 νmax
24.8, 22.6, 14.0; IR (CHCl
cm−1): 3652, 3423, 2925,
2854, 1958, 1735, 1639, 1518, 1470, 1215, 994, 769, 667;HR-
MS (ESI) m/z [M+H+]: calc for C26H46N4S is 446.36724,
found 447.36692 (C26H47N4S).
2.2 General procedure for biological evaluation
2.2a Antibacterialandantifungalassays: Theantibac-
terial and antifungal activities of the synthesized compounds
were determined using well diffusion method36 against dif-
ferent pathogenic bacterial strains such as Micrococcus luteus
MTCC2470, StaphylococcusaureusMTCC96, Staphylococ-
cus aureus MLS-16 MTCC 2940, Bacillus subtilis MTCC
121, Escherichia coli MTCC 739, Pseudomonas aeruginosa
MTCC 2453 and Klebsiella planticola MTCC 530 along with
different Candida strains such as Candida albicans MTCC
183, C. albicans MTCC 227, C. albicans MTCC 854, C.
albicans MTCC 1637, C. albicans MTCC 3017, C. albicans
MTCC 3018, C. albicans MTCC 3958, C. albicans MTCC
4748, C. albicans MTCC 7315, C. parapsilosis MTCC 1744,
C. aaseri MTCC 1962, C. glabrata MTCC 3019, C. kru-
sei MTCC 3020 and Issatchenika hanoiensis MTCC 4755.
All these bacterial and fungal strains were procured from the
Microbial Type Culture Collection and Gene Bank (MTCC),
CSIR-Institute of Microbial Technology, Chandigarh, India.
Thepathogenicreferencestrainswereseededonthesurfaceof
the media Petri plates, containing Muller-Hinton agar with 0.1
mLofpreviouslypreparedmicrobialsuspensionsindividually
containing 1.5×108 cfu mL−1 (equal to 0.5 McFarland stan-
dard). Wells of 6.0 mm diameter were prepared in the media
plates using a cork borer and the synthesized compounds dis-
solved in 10% DMSO at a dose range of 125–0.97 μg were
added in each well under sterile conditions in a laminar air
flow chamber. Standard antibiotic solutions of ciprofloxacin
(bacterial strains) and miconazole (Candida strains) at a dose
range of 125–0.97 μg well−1, served as positive controls,
while the well containing DMSO served as negative control.
The plates were incubated for 24 h at 30 ◦C and the well con-
taining the least concentration showing the inhibition zone
is considered as the minimum inhibitory concentration. All
experiments were carried out in duplicates and mean values
are represented.
2.1m 3-(Dec-9-en-1-yl)-6-pentadecyl-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazole (6h): The crude compound
was subjected to silica gel column chromatography and the
required product was eluted in a solvent mixture (Hexane:
EtOAc, 20: 80, v/v) as an off white solid with 76% yield.
1H NMR (500 MHz, CDCl3): δ(ppm) = 5.76−5.84(m, 1H,
CH2-CH = CH2), 4.91–5.01(m, 2H, CH2-CH = CH2),
3.02–3.05(t, J = 7.62 Hz, 2H, CH2), 2.94–2.97(t, J =
7.62 Hz, 2H, CH2), 2.01-2.05(m, 2H, CH2), 1.77–1.89(m,
4H, CH2), 1.25-1.45(m, 34H, (-CH2-)17) 0.86-0.89(t, J =
6.71 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ(ppm) =
169.9, 153.3, 148.1, 139.0, 114.1, 34.0, 33.7, 31.8, 29.6, 29.3,
3 νmax
29.0, 28.8, 28.4, 26.6, 22.6, 14.0; IR (CHCl
cm−1):
3418, 2925, 2854, 1640, 1518, 1471, 1215, 769, 667; HR-
MS (ESI) m/z [M+H+]: calc for C28H51N4S is 475.38289,
found 475.38266 (C28H51N4S).
2.1n 3-(Dec-9-en-1-yl)-6-heptadecyl-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazole (6i): The crude compound was
subjected to silica gel column chromatography and the
required product was eluted in a solvent mixture (Hexane:
EtOAc, 20: 80, v/v) as an off white solid with 72% yield.
1H NMR (400 MHz, CDCl3): δ(ppm) = 5.75−5.85(m,
1H, CH2-CH = CH2), 4.91–5.01(m, 2H, CH2-CH
=
CH2), 3.02–3.06(t, J = 7.58 Hz, 2H, CH2), 2.93–2.97(t,
J = 7.58 Hz, 2H, CH2), 2.00–2.05(m, 2H, CH2), 1.76–
1.90(m, 4H, CH2), 1.25–1.45(m, 38H, (-CH2-)19) 0.86–
0.89(t, J = 6.60 Hz, 3H, CH3); 13C NMR (100 MHz,
CDCl3): δ(ppm) = 169.6, 153.3, 148.1, 139.0, 114.1,
34.0, 33.7, 32.3, 31.8, 29.6, 29.3, 29.0, 28.8, 28.4, 26.6,
3 νmax
24.9, 22.6, 14.0; IR (CHCl
cm−1): 3418, 2925, 2854,
1958, 1732, 1640, 1470, 1215, 993, 769, 666; HR-MS (ESI)
m/z [M+Na+]: calc for C28H56N4SNa is 503.41179, found
503.41393(C28H56N4SNa).
2.2b Minimum bactericidal and fungicidal concen-
tration (MBC) assays: Bactericidal and fungicidal assay
2.1o 3-(Dec-9-en-1-yl)-6-(heptadec-8-en-1-yl)-[1,2,
4]triazolo[3,4b][1,3,4]thiadiazole(6j): Thecrudecom- (NCCLS, 2000) was performed in sterile 2.0 mL microfuge
pound was subjected to silica gel column chromatography and tubes against a panel of pathogenic bacterial strains, includ-
the required product was eluted in a solvent mixture (Hexane: ing Micrococcus luteus MTCC 2470, Staphylococcus aureus
EtOAc, 20: 80, v/v) as an off-white semi solid with 78% yield. MTCC 96, Staphylococcus aureus MLS-16 MTCC 2940,
1H NMR (500 MHz, CDCl3): δ(ppm) = 5.76−5.84 (m, 1H, Bacillus subtilis MTCC 121, Escherichia coli MTCC 739,
CH2-CH = CH2), 5.34–5.35 (m, 2H, -CH = CH-), 4.91– Pseudomonas aeruginosa MTCC 2453, Klebsiella planti-
5.01(m, 2H, CH2-CH = CH2), 3.02–3.05(t, J = 7.58 Hz, cola MTCC 530 and for fungicidal activity different Candida
2H, CH2), 2.93–2.96(t, J = 7.58 Hz, 4H, CH2), 1.94–2.05(m, strainswereculturedovernightinMuellerHintonbroth. Serial
4H, CH2), 1.77–1.89 (m, 4H, CH2), 1.25–1.42(m, 30H, (- dilutions of test compounds were prepared in Mueller Hin-
CH2-)15) 0.86–0.89(t, J = 6.60Hz, 3H, CH3); 13C NMR ton broth with different concentrations ranging from 0 to
(100 MHz, CDCl3): δ(ppm) = 169.6, 153.3, 148.1, 139.1, 150 μg mL−1. To the test compounds, 100 μL of overnight

J. Chem. Sci. (2018) 130:23
Page 5 of 8 23
cultured bacterial and Candida suspensions were added to dissolve the insoluble formazan crystals 100 μL DMSO was
reach a final concentration of 1.5 × 108 cfu mL−1 (equal to added to each well. Finally, the absorbance of the plates was
0.5 McFarland) and incubated at 37 ◦C for 24 h. After 24 h of measured using a Synergy H1 multi-mode plate reader, USA.
incubation, the minimum bactericidal or fungicidal concen- Doxorubicin was used as a positive control for the compari-
tration (MBC/MFC) was determined by sampling 10 μL of son.
suspension from the tubes onto Mueller Hinton agar plates
and were incubated for 24 h at 37 ◦C to observe the growth
of test organisms. MBC/MFC are the lowest concentration of
test compound required to kill a particular bacterium/Candida
strain. All the experiments were carried out in duplicates.
3. Results and Discussion
The target compounds were synthesized as outlined in
Scheme 1. Undec-10-enoic acid was converted to the
methyl ester by using methanol and few drops of con-
centrated sulphuric acid. Undec-10-enoic acid methyl
2.2c Biofilminhibitionassay: Thetestcompoundswere
screened in sterile 96 well polystyrene microtiter plates using
the modified biofilm inhibition assay,37 against a panel of
pathogenic bacterial strains including Staphylococcus aureus
MTCC 96, Staphylococcus aureus MLS-16 MTCC 2940 ester was treated with hydrazine hydrate to get Undec-
and Klebsiella planticola MTCC 530, which were cultured
overnight in tryptone soy broth (supplemented with 0.5% glu-
cose). The test compounds of predetermined concentrations
ranging from 0 to 250 μg/mL were mixed with the bacte-
rial suspensions having an initial inoculum concentration of
5 × 105 CFU/mL. Aliquots of 100 μL were distributed in
each well and then incubated at 37 ◦C for 24 h under static
conditions. The medium was then discarded and washed with
phosphate buffered saline to remove the non-adherent bacte-
ria. Each well of the microtiter plate was stained with 100 μL
of 0.1% crystal violet solution followed by 30 min incubation
at room temperature. Later the crystal violet solution from the
10-enehydrazide, which was further reacted with carbon
disulfide in ethanolic potassium hydroxide to yield
corresponding dithiocarbazinate in good yield. Dithio-
carbazinate was directly reacted with hydrazine hydrate
under refluxing conditions to yield triazole. Condensa-
tion of triazole with various fatty acids in presence of
POCl3 yielded triazolothiadiazoles (6a-t). The synthe-
sized compounds were characterized by 1H, 13C NMR,
ESI-MS, HRMS and IR spectral analysis.
All the synthesized compounds 6a–j were directly
screened for their in vitro antimicrobial activity against
plates was discarded, thoroughly washed with distilled water different gram-positive strains such as Staphylococ-
for 3 to 4 times and air dried at room temperature. The crystal
violetstainedbiofilmwassolubilisedin95%ethanol(100 μL)
and the absorbance was recorded at 540 nm using TRIAD
multimode reader (Dynex Technologies, Inc, Chantilly, VA,
USA). Blank wells were employed as background check.
The inhibition data were interpreted from the dose-response
curves, where IC50 value is defined as the concentration of
inhibitor required to inhibit 50% of biofilm formation under
the above assay conditions. All the experiments were carried
out in triplicates and the values are indicated as mean S.D.
cus aureus MTCC 96, Bacillus subtilis MTCC 121,
S. aureus MLS16 MTCC 2940, Micrococcus luteus
MTCC 2470, and gram-negative bacterial strains such
as Klebsiella planticola MTCC 530, Escherichia coli
MTCC 739, Pseudomonas aeruginosa MTCC 2453, as
well as fungi such as Candida albicans MTCC 3017.
Compound6a(againstStaphylococcusaureusMTCC
96, Bacillus subtilis MTCC 121, S. aureus MLS16 MT-
CC 2940, Micrococcus luteus MTCC 2470, Escherichia
coli MTCC 739, Pseudomonas aeruginosa MTCC 2453
strains) exhibited the most promising activity with MIC
value 3.9 μg/mL. Compounds, 6b (against Staphylo-
coccus aureus MTCC 96 and Pseudomonas aeruginosa
2.2d Cytotoxicity assay: Cytotoxicity assay (MTT) was
evaluated for the synthesized compounds as per the method
reported in the earlier study.38 Four different cancer cell lines
and one normal cell line namely, HeLa Homo sapiens cervix MTCC 2453 strains) and 6f (against Staphylococcus
adenocarcinoma (ATCC® CCL-2.1tm), B16-F10 Mouse skin
aureus MTCC 96, Bacillus subtilis MTCC 121 and
melanoma (ATCC® CRL¬6475tm), SKOV3 Human Ovarian
Micrococcus luteus MTCC 2470 strains) exhibited good
cancer (ATCC® HTB¬77tm), MCF7 Human Breast Ade-
activity with MIC value 7.8 μg/mL. Compound 6a
nocarcinoma ((ATCC® HTB-22tm) and CHO-K1-Chinese
showed antifungal activity against Candida albicans
hamster ovary cells, Normal Cell line (ATCC® CCL-61tm
)
MTCC 3017 with the MIC value 3.9 μg/mL. It is note-
worthy that, the antimicrobial activities in this study
depend on the alkyl chain lengths of triazolothiadiazole
at C-6 position. Interestingly, our results corroborate
with the previous findings where heterocyclic com-
pounds having alkyl chain moiety showed significant
biological activities.39 Compound 6a with a basic 1,2,4-
were obtained from the ATCC (Bethesda, MD, USA) and
maintained in DMEM supplemented with 10% FBS, 2 mM
l-glutamine, 100 U/mL penicillin, and 100 μg/mL strepto-
mycin at 37 ◦C in a 5% CO2 incubator. After seeding of cells
in 96 well culture plate, allowed to attach properly. Test com-
pounds of different concentrations ranging from 1 to 50 μM
were added in triplicates and incubated for 24 h. The cells
were then incubated with MTT (0.5 mg/mL) for 3 h and to triazolo[3,4-b]1,3,4-thiadiazole scaffold having butyl

23 Page 6 of 8
J. Chem. Sci. (2018) 130:23
Scheme 1. Reagents and conditions: (a) MeOH, H2SO4, reflux, 10 h; (b) Hydrazine hydrate,
EtOH, reflux, 10 h; (c) CS2, KOH, EtOH, RT, 8 h; (d) Hydrazine hydrate, reflux, 5 h; (e) R-COOH,
POCl3, reflux, 6 h.
moiety at C-6 position and undecyl moiety at C-3 posi- namely, HeLa Homo sapiens cervix adenocarcinoma
tion showed the prominent activity. Compounds having (ATCC® CCL-2.1tm), B16-F10 Mouse skin melanoma
hexyl (6b) and lauryl (6f) at C-6 position and undecyl (ATCC® CRL¬6475tm), SKOV3 Human Ovarian can-
moiety at C-3 position displayed good activities.
cer(ATCC®HTB¬77tm), MCF7HumanBreastAdeno-
Compound 6a (butyl) showed potent minimum bacte- carcinoma ((ATCC® HTB-22tm) and CHO-K1-Chinese
ricidal concentration activity with MIC value 7.8 μg/mL hamster ovary cells, Normal Cell line (ATCC® CCL-
against the tested strains. Compounds 6b (hexyl) against 61tm) using MTT assay. Doxorubicin was used as a pos-
Staphylococcus aureus MTCC 96 (MIC value itive control. IC50 values of the test compounds for 24 h
7.8 μg/mL) and Pseudomonas aeruginosa MTCC 2453 on each cell line was calculated and presented in Table 4.
(MIC value 7.8 μg/mL) and 6f (lauryl) against Staphy-
Majority of the synthesized compounds exhibited
lococcus aureus MTCC96, Bacillus subtilis MTCC cytotoxicity on all the tested cell lines (Table 4). Com-
121 and Micrococcus luteus MTCC 2470 (MIC value pounds 6b, 6d, 6f, 6g, 6h and 6i showed significant
7.8 μg/mL) displayed promising activities.
activities against SKOV3 with the IC50 values ranged
Further, all these compounds were screened for between 13.80 to 18.62 μM. Compounds 6d, 6e, 6f,
antibiofilm activity against various strains, which are 6g, 6h, 6i and 6j displayed significant activities against
important nosocomial pathogens encountered in medi- MCF-7 cell line with IC50 values ranged between 13.67
cal establishments and devices and have the ability to to 18.02 μM and compounds 6c, 6d, 6e, 6g, 6h,
form biofilms. The results to this regard are summa- 6i and 6j exhibited significant activities against B16-
rized in Table 3, which clearly reveal that few of the F10 cell line with IC50 values ranged from 13.80 to
compounds exhibited moderate activities towards all the 18.49 μM. Remaining compounds showed moderate
tested strains.
activity against HeLa cell line (IC50 values, 22.56 to
The cytotoxicity of all the synthesized compounds 57.50 μM). All the compounds were non toxic against
were screened against four human cancer cell lines Chinese hamster ovary cell (CHO-K1) normal cell.

J. Chem. Sci. (2018) 130:23
Page 7 of 8 23
4. Conclusions
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crobial activity and cytotoxicity were investigated. It
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Supplementary Information
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Acknowledgements
Venepally Vijayendar acknowledges the University Grants
Commission (UGC), New Delhi, India, for the financial sup-
port extended in the form of Senior Research Fellowship
(SRF).
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