Design and Synthesis of 1,3,4-Thiadiazole Derivatives as Novel Anticancer and Antitubercular Agents

Article abstract of DOI:10.1134/S1070363219040224

A series of novel 5-phenyl-substituted 1,3,4-thiadiazole-2-amines were designed, synthesized, and screened for their antitumor and antitubercular activities. The target compounds were synthesized starting from isocyanates and acid hydrazides by convention

Full text of DOI:10.1134/S1070363219040224

ISSN 1070-3632, Russian Journal of General Chemistry, 2019, Vol. 89, No. 4, pp. 770–779. © Pleiades Publishing, Ltd., 2019.
Design and Synthesis of 1,3,4-Thiadiazole Derivatives
as Novel Anticancer and Antitubercular Agents1
D. Chandra Sekhara,b, D. V. Venkata Raoa, A. Tejeswara Raoc, U. Lav Kumarb, and Anjali Jhaa*
a Department of Chemistry, GIS, GITAM University, Rushikonda, Visakhapatnam, Andhra Pradesh, 530045 India
*e-mail: anjalimanishjha@yahoo.com
b Shilpa Medicare, API, R and D Unit, Modavalasa, Vizianagaram, Andhra Pradesh, 531162 India
c Centre for Chemical Sciences and Technology, Department of Chemistry, Institute of Science and Technology,
Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad, Telangana, 500085 India
Received December 31, 2017; revised October 12, 2018; accepted April 2, 2019
Abstract—A series of novel 5-phenyl-substituted 1,3,4-thiadiazole-2-amines were designed, synthesized, and
screened for their antitumor and antitubercular activities. The target compounds were synthesized starting from
isocyanates and acid hydrazides by conventional and microwave-assisted protocols. The structures of the
products were confirmed by 1H NMR, 13C NMR, high-resolution mass spectrometry, and IR spectroscopy and
elemental analysis. Some of the synthesized compounds showed significant invitro antitumor activities against
breast cancer and normal human cell lines. Among them, N-benzyl-5-(4-fluorophenyl)-, N-benzyl-5-(4-
nitrophenyl)-, and 5-phenyl-N-(p-tolyl)-1,3,4-thiadiazole-2-amines demonstrated higher inhibitory activities
against the MDA-MB-231 cell line than the cisplatin control (IC50 3.3 µM). N-Benzyl-5-(4-methoxyphenyl)-,
5-phenyl-N-{[4-(trifluoromethyl)phenyl]methyl}-, N-benzyl-5-(4-fluorophenyl)-, and N-benzyl-5-(4-
nitrophenyl)-1,3,4-thiadiazole-2-amines exhibited high inhibitory activities against the HEK293T cell line (IC50
52.63, 42.67, 34.71, and 33.74 µM, respectively), which were higher compared to the cisplatin control. In
antitubercular activity testing against mycobacterium smegmatis MC155, 5-phenyl-N-{[4-(trifluoromethyl)-
phenyl]methyl}-1,3,4-thiadiazole-2-amine proved to be a more potent agent (MIC 26.46 µg/mL) compared to
the Isoniazid control (12 µg/mL). Potential bioactivities of the synthesized compounds were computed using
Molinspiration and Molsoft software tools.
Keywords: 1,3,4-thiadiazoles, anticancer activity, antitubercular activity, Molinspiration, Molsoft
DOI: 10.1134/S1070363219040224
INTRODUCTION
the ability of thiadiazole molecules for binding with
receptors [4, 5].
Heterocyclic compounds are commonly used
scaffolds for pharmacophores for the design of potent
and selective drugs. 1,3,4-Thiadiazole represent a key
motif in heterocyclic chemistry and a privileged
structure in medicinal chemistry due to its broad-range
pharmacological activity [1, 2]. Despite the progress in
medicine over the past century, cancer and tuberculosis
(TB) still remain the leading causes of death in the
world. This makes critically important to continue
search for new anticancer and anti-TB drug candidates.
Thiadiazole is a 5-membered planar aromatic motif
comprising a sulfur atom, which improves the lipid
solubility of thiadiazole derivatives and hence their
pharmacokinetics [3]. The two-electron donor nitrogen
system (N=C–S) and hydrogen-binding domain enhance
1,3,4-Thiadiazoles have been reported to exhibit
antifungal [6], antibacterial [7], inflammatory [8], anti
anxiety [9], and anti-tubercular activities [10]. Dif-
ferent mechanisms of the antitumor activity of 1,3,4-
thiadiazoleshave been reported, including specific
inhibition of DNA and RNA syntheses without
appreciable impact on protein synthesis [11], inhibition
of carbonic anhydrase [12], phosphodiesterase-7
(PDE7) [13], and histonedeacetylase [14], or adenosine
A3 receptor antagonism [15]. The list of thiadiazole-
containing clinically used drugs or drug candidates
includes Acetazolamide, Zibotentan, Sulfamethizole,
Methazolamide, Megazol, and Globucid. In view of
the aforesaid, we designed (Fig. 1, Scheme 1) and syn-
thesized novel thiadiazole molecules and performed
their anticancer and anti-TB activity testing.
1
Supporting materials are available from authors.
770

DESIGN AND SYNTHESIS OF 1,3,4-THIADIAZOLE DERIVATIVES
771
core nucleus
RESULTS AND DISCUSSION
Synthesis and structure. Novel 1,3,4-thiadiazole
derivatives 4а–4m were synthesized by the reaction of
substituted isocyanates 1 with acid hydrazides 2 by
conventional (Scheme 1, path a) and microwave-
assisted protocols (Scheme 1, path b). The
conventional synthesis involved two stages: (1) the
reaction of compounds 1 with compounds 2 in the
presence of triethyl amine (TEA) in dry THF to obtain
intermediate derivatives 3 and (2) the reaction of
intermediates 3 with TEA and p-TsCl in N-methyl-2-
pyrrolidone (NMP) under heating to obtain the target
1,3,4-thiadiazole derivatives 4а–4m (Scheme 1). The
microwave-assisted synthesis involved a one-pot
reaction of compound 1 with compound 2 in presence
of TEA, Dry THF, P-TsCl, NMP to obtained desired
products 4а–4m with excellent yields. Under non-
classical reaction condition (MWI), the reaction rates
were faster than classical heating conditions [16, 17].
For example, synthesis of 4b was obtained in 4.5 h by
traditional methods whereas under Mw condition, it
was obtained in 5 min (4b, Table 1). The structures of
all synthesized compounds were assigned on the basis
R1
N
N
proton varied for
better binding
R
N
S
H
for enhanced activity
Fig. 1. Design strategy for the synthesis of the target compounds.
signals in the 13C NMR spectra at δ 168.92 (C2),
162.18 (C5) ppm validated the construction of 1,3,4-
thiadiazole core. The IR spectrum of 4b displayed
characteristic absorption bands at νmax 3370 cm–1 (NH
str), 3058 cm–1 (Ar=CH str), 1399 cm–1 (C=N), and
1218 cm–1 (C–S–C) clearly evidenced the presence of
cyclic C–S–C linkage and N–N=C in the thiadiazole
frame work. And finally the molecular weight of the
4b is m/z = 284 [M – 1].
1
of the IR, mass, H NMR, 13C NMR spectral data. For
Biological activity testing
.
The in vitro
the compound 4b,on the 1H NMR timescale,
disappearance of labile protons (NHNH2) signals at δ
8.45 and 4.55 ppm for NH, CH2 protons as a triplet,
doublet and appearance of a signal at δ 7.83–7.25 ppm
for aromatic hydrogens asserted 4b. The resonance
cytotoxicity of compounds 4a–4m was assessed by
employing the MTT colorimetric assay as per ATCC
protocol at 25 µM against a panel of cancer cell lines,
namely, human breast adenocarcinoma (MDAMB231)
and normal cell lines (HEK293T). Cisplatin was used
Scheme 1. Design strategy for the synthesis of the target compounds.
O
O
N
H
H
NH2
TEA, Dry THF
R
C
N
N
R1
N
+
S
R1
N
H
R
H
room temperature,
16 h, 74−95 %
S
2
3
1
path b
TEA, Dry THF,
P-TsCl, NMP
90°C, 3−6 min
TEA, P-TsCl, NMP
path a
0°C−room temperature,
4−5 h
N
N
R1
R
S
N
H
4a−4m
R = PhCH2, R1 = Ph (a); R = PhCH2, R1 = p-FC6H4 (b); R = PhCH2, R1 = p-NO2C6H4 (c); R = PhCH2, R1 = p-ClC6H4 (d);
R = PhCH2, R1 = p-MeOC6H4 (e); R = PhCH2, R1 = p-MeC6H4Ph (f); R = p-MeOC6H4, R1 = Ph (g); R = p-CF3C6H4, R1 = Ph
(h); R = p-FC6H4, R1 = Ph (i); R = p-ClOC6H4, R1 = Ph (j); R = p-MeC6H4, R1 = Ph (k); R = p-NO2C6H4, R1 = Ph (l); R =
p-MeOC6H4, R1 = Ph (m).
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CHANDRA SEKHAR et al.
Table 1. Synthesis times and yields of compounds 4a–4m by two protocols
Comp. no
Microwave-assisted synthesis: min/yield, %
6.0/87
Conventional synthesis: h/yield, %
4.0/68
4a
4b
4c
4d
4e
4f
5.0/79
4.0/80
4.5/82
5.5/72
5.0/73
4.0/70
4.0/68
4.5/66
4.0/72
5.0/63
4.5/74
5.0/68
5.0/82
4.5/60
4.0/69
5.0/62
5.0/60
5.0/53
4.0/65
4.0/44
4.5/58
5.0/55
4.0/62
4.5/53
4g
4h
4i
4j
4k
4l
4m
as standard, and the results are summarized in Table 2.
Compounds 4k, 4b, and 4c (% inhibition 30.29, 22.44,
and 21.84 respectively) exhibited the highest
anticancer activity against MDAMB231 cells. Among
the test compounds, 4e (52.63 µM) displayed
promising activity, and products 4h, 4b, and 4c
showed moderate to good activity against HEK293T
cells with percentage inhibitions at 25 µM of 42.67,
34.71, and 33.74 respectively. Homologation of 4a to
4f in turn to 4l and then to 4g decreased the activity
Table 2. Сytotoxicities, lipophilicities, and antimycobacterial activities of compounds 4a–4m
Cytotoxicity (% inhibition at 25 µM)
Anti-TB activity (MIC, µM)
Lipophilicity
(log P/сlog P)
Comp. no.
M. smegmatis
MC 155 strain
MDAMB231 cell line HEK293T cell line
4a
–3.30
22.44
15.06
34.71
4.79/3.92
4.95/4.07
0.00/3.67
5.35/4.64
4.67/3.98
5.28/4.42
4.67/3.84
5.71/4.80
5.19/4.61
5.59/5.18
5.51/4.95
0.00/4.26
4.90/4.38
0.00/–2.50
–0.6/–0.66
>100
36.05
4b
4c
21.84
33.74
45.23
4d
11.64
20.14
>100
4e
8.04
52.63
45.09
4f
–12.27
–0.93
20.30
>100
4g
9.30
28.78
4h
5.59
42.67
26.46
4i
18.20
22.61
36.15
4j
20.10
17.45
>100
4k
30.29
24.58
78.72
4l
15.06
–5.40
>100
4m
5.18
2.70
>100
Cisplatin
Isoniazid
3.30
2.80
Not determined
12
Not determined
Not determined
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DESIGN AND SYNTHESIS OF 1,3,4-THIADIAZOLE DERIVATIVES
60
773
profile for both cell lines. For the series 4b (R1 = p-
FC6H4), 4c (R1 = p-NO2C6H4), 4h (R = p-CF3C6H4),
the activity against the lung cancer cell line increases
with decreasing –I effect of the substituent. Hence, it is
believed that the synthesized compounds may act as
effective anticancer agents. The synthesized
derivatives were also tested for cytotoxicity using
normal human cells HEK293T and were found to
exhibit mediocre safety profiles. Among the test cell
lines, HEK293T cells were more sensitive to all the
test compounds than MDAMB231 cells. As an
example, Fig. 2 presents the anticancer activity profile
for compound 4k.
52.63
49.45
40
20
36.55
21.40
9.82
0
1
5
10
15
20
25
Concentration, μg/mL
Fig. 2. Dose‒response curve for the most active compound
4k in the HEK293T cell line.
Additionally, the synthesized compounds were screened
for antimycobacterial capacity against M. smegmatis,
and the resulting data are summarized in Table 2. As
seen from the data in the table, some compounds from
the series showed promising antimycobacterial
properties. Compounds 4h, 4g and 4b exhibited the
highest antimycobacterial activity with MIC values of
26.46, 28.78, and 36.05 µM, respectively. Compounds
4e, 4c, and 4k showed a moderate anti-mycobacterial
activity with MIC values of 45.09, 45.13, and
78.72 µM, respectively. Compounds 4a, 4d, 4f, 4j, and
4l did not show any growth inhibition even at the
highest concentration tested.
describing drug permeability [21]. A poor permeation
or absorption is more likely, when the compound has
more than five H-bond donors and more than ten H-
bond acceptors. In the present study, 2,5-substituted-
1,3,4-thiadiazoles have a number of H-bond acceptors
(≤ 10) and a lower number of hydrogen bond donors
(≤ 5) (Table 3). The number of rotatable bonds relates
to conformational flexibility and is essential for
binding acceptors or passages. To meet the oral
bioavailability criteria, the number of rotatable bonds
should be less than 10. The title compounds in general
possess high number of rotatable bonds (0‒5) and
therefore, exhibit large conformational flexibility [22].
Absorption, polar surface area, druglikeness,
and pharmacokinetic properties. High oral
bioavailability is an important factor for the
development of bioactive molecules as therapeutic
agents. Good intestinal absorption, reduced molecular
flexibility, low polar surface area, or total hydrogen
bond count (some of donors and some of accepters) are
important predictors of this property [18].
Physicochemical characteristics play a vital role in the
generation and determination of the bioactivity of any
compound. Molecular properties, such as membrane
permeability and bioavailability, are always
confederate with some basic molecular descriptors
such as partition coefficient (log P), molecular weight
(Mw), or numbers of hydrogen bond acceptors and
donors in the molecule [19]. The rule states that most
molecules with good membrane permeability have
log P ≤ 5, Mw ≤ 500, number of hydrogen bond
acceptors ≤ 10, and number of hydrogen bond donors
≤ 5. This rule is widely used as a filter for drug-like
properties. In the present consideration, the percentage
absorption (ABS) was calculated by the equation ABS
= 109–0.345·PSA [20]. Hydrogen bonding capacity
was also recognized as an important parameter for
Molecular polar surface area (PSA) is a very useful
parameter for the prediction of drug transport
properties and PSA is a sum of surfaces of polar atoms
(usually oxygen, nitrogen and attached hydrogen) in a
molecule. Topological polar surface area (TPSA) was
determined by the fragment-based method of Ertl and
co-workers PSA has been used for characterizing drug
absorption,
including
abdominal
absorption,
bioavailability, CaCO2 permeability, and blood brain
barrier permeability. PSA and volume is inversely
proportional to percentage of absorption (%ABS)
Table 4. Number of rotatable bonds (nrotb) is a simple
topological parameter that measures molecular
flexibility. Rotatable bond is defined as any single non
ring bond, bounded to non terminal atom and it has
been shown to be a very good descriptor of oral
bioavailability of drug. The drug likeness score was a
consolidate effect of physicochemical properties,
pharmacokinetics and pharmaco dynamics of a
compound and is represented by a numerical value
computed by MolSoft (MolSoft, 2007). The drug
likeness score was calculated by considering Milog P
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774
CHANDRA SEKHAR et al.
Table 3. Molinspiration bioactivity scores for compounds 4a‒4m
Compound
GPCRL
–0.47
–0.39
–0.49
–0.41
–0.41
–0.46
–0.57
–0.21
–0.57
–0.59
–0.64
–0.63
–0.57
–1.39
ICM
–0.69
–0.68
–0.67
–0.67
–0.73
–0.75
–0.84
–0.48
–0.79
–0.78
–0.88
–0.76
–0.84
–1.45
KI
NRL
–0.42
–0.33
0.38
PI
EI
nON
3
nOHNH
Nviolations
4a
–0.06
–0.02
–0.13
–0.04
–0.03
–0.07
–0.10
–0.12
–0.06
–0.13
–0.17
–0.20
–0.10
–1.05
–0.60
–0.55
–0.57
–0.58
–0.53
–0.60
–0.73
–0.32
–0.78
–0.80
–0.82
–0.74
–0.73
–1.23
–0.13
–0.14
0.24
1
1
1
1
1
1
1
1
1
1
1
1
1
3
0
0
0
0
0
0
0
0
0
1
1
0
0
0
4b
3
4c
6
4d
–0.38
–0.32
–0.40
–0.55
–0.07
–0.59
–0.64
–0.65
–0.59
–0.55
–2.33
–0.16
–0.17
–0.19
–0.28
–0.10
–0.27
–0.29
–0.33
–0.33
–0.28
–0.66
3
4e
4
4f
4
4g
4
4h
3
4i
3
4j
3
4k
3
4l
6
4m
Isoniazid
4
4
(Logarithm of the octanol/water partition coefficient),
molecular weight, number of heavy atoms, number of
hydrogen donor, number of hydrogen acceptor and
number of violation, number of rotatable bonds,
volume. The maximum drug-likeness score (–0.21)
was found to be 4b in Table 4.The bioactivity scores of
the isolated compounds are compared with standard
drug on the basis of GPCR ligand (GPCRL), ion
channel modulator (ICM), nuclear receptor legend
(NRL), kinase inhibitor (KI), protease inhibitor (PI),
enzyme inhibitor (EI) in Table 3.
using the mobile phases indicated. The spots were
observed by exposure to iodine vapours, UV light or p-
anisaldehyde stain solution. Elemental analysis was
1
performed on a Carlo Erba 1108 analyzer. The H
NMR spectra were measured on a Bruker Advance
1
spectrometer at 400.1 and 100.6 MHz for H for 13C
respectively, in CDCl3 or DMSO-d6 against internal
TMS. Chemical shifts are given in ppm (δ) and
referenced to the residual proton resonances of the
solvents (1H NMR: TMS at 0.00 ppm, CDCl3 at
7.26 ppm, DMSO at 2.50 ppm; 13C NMR: CDCl3 at
77.16 ppm, DMSO at 40.00 ppm).
EXPERIMENTAL
General procedure for synthesis of substituted
hydrazine carbothioamides (3a‒3m). Aromatic iso
thiocyanates 1 (2.40 mmol) was added to a stirred
solution of aromatic substituted hydrazides 2 (2 mmol)
and triethylamine (2 mmol) in 10 mL of THF. The
reaction mixture was stirred at room temperature, and
then the solvents were removed on a rotary evaporator.
The residue was triturated with diethyl ether–ethyl
acetate (95 : 5) to afford 95% of the desired
thiosemicarbazide.
All reactions were carried out under argon in oven
dried glassware with magnetic stirring. Unless
otherwise noted, all materials were obtained from
commercial suppliers and used without further
purification. All solvents were reagent grade. Unless
otherwise noted, organic extracts were dried with
anhydrous Na2SO4, filtered through a fitted glass
funnel, and concentrated on a rotary evaporator (20–30
Torr). Commercial chemicals were treated as follows:
DMF was distilled over CaH2 and degassed (freeze and
thaw) three times prior to use; THF, ether, and hexane
were distilled over Na/benzophenone. Flash chromato-
graphy was performed on silica gel (200–300 mesh),
General procedure for synthesis of 2,5-substituted-
1,3,4-thiadiazoles (4a‒4m). a. Conventional method.
p-Toluene sulfonyl chloride (0.60 mmol) was added to
a stirred solution of substituted thiosemicarbazide 3
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DESIGN AND SYNTHESIS OF 1,3,4-THIADIAZOLE DERIVATIVES
775
Table 4. Calculated percentage absorptions, polar surface areas, Lipinski parameters, and drug-likeness model scores for
compounds 4a‒4m
Absorption,
%
Drug-likeness model
score
Compound
V
TPSA NROTB HBA HBD milog P
MW
4a
95.55
95.55
47.70
95.55
74.27
95.55
92.76
95.55
95.55
95.55
95.55
80.14
92.76
85.53
90.50
238.46
243.40
261.80
252.00
264.01
255.03
247.21
269.76
226.59
235.20
238.22
245.00
247.21
122.56
245.26
37.81
37.81
83.64
37.81
47.05
37.81
47.05
37.81
37.81
37.81
37.81
83.64
47.05
68.01
53.60
4
4
5
4
5
4
4
5
3
3
3
4
4
1
0
3
3
5
3
4
3
4
3
3
3
3
5
4
3
2
1
1
1
1
1
1
1
1
1
1
1
1
1
3
4
3.83
3.99
3.79
4.51
3.89
4.28
4.62
4.72
4.73
5.24
5.01
4.52
4.62
–9.70
–0.69
267.36
285.35
312.35
301.80
297.38
281.38
283.36
335.35
271.32
287.77
267.36
298.33
283.36
137.14
298.96
–0.77
–0.21
–0.50
0.02
4b
4c
4d
4e
–0.35
–0.55
–0.74
–0.93
–0.52
–0.27
–1.01
–1.17
–0.74
0.51
4f
4g
4h
4i
4j
4k
4l
4m
Isoniazid
Cisplatin
–1.12
(0.5 mmol) and triethylamine (1.1 mmol) in 4 mL of
NMP. The reaction mixture was stirred at room
temperature for 2 h and extracted with DCM (15 mL)
and distilled water (10 mL), after which the aqueous
layer was removed. The aqueous layer was back-
extracted with DCM (3 × 10 mL). The combined
organic layers were dried over Na2SO4 and evaporated
to leave the desired crude product, which was purified
by column chromatography on silica gel (hexane/ethyl
acetate) to obtain 2,5-substituted-1,3,4-thiadiazole
derivative 4 (yield 44–82%).
and the residue was treated with DCM (2×20 mL). The
combined organic extracts were dried over Na2SO4 and
evaporated under reduced pressure. The obtained crude
product was recrystallized from methanol to obtain
2,5-substituted-1,3,4-thiadiazole derivative 4 (yield
66–87%).
N-Benzyl-5-phenyl-1,3,4-thiadiazole-2-amine (4a).
Brown solid, mp 180–181°C. IR spectrum, ν, cm–1:
3438 (NH), 3070 (Ar=CH), 2958, 2868 (CH), 1597,
1
1555, 1488 (ArC=C), 1392 (C=N), 1217 (CSC). H
NMR spectrum, δ, ppm: 7.81–7.77 m (2H, ArH), 7.41–
7.31 m (8H, ArH), 5.72 br.s (1H, NH), 4.60 d (2H, J =
4 Hz, CH2). 13C NMR spectrum, δC, ppm: 169.46 (C5),
158.38 (C2), 136.97 (ArC), 130.98 (ArC), 129.85
(ArC), 128.90 (ArC), 128.89 (ArC), 128.06 (ArC),
127.81 (ArC), 126.92 (ArC), 50.75 (CH2). Found, %:
C 67.78; H 4.96; N 15.91; S 12.16. C15H13N3S.
Calculated, %: C 67.39; H 4.90; N 15.72; S 11.99.
[M]+ 266.
b. Microwave-assisted method. A mixture of com-
pound isothiocyanate 1 (1.0 equiv), aromatic sub-
stituted hydrazide 2 (0.8 equiv), and trimethyl amine
(0.8 equiv) in THF (5 vol %) was loaded into a closed
vessel and subjected to MW irradiation at 65°C for
18 min. Completion of the reaction to form product 3
was monitored by TLC monitoring. After that thios-
emicarbazide, tri ethylamine, and NMP were added to
the reaction mixture, and it was MW-irradiated at 90°C
for 3–6 min. The organic solvent was then distilled off,
N-Benzyl-5-(4-fluorophenyl)-1,3,4-thiadiazole-2-
amine (4b). White solid, mp 173–175°C. IR spectrum,
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CHANDRA SEKHAR et al.
ν, cm–1: 3370 (NH), 3058 (Ar=CH), 2932, 2867 (CH),
δC, ppm: 173.21 (C5), 163.89 (C2), 157.12 (ArC),
132.19 (ArC), 130.71 (ArC), 128.10 (ArC), 126.04
(ArC), 115.78 (ArC), 56.46 (OCH3), 42.13 (CH2).
Found, %: C 64.72; H 5.91; N 14.84; S 10.04.
C16H15N3OS. Calculated, %: C 64.62; H 5.08; N 14.13;
S 10.78. [M]+ 297.
1593, 1514 (ArC=C), 1399 (C=N), 1218 (CSC), 979
1
(C–F). H NMR spectrum, δ, ppm: 8.45 t (1H, J =
8 Hz, NH), 7.83–7.75 m (2H, ArH), 7.41–7.25 m (7H,
ArH), 4.55 d (2H, J = 4 Hz, CH2). 13C NMR spectrum,
δC, ppm: 168.92 (C5), 162.18 (1JCF = 248.5 Hz), 155.61
(C2), 139.02 (ArC), 119.91 (3JCF = 9.1 Hz), 128.92,
128.05 (ArC), 127.91 (4JCF = 3.0 Hz), 127.67 (ArC),
116.70 (2JCF = 22.2 Hz), 48.57 (CH2). Found, %: C
63.84; H 4.92; F 6.78; N 14.98; S 11.46. C15H12N3SF.
Calculated, %: C 63.14; H 4.24; F 6.66; N 14.73; S
11.24. [M]+ 284.
N-benzyl-5-(p-tolyl)-1,3,4-thiadiazol-2-amine (4f).
White solid, mp 170–171°C. IR spectrum, ν, cm–1:
3425 (NH), 3056 (Ar=CH), 2984 (CH), 1545, 1491
(ArC=C), 1331 (C=N), 1228 (C–S–C), 1136 (C–O–C).
1H NMR spectrum, δ, ppm: 8.24 d (2H, J = 7.5 Hz,
ArH), 7.45–7.40 m (3H, ArH), 7.28 d (2H, J = 8.2 Hz,
ArH), 7.16 d (2H, J = 8.2 Hz, ArH), 5.42 t (1H, J =
4 Hz, NH), 4.35 d (2H, J = 4 Hz, CH2), 2.95 s (3H,
CH3). 13C NMR spectrum, δC, ppm: 176.18 (C5),
154.19 (C2), 141.26 (ArC), 134.41 (ArC), 133.15
(ArC), 131.78 (ArC), 130.52 (ArC), 130.03 (ArC),
128.18 (ArC), 125.51 (ArC), 119.84 (ArC), 35.89
(CH2), 29.40 (CH3). Found, %: C 68.45; H 5.40; N
14.75; S 11.91. C15H13N3S. Calculated, %: C 68.30; H
5.37; N 14.93; S 11.40. [M]+ 281.
N-Benzyl-5-(4-nitrophenyl)-1,3,4-thiadiazole-2-
amine (4c). Yellow solid, mp 198–200°C.IR spectrum,
ν, cm–1: 3420 (NH), 3062 (Ar=CH), 2985, 2856 (CH),
1592, 1551, 1487 (ArC=C), 1345 (C=N), 1205 (C–S–C).
1H NMR spectrum, δ, ppm: 8.78 t (1H, J = 4 Hz, NH),
8.30 d (2H,J = 4 Hz, 2H, ArH), 8.03–8.01 d (2H, J =
8 Hz, ArH), 7.42–7.29 m (5H, ArH), 4.60 d (2H, J =
4 Hz, CH2).13C NMR spectrum, δC, ppm: 170.19 (C5),
154.19 (C2), 147.97 (ArC), 138.67 (ArC), 137.11
(ArC), 128.93 (ArC), 128.08 (ArC), 127.79 (ArC),
127.64 (ArC), 124.92 (ArC), 48.64 (CH2). Found, %:
C 57.70; H 3.90; N 18.14; S 10.30. C15H12O2N4S.
Calculated, %: C 57.68; H 3.87; N 17.94; S 10.27.
[M]+ 310.
N-(4-Methoxybenzyl)-5-phenyl-1,3,4-thiadiazole-
2-amine (4g). White solid, mp 227–229°C. IR spec-
trum, ν, cm–1: 3449 (NH), 3062 (Ar=CH), 2980, 2842
(CH), 1565, 1512 (ArC=C), 1348 (C=N), 1215 (CSC),
1
1189 (COC). H NMR spectrum, δ, ppm: 8.40 t (1H,
N-Benzyl-5-(4-chlorophenyl)-1,3,4-thiadiazole-2-
amine (4d). White solid, mp 188–190°C. IR spectrum,
ν, cm–1: 3405 (NH), 3052 (Ar=CH), 2945, 2850 (CH),
1590, 1548 (ArC=C), 1445 (C=N), 1198 (CSC), 826
J = 4 Hz, NH), 7.76–7.74 d.d (2H, J = 8 Hz, ArH),
7.47 d (3H, J = 8 Hz, ArH), 7.34 d (2H, J = 12 Hz,
ArH), 6.94–6.91 d.d (2H, J = 8.5 Hz, ArH), 4.47 d
(2H, J = 4 Hz, ArH), 3.74 s (3H, OCH3). 13C NMR
spectrum, δC, ppm: 172.45 (C5), 164.24 (C2), 158.94
(ArC), 133.78 (ArC), 133.38 (ArC), 129.12 (ArC),
128.79 (ArC), 127.94 (ArC), 114.9 (ArC), 55.8
(OCH3), 46.8 (CH2).Found, %: C 64.84; H 5.78; N
14.23; S 10.94. C16H15N3OS. Calculated, %: C 64.62;
H 5.08; N 14.13; S 10.78. [M]+ 296.
1
(CCl). H NMR spectrum, δ, ppm:8.21 d (2H, J =
8.0 Hz, ArH), 7.61 d (2H, J = 8.0 Hz, ArH), 7.45–7.36
m (5H, ArH), 5.21 t (1H, J = 3.9 Hz, NH), 4.60 d (2H,
J = 4.1 Hz, CH2).13C NMR spectrum, δC, ppm: 178.61
(C5), 168.41 (C2), 143.13 (ArC), 137.71 (ArC), 135.12
(ArC), 131.85 (ArC), 130.19 (ArC), 129.21 (ArC),
128.61 (ArC), 127.92 (ArC), 127.21 (ArC), 55.61
(CH2). Found, %: C 60.45; H 4.91; Cl, 11.82; N 14.12;
S 10.71. C15H12ClN3S. Calculated, %: C 59.70; H 4.01;
Cl, 11.75; N 13.92; S 10.62. [M]+ 300.
N-[4-(Trifluoromethyl)benzyl]-5-phenyl-1,3,4-
thiadiazole-2-amine (4h). White solid, mp 153–155°C.
IR spectrum, ν, cm–1: 3478 (NH), 3067 (Ar=CH),
2982, 2835 (CH), 1570, 1513 (ArC=C), 1345 (C=N),
1
N-benzyl-5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-
amine (4e). White solid, mp 229–230°C. IR spectrum,
ν, cm–1: 3440 (NH), 3052 (Ar=CH), 2976 (CH), 1585,
1541 (ArC=C), 1384 (C=N), 1218 (CSC), 1161
1189 (CSC), 942 (C–F). H NMR spectrum, δ, ppm:
8.57 t (1H, J = 4 Hz, NH), 7.75–7.72 m (4H, ArH),
7.52–7.50 d (2H, J = 8 Hz, ArH), 7.50–7.41 m (3H,
ArH), 4.67 d (2H, J = 4 Hz, CH2). 13C NMR spectrum,
δC, ppm: 168.68 (C5), 157.13 (C2), 144.17 (ArC),
131.28 (ArC), 130.25 (ArC), 129.57 (ArC), 128.56
(ArC), 126.93 (ArC), 128.21 (2JCF = 31.3 Hz), 125.72
(3JCF = 3.8 Hz), 124.86 (1JCF = 273.7 Hz), 47.92
(CH2). Found, %: C 57.78; H 3.92; F 17.76; N 12.46; S
1
(COC). H NMR spectrum, δ, ppm: 8.42 t (1H, J =
4 Hz, NH), 7.78–7.75 d.d (2H, J = 8 Hz, ArH), 7.45 d
(3H, J = 8 Hz, ArH), 7.36 d (2H, J = 12 Hz, ArH),
6.85–6.83 d.d (2H, J = 8.5 Hz, ArH), 4.47 d (2H, J =
4 Hz, CH2), 3.65 s (3H, OCH3). 13C NMR spectrum,
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DESIGN AND SYNTHESIS OF 1,3,4-THIADIAZOLE DERIVATIVES
777
9.91. C16H12F3N3S. Calculated, %: C 57.31; H 3.61; F
ArH), 7.58–7.52 m (3H, ArH), 7.12 d (2H, J = 8.2 Hz,
ArH), 4.52 s (1H, NH). 13C NMR spectrum, δC, ppm:
175.23 (C5), 159.41 (C2), 141.13 (ArC), 138.24 (ArC),
137.91 (ArC), 134.85 (ArC), 132.16 (ArC), 130.92
(ArC), 130.34 (ArC), 129.22 (ArC), 128.23 (ArC).
Found, %: C 56.68; H 3.92; N 18.92, S 10.92.
C14H10N4O2S. Calculated, %: C 56.37; H 3.38; N
18.78, S 10.75. [M]+ 297.
17.00; N 12.53; S 9.56. [M]+ 333.
N-(4-Fluorophenyl)-5-phenyl-1,3,4-thiadiazole-2-
amine (4i). White solid, mp 247–250°C. IR spectrum,
ν, cm–1: 3476 (NH), 3048 (Ar=CH), 1561, 1520
1
(ArC=C), 1341 (C=N), 1195 (CSC), 938 (C–F). H
NMR spectrum, δ, ppm: 9.34 br.s (1H, NH), 7.86–7.84
m (2H, ArH), 7.47–7.25 m (5H, ArH), 7.14–7.09 m
(2H, ArH. 13C NMR spectrum, δC, ppm: 169.14 (C5),
162.58 (C2), 161.25 d (1JCF = 248.0 Hz), 152.05 (ArC),
N-(4-Methoxyphenyl)-5-phenyl-1,3,4-thiadiazole-
2-amine (4m). White solid, mp 227–229°C. IR
spectrum, ν, cm–1: 3423 (NH), 3048 (Ar=CH), 2921
(CH), 1578, 1489 (ArC=C), 1342 (C=N), 1226 (CSC),
131.68 d (3JCF = 9.4 Hz), 131.34 d (4JCF
=
3.1 Hz), 130.86 (ArC), 129.02 (ArC), 128.85 (ArC),
126.28 (ArC), 116.69 d (2JCF = 22.6 Hz). Found, %: C
62.19; H 3.87; F 7.67; N 15.78; S 12.12. C14H10FN3S.
Calculated, %: C 61.98; H 3.72; F 7.00; N 15.49; S
11.82. [M]+ 269.
1
1165 (COC). H NMR spectrum, δ, ppm: 8.86 s (1H,
NH), 7.83 d.d (2H, J = 8 Hz, ArH), 7.47–7.42 m (3H,
ArH), 7.25 m (2H, ArH), 6.95 d (2H, J = 8.5 Hz, 2H,
ArH), 3.83 s (3H, OCH3).13C NMR spectrum, δC, ppm:
169.32 (C5), 159.94 (C2), 151.12 (ArC), 130.68 (ArC),
130.14 (ArC), 129.12 (ArC), 128.56 (ArC), 127.60
(ArC), 126.44 (ArC), 114.87 (ArC), 55.78 (OCH3).
Found, %: C 60.67; H 5.02; N 14.92, S 11.92. C15H13N3OS.
Calculated, %:C, 63.58; H 4.62; N 14.83,S, 11.32.
[M]+ 282.
N-(4-Chlorophenyl)-5-phenyl-1,3,4-thiadiazole-2-
amine (4j). White solid, mp 168–170°C. IR spectrum,
ν, cm–1: 3478 (NH), 3038 (Ar=CH), 1546, 1489
1
(ArC=C), 1410 (C=N), 1174 (CSC), 886 (C–Cl). H
NMR spectrum, δ, ppm: 8.11 d (2H, J = 7.8 Hz, ArH),
7.61–7.56 m (3H, ArH), 7.45 d (2H, J = 8.2 Hz, ArH),
7.39 d (2H, J = 8.2 Hz, ArH), 4.60 s (1H, NH). 13C
NMR spectrum, δC, ppm: 172.16 (C5), 162.44 (C2),
142.13 (ArC), 139.18 (ArC), 135.91 (ArC), 133.85
(ArC), 132.11 (ArC), 130.91 (ArC), 130.12 (ArC),
129.92 (ArC), 127.23 (ArC). Found, %: C 58.78; H
3.98; Cl, 12.46; N 14.78; S 11.79. C15H10ClN3S.
Calculated, %: C 58.43; H 3.50; Cl, 12.32; N 14.60; S
11.14. [M]+ 270.
Biological activity screening. Cytotoxicity. The in
vitro anti-cancer activity of compounds 4а–4m was
tested using the MTT colorimetric assay following the
ATCC protocol [23,24]. The cell lines used for in vitro
cytotoxicity testing included MDAMB231 derived
from human lung carcinoma cells (ATCC No. CCL-
185) and HEK293T derived from human embryonic
kidney cells (non-cancerous; ATCC No. CRL-1573),
purchased from American Type Culture Collection
(Manassas, VA, USA). The breast cancer cell line
A549 was maintained in DMEM medium supple-
mented with 10% newborn calf serum (NBCS), 1%
nonessential amino acids, 0.2 % sodium bicarbonate,
1% sodium pyruvate, and 1% antibiotic mixture (10
000U of penicillin and 10 mg of streptomycin per mL).
MDAMB231 and HEK293T were maintained in RPMI-
1640 medium supplemented with 10% NBCS,
100 IU/mL penicillin, 100 mg/mL streptomycin, and 2
mM glutamine. Cell lines were maintained at 37°C in a
humidified 5% CO2 incubator (Thermo Scientific).
Cell lines were processed by initial trypsinization to
detach the adhered cells followed by centrifugation to
obtain a cell pellet. In the fresh medium the pellet of a
cell count using haemocytometer and take in 96-well
plate added to 100 μL of medum with range from
5.000–6.000 per well. The plate was incubated
overnight in CO2 incubator for cells to adhere and
regain their shape. After 24 h, cells were treated with
N-(p-Tolyl)-5-phenyl-1,3,4-thiadiazole-2-amine (4k).
White solid, mp 168–170°C. IR spectrum, ν, cm–1:
3412 (NH), 3047 (Ar=CH), 2972, 2845 (CH), 1572,
1483 (ArC=C), 1340 (C=N), 1215 (C–S–C), 1148
1
(C–O–C). H NMR spectrum, δ, ppm: 8.09 d (2H, J =
7.5 Hz, ArH), 7.52–7.45 m (3H, ArH), 7.38 d (2H, J =
8.2 Hz, ArH), 7.32 d (2H, J = 8.2 Hz, ArH), 4.92 s
(1H, NH), 2.97 s (3H, CH3).13C NMR spectrum, δC,
ppm: 174.12 (C5), 155.40 (C2), 142.15 (ArC), 134.16
(ArC), 133.85 (ArC), 131.12 (ArC), 130.98 (ArC),
130.05 (ArC), 129.91 (ArC), 126.26 (ArC), 120.32
(ArC), 28.49 (CH3). Found, %: C 68.12; H 5.26; N
15.98; S 12.24. C15H13N3S. Calculated, %: C 67.39; H
4.90; N 15.72; S 11.99. [M]+ 266.
N-(4-Nitrophenyl)-5-phenyl-1,3,4-thiadiazole-2-
amine (4l). White solid, mp 189–191°C. IR spectrum,
ν, cm–1: 3426 (NH), 3098 (Ar=CH), 1601, 1542 (ArC=C),
1
1340 (C=N), 1214 (CSC). H NMR spectrum, δ, ppm:
8.19 d (2H, J = 7.4 Hz, ArH), 8.11 d (2H, J = 8.2 Hz,
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 89 No. 4 2019

778
CHANDRA SEKHAR et al.
the test compounds (25 μM in the medium) and
incubated for 48 h to assay the effect of the test
compounds on different cell lines. Zero hour reading
was taken with untreated cells and also control with
1% DMSO to subtract further from the 48 h reading.
After 48 h incubation, cells were treated by the MTT
reagent [4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide] dissolved in PBS (5 mg/mL) and
incubated for 3–4 h at 37 ˚C. The formazan crystals
thus formed were dissolved in 100 μL of DMSO, and
viability was measured at 540nm on a Spectra Max
multimode reader.
Lipinski’s rule of five using the Molinspiration online
property calculation toolkit [28]. The percentage
absorption was calculated by the equation [20]:
ABS, % = 109–0.345·PSA.
CONCLUSIONS
In summary, 2,5-substituted-1,3,4-thiadiazoles were
designed and synthesized. The antitubercular and
cytotoxic activities of the synthesized 1,3,4-thiadiazole
derivatives were evaluated against M. smegmatis and
breast cancer cell lines and one normal cell line. The
SAR analysis showed that a smaller aromatic ring and
electron-withdrawing groups favor both activities, and
this paves the way for further design and chemical
modification of thiadiazoles. The molecular and
pharmacokinetic properties, and drug likeness model
scores were calculated by Molinspiration and MolSoft.
In terms of the Lipinski’s rule of five, compounds 4b
and 4j displayed the highest drug likeness (–0.21,
–0.27) as potential orally bioavailable antitubercular
and anticancer agents.
Antitubercular activity. The antitubercular activity
of the synthesized compounds was tested using the M.
smegmatis MC-155strain obtained from the Tuber-
culosis Research Center, Indian Council of Medical
Research, Chennai, India. The assay was performed in
a 96-well microtiter format plate. Initially, 10 mM
stock concentrations of the test compounds were
diluted with DMSO to achieve 1.5 mM (2%) working
concentrations and 30 µM final concentrations. The
agar dilution assay was performed using Middlebrook
7H9 broth supplemented with 0.1% Tween 80 and
0.5% glycerol [25–27]. At the time of inoculation,
10% albumin dextrose and saline (ADS) was added.
When the optical density of the inoculum reached
~0.4, the inoculum was diluted 1 : 1000. A 2µL aliquot
of a 1.5 mM concentration was added in triplicate to
each well, after which 98 µL of inoculum (1 : 1000)
was added, and the cultures were incubated for 30–
32 h in a shaker incubator at 37°C and 200 rpm. After
the incubation period, absorbance of the inoculum in
each well was observed at 600nm in a Multimode
Reader, and assays were usually completed in 5–
8 days. The percentage inhibition was measured as GI
(growth inhibition) of test sample/GI of control)
×100%. The minimum inhibitory concentration of a
molecule, that caused a reduction of daily GIrate
smaller than that observed with a 1 : 1000 dilute
control culture on the next day, reached a GI of at least
30. Isoniazid was used as a positive control of
inhibition of M. smegmatis.
ACKNOWLEDGMENTS
We are grateful to our research supervisor Dr.
Anjali Jha and Dr. Lav Kumar for providing the
required facilities and motivation for completion of the
research work. We also extend our gratitude toward
Department of Chemistry, GIS, GITAM University.
CONFLICT OF INTEREST
No conflict of interest was declared by the authors.
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