Synthesis of 5-Arylidine amino-1,3,4-thiadiazol-2-[(N-substituted benzyol)]sulphonamides endowed with potent antioxidants and anticancer activity induces growth inhibition in HEK293, BT474 and NCI-H226 cells

Article abstract of DOI:10.1007/s00044-013-0890-z

A series of imines 5-Amino-1,3,4-thiadiazol-2-[(N-substituted benzyol)]sulphonamide derivatives were synthesized from various aromatic aldehydes and substituted with benzoyl acetazolamides under different reaction conditions and were evaluated for their antioxidant and free radical scavenging, antimitotic activity by Allium cepa meristem root model and cytotoxicity activity against HEK 293 (human epidermal kidney cell line), BT474 (breast cancer cell line) and NCI-H226 (lung cancer cell line) by MTT assay. Some of the synthesized compounds showed moderately potent cytotoxicity compared to indisulam. Graphical abstract: A series of imines 5-Amino-1,3,4-thiadiazol-2- [(N-substituted benzyol)]sulphonamide derivatives (9a-j); 5-Amino-1,3,4- thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (4a-g); 5-(4-Acetamido phenyl sulphonamido)-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (6a-g); and 5-(4-Amino phenyl sulphonamido)-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (7a-g) were synthesized from acetazolamide and were investigated for the in vitro anticancer by MTT assay, free radical scavenging and antimitotic activity by Allium cepa root meristem model. Experimental observations indicate that synthesized compounds were moderately potent anticancer agents. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-013-0890-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0890-z
ORIGINAL RESEARCH
Synthesis of 5-arylidine amino-1,3,4-thiadiazol-2-[(N-substituted
benzyol)]sulphonamides endowed with potent antioxidants
and anticancer activity induces growth inhibition in HEK293,
BT474 and NCI-H226 cells
•
Mahavir Chhajed Anil Kumar Shrivastava
•
Vijay Taile
Received: 19 August 2013 / Accepted: 11 December 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract A series of imines 5-amino-1,3,4-thiadiazol-2-
[(N-substituted benzyol)]sulphonamide derivatives were
synthesized from various aromatic aldehydes and substi-
tuted with benzoyl acetazolamides under different reaction
conditions and were evaluated for their antioxidant and free
radical scavenging, antimitotic activity by Allium cepa
meristem root model and cytotoxicity activity against HEK
293 (human epidermal kidney cell line), BT474 (breast
cancer cell line) and NCI-H226 (lung cancer cell line) by
MTT assay. Some of the synthesized compounds showed
moderately potent cytotoxicity compared to indisulam.
DNA base sequences. Development of anticancer drugs
with fewer or no side effects is important for the treatment
for cancer. The search for such potential anticancer drugs
has led to the discovery of synthetic small molecules with
anti-carcinogenic activity and limited harmful side effects
particularly with respect to the immune system. Research
in this area is expanding rapidly, and some promising leads
have emerged. Heterocyclic moieties can be found in a
large number of compounds, which display biological
activity. The biological activity of the compounds is
mainly dependent on their molecular structures (Salimon
et al., 2010). A vast number of 1,3,4-thiadiazoles have been
reported as potential pharmacologically active compounds
with antimicrobial (Patil and Biradar, 2001; Zamani et al.,
2004; Sharma et al., 2006), antiviral (Pandey et al., 2004),
antitubercular (Oruc et al., 2004; Desai et al., 1984),
anticonvulsant (Shrivastava et al., 1999; Kumar et al.,
2003; Gupta et al., 2008; Stillings et al., 1986; Jatav et al.,
2008), CNS depressant (Jatav et al., 2008), hypoglycaemic
(Hanna et al., 1995; Pattan et al., 2009), anti-inflammatory
(Sharma et al., 2008; Varandas et al., 2005) and anticancer
(Noolvi et al., 2011; Kumar et al., 2010) properties. At the
same time, the 1,3,4-thiadiazole fragment appears in a
number of clinically used drugs such as acetazolamide;
methazolamide; butazolamide (diuretic); sulfamethiazole
(antibacterial); cefazolin, cefazedone (antibiotic); atibe-
prone (anti-depressant); glybuthiazole, glybuzole (antidia-
betic); and tebuthiuron (insecticide) (Wilson and Gisvold,
1991; Abrahum, 2003; Supran et al., 2003).
Keywords 1,3,4-Thiadiazole ꢀ Antimitotic ꢀ
Antioxidants ꢀ Cytotoxicity ꢀ MTT assay
Introduction
The rapid spread of cancer has sparked an intense world-
wide search for new compounds, which may be used in
designing anticancer drugs. The search of more effective
anticancer agent has focused to a large extent on the design
of molecules capable of recognizing and binding to target
M. Chhajed (&)
Department of Pharmaceutical Chemistry, Suresh Gyan Vihar
University, Mahal Jagat Pura, Jaipur, India
e-mail: mahavirchhajed@rediffmail.com
Schiff bases, contain an azomethine group, derived from
aromatic aldehydes and aromatic amines, have potential for
both chemical and biological activities (Dhar and Taploo,
1982; Pacheco et al., 1970). This is due to the presence of
carbon–nitrogen double bond having potential receptor-
binding ability. Schiff bases are also one of the intensively
A. K. Shrivastava
Nandini Nagar Mahavidyalaya College of Pharmacy,
Nawabganj, Gonda, Uttar Pradesh, India
V. Taile
Department of Chemistry, RTM Nagpur University, Nagpur,
India
123

Med Chem Res
investigated classes of aromatic and heteroaromatic com-
pounds. This class of compounds showed a variety of
applications ranging from anticancer (Sharma et al., 1998;
Kuzmin et al., 2005), antibacterial (More et al., 2002;
Vaghasiya et al., 2004), diuretic (Supran et al., 1996),
antifungal (Manrao et al., 1982, 1995, 2001) and antipar-
asitic activity (Rathelot et al., 2002). They have also
medicinal importance and are used in drug design due to
their activity against a wide range of organisms (Khan
et al., 2002; Verma et al., 2004). Schiff bases are used as
substrates in the preparation of a number of industrially and
biologically active compounds via closure, cycloaddition
and replacement reactions (Taggi et al., 2002).
this reduced pHe are associated with tumour progression and
poor prognosis (Parkkila et al., 2000; Svastova et al., 2004).
Aromatic sulphonamide compounds have been shown to
reverse the effect of tumour acidification, to inhibit the
growth of cancer cells and to suppress tumour invasion
mediated by these CAs (Tureci et al., 1998; Wykoff et al.,
2000; Parkkila et al., 2000; Svastova et al., 2004; Cecchi
et al., 2005; Brzozowski et al., 2010).
Thus, the data from these many physiological studies
appear to have identified a CA-mediated, hypoxic tumour-
specific pathway. This provides firm grounds for exploring
the effects of this class of compounds as a novel approach
to discriminate between healthy cells and cancerous cells,
specifically targeting hypoxic tissues, an attractive attribute
that is lacking in many existing cancer therapies (Minch-
inton and Tannock 2006; Kamb, 2005).
Sulphonamides are a significant class of compounds in
medicinal and pharmaceutical chemistry with several bio-
logical applications (Tilles, 2001; Slatore and Tilles, 2004;
Brackett et al., 2004; Harrison, 1994; Eroglu, 2008).
There are many connections between carbonic anhydrase
(CA) and cancer (Supuran, 2008; Supuran and Scozzafava,
2000; Pastorek et al., 1994; Pastorekova et al., 1997; Cheg-
widden et al., 2001). It is well known that some CA isozymes
are predominantly found in cancer cells and are lacking from
their normal counterparts (Pastorek et al., 1994; Pastorekova
et al., 1997; Chegwidden et al., 2001), and these are two
transmembrane isozymes CA IX and CA XII. Isozyme CA
XIV was the last one to be discovered among the 15 CA
isoforms of this widespread metalloprotein known up to now
in human (Supuran et al., 2004). Kaunisto et al. (2002) and
Parkkila et al., (2001, 2002) revealed CA XIV distribution in
the human body as well as potential physiological/patholo-
gical roles. It has been observed that hCA XIV is highly
abundant in the brain, kidney, colon, small intestine, urinary
bladder, liver and spinal cord (Kaunisto et al., 2002; Parkkila
et al., 2001, 2002; Fujikawa-Adachi et al., 1999; Ashida
et al., 2002). Similar to isozymes CA IX and CA XII, CA XIV
is a transmembrane protein with the active site oriented
extracellularly, but unlike the first two proteins, isozyme XIV
is not associated with tumour cells (Pastorek et al., 1994;
Kaunisto et al., 2002; Parkkila et al., 2001, 2002; Ashida
et al., 2002). Membrane-associated human carbonic anhy-
drase (hCAs) isozymes IX, XII and XIV (Fujikawa-Adachi
et al., 1999; Tureci et al., 1998) like other hCAs regulate pH
and carbon dioxide (CO2)–bicarbonate anion (HCO3) ho-
moeostasis, through the catalysis of the reversible hydration
ofCO2 togiveHCO3 andproton(Hþ). Theexpressionlevelof
isozymes hCA IX and XII is elevated in response to hypoxia,
and research on the involvement of these isozymes in cancer
has progressed considerably in recent years, particularly for
hCA IX (Tureci et al., 1998; Wykoff et al., 2000; Parkkila
et al., 2000; Svastova et al., 2004; Cecchi et al., 2005). It has
been confirmed that hCA IX is a high-activity CA isozyme
responsible for the extracellular acidification (pHe) of the
tumour microenvironment. Multiple downstream effects of
These findings prompted us to the synthesis of 5-arylidine
amino-1,3,4-thiadiazol-2-[(N-benzoyl)]sulphonamide deriv-
atives (9a–j) from carbonic anhydrase inhibitor drug aceta-
zolamide. The synthesized compounds reported previously
(Chhajed et al., 2007, 2013), such as 5-amino-1,3,4-thia-
diazol-2-[N-(substituted benzoyl)]sulphonamide (4a–g),
5-(4-acetamido phenyl sulphonamido)-1,3,4-thiadiazol-2-
[N-(substituted benzoyl)]sulphonamide (6a–g), and 5-(4-
amino phenyl sulphonamido)-1,3,4-thiadiazol-2-[N-(substi-
tuted benzoyl)]sulphonamide (7a–g) from acetazolamide by
modified Schotten–Bauman synthesis method, and com-
pounds (9a–j) reported herein are evaluated for anticancer
activity, having better therapeutic index for free radical
scavenging, antimitotic activityandin vitro cytotoxicactivity
by MTT assay for establishing their possible therapeutic
value. The synthesized molecules have been characterized by
various techniques such as NMR, FTIR and LCMS.
Results and discussion
Chemistry
5-Amino-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sul-
phonamides (4a–g) were prepared by hydrolysis of the
benzoylated acetazolamides (3a–g), which was prepared
from the acetazolamide (1) by benzoylation with substi-
tuted benzoyl chlorides (2a–g). Compound (4) was
refluxed with substituted aromatic aldehydes (8a–j) using
concentrated sulphuric acid as a catalyst to obtain the
Schiff bases (Scheme 1).
The FT-IR spectra showed stretching at 3,485–3,265 cm-1
(sulphonamide N–H), 3,037.4 (Ar C–H), 1,676–1,645 (C=O),
1,625–1,594 cm-1 (C=N), 1,517–1,530.9 (Ar C–C),
1,270 cm-1 (C–N), 1,177–1,125 cm-1 (sulphonamide),
1,128–1,030 cm-1 (S=O) and 756–662 cm-1 (thiadiazole
C–S). The 1H-NMR spectra of all compounds indicated
123

Med Chem Res
O
O
O
O
N
N
N
N
O
S
O
S
Benzoylation
NaOH
+
H3C
N
H3C
N
Cl
NH2
S
N
H
S
H
R1
R1
H
O
O
(1)
(3a-g)
(2a-g)
Hydrolysis
10% NaOH
O
O
S
Cl
H3C
O
O
O
N
N
O
N
N
O
(5)
H
N
O
S
N
S
N
S
S
H2N
H
N
H
R1
S
H
O
O
O
R1
H3C
O
(6a-g)
(4a-g)
Hydrolysis
10% NaOH
Ar-CHO
(8a-j)
H2SO4
O
O
N
N
N
N
O
S
O
S
H
O
S
N
H
NH2
N
H
Ar
N
N
S
S
H
O
O
O
R1
R1
(7a-g)
(9a-j)
Compd. No.
R1
Ar
Compd. No.
R1
9a
9b
9c
9d
9e
9f
H
H
H
H
H
H
H
H
H
H
C6H5
2a 3a 4a 6a 7a
2b 3b 4b 6b 7b
2c 3c 4c 6c 7c
2d 3d 4d 6d 7d
2e 3e 4e 6e 7e
2f 3f 4f 6f 7f
2g 3g 4g 6g 7g
-H
p-ClC6H4
o-NH2
p-NH2
o-OH
p-NO2
m-NO2
p-Cl
o-OCH3C6H4
p-OCH3 C6H4
p-OH C6H4
o-OH C6H4
9g
9h
9i
m-OCH3, p-OH C6H3
p-N(CH3)2 C6H4
p-NO2 C6H4
9j
O
Scheme 1 Synthesis of 5-amino-1,3,4-thiadiazol-2-[N-(substituted
benzoyl)]sulphonamide (4a–g), 5-(4-acetamido phenyl sulphonami-
do)-1,3,4-thiadiazole-2-[N-(substituted benzoyl)]sulphonamide (6a–
g), 5-(4-amino phenyl sulphonamido)-1,3,4-thiadiazole-2-[N-(substi-
tuted benzoyl)]sulphonamide (7a–g), and 5-arylidine amino-1,3,4-
thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (9a–j)
Pharmacological evaluation
expected peaks in the region of 1.249–1.254 d ppm (s, Ar–
SO2NH), 3.569–4.116 d ppm (s, Schiff base CH=N) and
8.24–8.523 d ppm (s, amide C(=O)N–H), while multiplets of
aromatic ring are in the range of 6.6–8.2 d ppm. Thin-layer
chromatography (TLC) was run throughout the reaction to
optimize the reaction for purity and completion.
Antioxidant and free radical scavenging activity
ABTSꢀ1 radical, lipid peroxidation, DPPH radical, super-
oxide anion and nitric oxide anion radical scavenging
123

Med Chem Res
Table 1 Comparative IC50 inhibitory concentration of synthesized compounds and standards against different free radicals
Compound no.
IC50 inhibitory concentration (nM/mL)a
ABTS? radicalb
Lipid peroxidationc
DPPH radicald
Superoxide anione
Nitric oxide radicalf
9a
9b
9c
9d
9e
9f
73.30 7.05*
[4.07]
121.63 18.60
[10.74]
134.07 12.90*
[22.34]
151.89 14.42*
[24.97]
103.67 7.50*
[12.99]
93.30 10.67*
[6.16]
133.02 11.53*
[6.65]
88.19 11.09*
[6.40]
76.31 11.80*
[6.81]
52.57 16.73*
[9.66]
196.17 16.60*
[9.58]
101.78 14.51**
[8.38]
41.27 4.23**
[2.44]
128.09 21.74*
[12.55]
81.90 10.44*
[6.02]
55.61 6.98*
[4.03]
164.49 14.56*
[8.41]
63.56 8.35**
[4.82]
74.52 8.3*
[4.79]
53.03 6.74*
[3.89]
47.89 9.90*
[5.72]
134.34 14.70**
[8.49]
107.28 18.13**
[10.46]
135.52 22.55*
[13.02]
155.21 17.64*
[10.19]
207.14 17.41*
[10.05]
203.74 20.11**
[11.61]
80.63 11.38**
[6.57]
36.6604 14.39*
[8.31]
38.00 11.77*
[6.79]
Std
84.54 9.39*
[5.42]
150.12 16.93**
[9.77]
187.20 35.38*
[19.96]
171.36 9.10**
[5.25]
73.67 9.44*
[5.45]
* P \ 0.05; ** P \ 0.01
a
IC50 value reported as Conc. SD [SEM]; SEM of three independent experiments performed in duplicate
b
c
d
e
f
Standard used was trolox
Standard used was ascorbic acid
Standard used was ascorbic acid
Standard used was catechin
Standard used was curcumin
Antimitotic activity
activity has been used as a quick and reliable parameter to
assess the in vitro antioxidant activity. Each method relates
to the generation of a different radical, acting through a
variety of mechanisms and the measurement of a range of
end points at a fixed time point or over a range (Miller and
Rice-Evans, 1994, 1996). The different concentrations of
the synthesized compounds showed antioxidant activities
in a dose-dependent manner. Comparative IC50 (nM/mL)
inhibitory concentrations of synthesized compounds
against different free radicals are reported in Table 1. All
the tested compounds showed statistically significant
(P \ 0.05) IC50 values. Among the tested compounds, (9c)
is the most potent compound and had lowest IC50 (nM/mL)
value against DPPH radical, nitric oxide anion and lipid
peroxidation, while (9e) and (9f) showed maximum
potency against ABTSꢀ1 radical and superoxide anion
radical, respectively. The study also indicates that the
compounds (9c), (9d) and (9f) showed the smaller IC50
(nM/mL) values even than respective standards, indicating
that these compounds are more potent than the standard,
and reveals that the electron-donating functional group like
–OCH3 (9c and 9d) or the functional group like –OH
having the ability to bind with free radical (9f) is respon-
sible for the potency.
The levels of the physicochemical parameters of Allium
cepa (root number and root length) were recorded after
treatment with various drugs at 0, 48 and 72 h and found to
cause significant inhibition in the growth of roots in com-
parison with negative control and positive control. From
the observations, it has been revealed that average root
length in (9f) treatment group was decreased significantly
(1.06 cm) compared with that of the negative control
(3.93 cm) after 72 h of treatment. The root morphology
was nearly normal during the negative control treatment,
but at positive control and synthesized compound groups,
the roots morphology showed an obvious difference in its
appearance in that it turned to slightly yellowish to
brownish in colour. Its cytotoxic effect was evident in the
form of shortening and decaying of roots, while progres-
sive increases in root length and root numbers were
observed in control group. The cytotoxic effect of tested
compounds inhibits root growth and mitosis to a significant
extent. The compound 9f showed lowest mitotic index
(0.41 %) with highest activity among all the treatment
groups, and it was also observed that the number of non-
dividing cells increased in all treatment groups other than
123

Med Chem Res
Table 2 Mitotic index and chromosome and mitotic aberrations in the root meristem cells of Allium cepa after the synthesized compounds
treatment
Treatment Dose
groups
MI
Chromosome breaks
Stickiness
(%) SEMb
Polar deviations
(%) SEMb
Aberrant cells
(%) SEMb
MNC
(%) SEMb
(%) SEMa,b (%) SEMb
NCc
PCd
9a
9b
9c
9d
9e
9f
–
6.22 0.32
–
0.92 0.32
36.31 9.84
6.64 2.38
9.12 1.33
28.04 6.34
14.48 2.52
9.88 1.46
21.96 7.22
32.24 6.92
16.50 3.23
7.35 2.06
6.89 1.32
12.36 3.36
8.62 2.16
7.32 1.24
7.22 2.61
9.15 6.92
8.41 1.35
7.33 2.52
10.26 2.13
8.91 1.56
6.57 1.33
10.12 1.58
43.20 7.10
19.28 5.22
24.64 7.01
38.54 8.18
25.33 9.42
26.74 6.56
33.41 9.47
40.48 12.94
29.83 5.03
22.41 6.18
0.35 0.12
0.59 0.09
0.34 0.15
0.42 0.18
0.36 0.14
0.51 0.17
0.21 0.06
0.39 0.20
0.48 0.32
0.31 0.14
0.61 0.15
2 9 10-2 M 1.86 0.23
–
1 mg/mL
1 mg/mL
1 mg/mL
1 mg/mL
1 mg/mL
1 mg/mL
1 mg/mL
1 mg/mL
1 mg/mL
3.22 0.16
2.77 0.19
0.53 0.03
2.34 0.19
1.27 0.11
0.91 0.13
0.41 0.04
1.07 0.13
3.07 0.22
6.22 1.02
3.36 0.57
–
0.96 0.46
2.72 0.94
1.47 0.13
–
9g
9h
9i
2.43 0.67
7.33 2.06
a
The MI values indicates that lower the MI value higher the activity
b
c
d
All the values are expressed as mean SEM ‘‘data are the mean, SEM of 3 independent experiments performed in duplicate’’
Distilled water was used as negative control (NC)
Ethyl methanesulphonate (EMS) was used as positive control (PC)
negative control. As there is no antimitotic principle in
water, it was considered as negative control. Ethyl
methanesulphonate (EMS) was treated as positive control
treatment group and induces DNA damage by a direct
mechanism, acting at various sites as a monofunctional
ethylating agent of nucleotides (Budavari, 1989; Sega,
1984).
tested synthesized compounds. In addition to the chromo-
some fragments, sticky metaphase and polar deviations
(wrong directions of chromosome movement) were also
observed. In general, it is possible to observe an increase in
different abnormalities as the nucleophilic functional group
concentration increased. In Allium test, a strong toxic effect
of tested compounds was observed, supported by great
occurrence of sticky metaphases, leading to cellular death
(mitotic index decrease). All the tested compounds pro-
duced a significant decrease in mitotic index were time
dependent at the treatment of 1 mg/mL. There was a sta-
tistically significant increase in total aberrant cells
(P \ 0.05) (aberrant cells include chromosome breaks,
thickness and polar deviation) as compared with the neg-
ative control (Table 2); however, the highest value of
aberrant cells is shown by the positive control. Statistical
analysis showed that the genotoxic activities of the tested
compounds induced micronuclei in the root tip meristem
cells of A. cepa. Micronucleus formation in 1,000 cells per
slide (%MNC value) was also increased in tested
compounds and in positive control EMS compared with
negative control, which is statistically significant
(P \ 0.05).
Cytogenetic analysis
With the objective of investigating the possible mechanism
involved in root growth inhibition, cytogenetic analysis
was performed (Angayarkanni et al., 2007; Auti et al.,
2010; Pavlica et al., 2000). All the tested compounds
provoked strong inhibition of the mitotic index, where a
statistically significant difference in relation to the control,
and the decrease in the mitotic index was positively cor-
related with the electron-releasing group (Table 2). Chan-
ges in chromosome and cellular morphology were observed
with increasing time. Partial c-mitosis (colchicine-like
mitosis) and full c-mitosis, with partially functional spin-
dles and completely normal mitotic phases, were seen in
the various cells of the same root tip between 6- and 72-h
time period. Cytogenetic alterations were investigated, and
the results are depicted in Table 2. All the tested com-
pounds induced chromosome and cytological alterations in
treatment groups. An analysis of chromosome aberrations
showed that most of the fragments detected in the different
treatments were of chromosome type. The observation of
chromosome breaks showed the clastogenic effect of tested
compounds. The occurrence of chromosome fragments
allows observation of statistically significant differences at
In the light of the results obtained in the present study,
these observations above may be due to the genotoxic and
nucleotoxic action of the compounds or the disturbance of
the formation of spindle fibres during cell division, which
leads to chromosomal aberrations. Stickiness and clumping
of the chromosomes were some of the most common
effects of these tested compounds on the treated root tips.
Stickiness usually leads to the formation of anaphase
and telophase bridges, and this ends up inhibiting
123

Med Chem Res
Table 3 Anticancer activity (% cytotoxicity) and CTC50 values of synthesized compounds on HEK 293 (human epidermal kidney cell line)
Treatment
% cytotoxicity (100 - % cell survival) of HEK 293 cell line at conc. (lM)
CTCC50 (lM)a
100
Log conc.
33.33
11.11
3.7
1.23
0.41
0.13
0.045
0.015
0.005
2.00
1.52
1.05
0.57
0.09
-0.39
-0.89
-1.35
-1.82
-2.30
4a
4b
4c
4d
4e
4f
32.96
65.41
49.12
48.13
40.20
31.97
50.18
35.42
48.23
38.78
41.30
54.97
62.43
31.97
35.69
51.86
49.93
29.58
39.76
43.78
42.87
50.59
40.72
52.34
38.89
39.61
42.81
38.61
37.59
43.48
38.91
69.39
31.71
63.14
47.84
47.57
40.04
31.19
48.71
35.16
46.83
38.22
40.73
51.16
59.31
28.73
34.15
50.68
49.17
29.03
38.78
41.25
40.29
46.23
38.89
47.41
38.22
37.65
39.79
34.14
36.90
39.51
36.86
61.24
29.48
62.32
46.53
47.04
39.12
30.74
47.08
34.98
45.29
37.79
39.29
49.87
58.65
26.15
33.49
48.17
49.15
27.25
38.08
40.59
38.13
45.62
38.60
45.94
36.31
34.24
37.94
33.55
36.25
38.84
36.12
57.83
28.87
59.72
42.12
44.62
38.89
30.04
46.35
33.56
43.99
36.59
38.41
49.15
54.16
24.22
32.54
47.80
47.06
26.57
36.42
39.53
37.17
44.17
38.21
44.29
35.84
31.41
37.43
32.77
35.73
37.19
35.26
55.37
28.54
58.13
40.66
42.39
37.12
29.17
45.62
32.17
43.13
35.72
37.16
47.06
51.24
20.81
32.45
46.53
45.27
25.26
35.48
38.74
36.52
43.11
38.04
43.13
35.51
30.29
37.11
32.09
35.68
37.03
35.02
52.22
28.18
57.56
39.93
42.08
35.43
28.85
45.14
30.14
42.63
34.75
36.73
45.27
49.12
20.09
30.16
45.26
43.36
24.12
34.68
37.52
35.91
42.42
37.73
42.92
34.78
29.81
36.42
31.15
35.06
36.69
34.51
51.07
26.93
53.61
39.10
40.54
34.75
28.43
43.74
29.88
39.91
33.58
35.94
43.36
47.20
18.32
28.58
43.99
42.66
22.18
32.12
36.99
35.14
40.73
36.59
42.06
34.75
28.32
35.14
30.32
34.82
36.32
34.31
50.12
26.64
50.42
38.24
39.42
34.13
28.12
41.18
28.19
37.86
32.94
35.10
42.66
45.35
18.01
26.39
40.45
40.65
20.28
30.19
36.04
33.26
39.83
34.57
40.33
33.85
26.59
34.03
28.54
34.54
35.12
33.73
48.56
25.82
47.02
37.87
38.30
31.57
26.39
40.53
26.78
36.22
32.05
34.80
41.98
42.21
16.52
25.75
39.24
38.21
19.87
28.97
35.11
31.16
38.24
34.08
38.16
32.57
26.66
33.12
27.57
32.93
34.46
32.81
46.89
25.57
41.45
36.34
37.27
30.58
24.28
39.32
26.51
35.64
30.46
33.32
39.12
39.29
15.14
23.69
37.78
36.49
18.85
26.94
33.19
29.12
37.35
33.23
36.83
30.64
25.27
32.53
25.40
32.02
33.04
31.41
42.28
64.363
0.922
4.563
10.347
1.8846
120.951
2.798
97.475
4.324
187.19
31.793
3.937
1.122
6.658
5.525
2.268
4.621
31.443
2.337
0.754
1.261
24.642
1.162
2.413
12.77
16.044
7.428
22.12
1.829
41.71
2.934
0.217
4g
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
7f
7g
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
ISL
a
CTC50 cytotoxicity concentration (lM) determined experimentally
post-telophase, metaphase and cytokinesis, respectively,
and thus hampering cell division.
compound with the help of MTT assay HEK 293 (epider-
mal kidney cell line), BT474 (breast cancer cell line) and
NCI-H226 (lung cancer) cell lines by MTT method
(Freshney, 2000; Edmondson et al., 1988; Prasad et al.,
2005; Chiruvella et al., 2008). The viability of control cells
was designated as 100 %, and the others were expressed as
percentage compared to the control. The results were
compared with standard drug indisulam (ISL). The results
demonstrated a strong dose-dependent growth inhibition in
treated cell lines. It showed that different cells had a
In vitro cytotoxicity activity by MTT assay method
All the synthesized compounds prepared by Scheme I and
previously reported (Chhajed et al., 2007, 2013) com-
pounds were subjected to anticancer activity. CTC50
(cytotoxic concentration at which 50 % of the cells are
dead after drug exposure) determined for test and standard
123

Med Chem Res
Table 4 Anticancer activity (% cytotoxicity) and CTC50 values of synthesized compounds on BT474 (breast cancer cell line)
Treatment
% cytotoxicity (100 - % cell survival) of BT474 cell line at conc. (lM)
CTC50 (lM)a
100
Log conc.
33.33
11.11
3.7
1.23
0.41
0.13
0.045
0.015
0.005
2.00
1.52
1.05
0.57
0.09
-0.39
-0.89
-1.35
-1.82
-2.30
4a
4b
4c
4d
4e
4f
28.73
26.66
41.35
32.09
40.37
59.31
38.22
32.69
31.97
39.44
33.85
37.27
50.81
46.38
46.32
36.61
27.87
38.89
51.16
64.14
40.06
65.97
64.99
67.11
39.40
56.21
38.66
38.14
47.67
41.29
61.43
73.52
28.11
23.31
40.32
30.34
38.91
55.26
37.84
32.09
30.32
38.21
33.29
34.77
45.31
44.19
43.67
35.52
26.43
37.95
50.38
60.28
38.46
41.46
62.26
58.80
38.00
47.52
38.22
36.17
41.55
40.50
56.93
66.14
27.45
22.19
39.37
29.44
37.21
52.38
36.21
31.26
29.34
37.91
32.92
32.45
42.19
42.44
41.82
34.59
25.71
36.07
49.11
58.64
37.71
40.56
60.68
54.83
37.37
41.77
36.12
34.74
38.42
39.19
52.13
62.46
26.28
20.47
38.82
28.10
36.96
50.12
35.19
30.89
28.72
37.09
32.11
31.08
40.62
39.51
40.72
33.33
24.22
35.68
48.46
56.72
34.74
40.2
25.92
19.85
37.56
27.13
35.73
48.54
34.87
30.38
28.14
36.69
31.02
30.13
37.19
38.20
39.54
32.16
22.81
34.42
47.56
54.23
33.24
38.97
50.12
50.42
36.75
31.92
35.51
32.82
34.21
36.73
45.14
52.94
25.37
18.14
36.26
26.82
33.14
45.32
34.15
29.83
27.13
35.37
30.56
29.38
35.84
37.56
38.21
31.36
20.98
33.11
47.13
52.17
32.73
38.05
46.10
47.02
34.22
29.89
34.78
31.42
33.76
36.12
43.57
50.79
24.64
17.99
35.55
26.23
32.29
43.76
33.18
28.61
26.25
34.95
29.44
28.67
33.41
34.12
37.77
30.24
20.13
31.92
46.28
50.09
31.29
37.05
42.01
44.37
33.96
28.93
34.75
29.23
32.92
35.42
40.13
49.03
23.12
17.37
34.19
25.34
31.76
41.28
32.07
27.96
25.78
34.13
28.93
28.11
32.15
33.86
36.69
29.47
19.76
30.64
45.39
47.21
29.98
36.38
41.47
42.60
33.52
27.27
33.86
28.71
30.64
34.59
37.35
46.42
22.64
16.56
32.11
24.24
31.02
39.05
31.45
27.18
25.06
33.27
27.72
28.01
30.07
32.75
34.95
28.13
19.43
29.31
44.21
45.80
28.39
35.84
39.42
41.45
33.42
26.43
32.57
28.02
29.11
33.31
34.64
44.97
20.06
16.18
30.65
23.19
30.89
37.60
30.59
26.01
24.32
33.11
26.34
27.14
29.13
30.46
34.13
27.42
18.80
28.53
43.90
42.38
27.27
35.26
38.81
38.13
33.28
25.17
30.64
27.38
29.02
31.52
30.38
42.23
61.336
5.496
8.743
1.746
2.798
1.561
2.346
11.147
3.656
11.552
127.620
2.418
1.007
1.028
9.215
1.884
10.336
1.195
2.349
0.751
1.473
13.723
2.414
0.794
11.557
12.770
112.202
18.345
1.281
6.324
1.361
0.348
4g
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
7f
7g
9a
9b
9c
9d
9e
9f
56.34
53.61
36.80
37.86
35.80
33.23
35.17
37.56
49.34
54.71
9g
9h
9i
9j
ISL
a
CTC50 cytotoxicity concentration (lM) determined experimentally
different sensitivity to the inhibition effect of tested com-
pounds. The results are given in Tables 3, 4 and 5 for HEK
293, BT474 and NCI-H226. Thus, from the data, it can be
concluded that all test compounds are potent cytotoxic
agents because of higher CTC50 at lower concentrations,
and moreover, the compound (4b) (CTC50 = 0.922) and
compound (7f) (CTC50 = 0.754) were found to be most
potent agent among all the compounds tested against HEK
293. While compounds (9c) (CTC50 = 0.751) and (9j)
(CTC50 = 0.913) were found to be most potent agent
among all the compounds tested against BT474 and NCI-
H226 cell lines, respectively. But none of tested compound
was found to be potent compared to standard drug in-
disulam. From above all cell lines such as HEK 293, M468
and NCI-H226, it has been concluded that compounds (7f),
(6f), (9b), (9c) and (9j) are more potent than all synthesized
compounds. Compounds (6e) and (6b) have moderate
activity than all synthesized compounds. Compounds (4a)
and (9 g) have less activity than all synthesized compounds
on all cell lines. Structure activity relationship of
123

Med Chem Res
Table 5 Anticancer activity (% cytotoxicity) and CTC50 values of synthesized compounds on NCI-H226 (lung cancer cell line)
Treatment
% Cytotoxicity (100 - % cell survival) of NCI-H226 cell line at conc. (lM)
CTC50 (lM)a
100
Log conc.
33.33
11.11
3.7
1.23
0.41
0.13
0.045
0.015
0.005
2.00
1.52
1.05
0.57
0.09
-0.39
-0.89
-1.35
-1.82
-2.30
4a
4b
4c
4d
4e
4f
27.86
39.46
54.16
42.56
34.59
52.87
41.75
40.92
49.15
38.98
49.15
54.52
65.97
46.01
36.94
42.44
40.27
38.92
36.05
67.99
38.99
42.36
37.99
43.51
53.02
51.36
40.39
42.47
39.98
38.66
52.43
59.26
27.49
37.21
51.24
42.06
33.33
50.14
41.35
38.89
47.06
38.32
47.26
51.14
59.62
43.19
36.21
41.12
38.88
38.50
35.80
65.83
38.74
41.13
37.72
40.34
48.22
49.32
38.72
39.75
39.25
38.57
45.35
44.69
27.09
36.30
49.12
39.73
32.16
48.31
40.29
38.22
45.27
36.52
45.31
50.83
57.09
42.63
35.13
40.65
38.60
37.91
35.53
60.68
37.12
39.07
37.02
38.19
47.78
48.22
37.14
39.20
37.94
36.72
42.72
38.58
26.55
35.96
47.20
38.26
31.26
46.52
39.37
36.05
43.36
35.08
43.41
49.22
55.18
41.32
34.55
39.07
38.21
35.98
34.87
56.43
36.26
38.10
36.62
37.73
43.14
47.61
36.91
38.61
37.46
35.27
39.13
36.46
26.13
35.11
45.35
38.02
30.59
45.78
38.14
35.61
42.66
34.91
41.96
48.64
54.64
40.65
32.17
38.79
38.04
35.37
34.52
52.12
36.11
37.89
36.47
36.15
41.21
45.79
35.67
37.51
37.24
34.95
37.04
34.12
25.46
34.69
42.21
37.34
29.89
42.90
37.84
33.65
41.98
34.79
41.18
47.65
51.26
39.82
30.41
37.41
37.79
35.66
33.48
46.10
35.72
37.01
36.11
35.87
40.59
43.35
34.95
36.33
36.39
34.59
36.06
32.98
24.94
34.05
39.29
36.29
29.55
41.57
37.13
32.94
39.12
34.15
39.12
45.39
48.28
37.34
29.35
37.05
36.59
35.17
31.75
42.62
35.32
36.15
35.72
35.12
38.31
42.54
33.42
35.06
36.32
34.14
35.27
31.11
24.22
33.46
38.91
35.23
28.93
39.72
36.95
32.17
38.44
33.59
37.05
42.38
46.54
36.75
29.17
35.48
34.75
34.59
30.46
40.07
33.62
35.32
35.43
34.15
37.46
41.86
32.39
34.11
35.35
33.97
34.62
30.20
23.14
32.87
38.19
35.11
28.11
38.63
36.10
31.57
37.26
32.75
36.38
41.25
44.85
34.95
28.36
33.62
34.03
34.13
29.97
39.26
32.79
34.84
29.46
33.25
36.27
40.27
31.24
33.17
35.01
33.92
33.23
28.42
22.41
32.12
37.65
34.16
27.31
37.24
35.77
30.46
36.29
30.41
35.51
38.76
41.28
33.52
27.44
33.48
33.23
33.72
29.04
38.76
30.66
33.29
27.75
31.49
35.65
39.11
30.26
32.72
32.85
33.61
32.98
26.37
145.347
33.268
2.527
17.482
7.965
1.197
4g
6a
6b
6c
6d
6e
6f
22.274
52.953
1.119
12.829
1.816
1.018
0.978
6g
7a
7b
7c
7d
7e
7f
3.108
10.735
13.829
1.164
6.342
12.729
1.784
7g
9a
9b
9c
9d
9e
9f
10.215
5.674
1.487
5.726
2.268
12.763
17.327
166.376
1.467
9g
9h
9i
9.215
9j
0.913
ISL
0.313
a
CTC50 cytotoxicity concentration (lM) determined experimentally
compounds showed that the presence of NH linker between
aryl moiety which is substituted by electron-withdrawing
group and 1,3,4-thiadiazole ring has been recognized as
potent anticancer agent. Substitution on phenyl ring with
chloro, methoxy and nitro group gives better anticancer
activity.
Conclusion
Thiadiazoles are mesoionic system, a poly-heteroatomic
system containing a five-membered heterocycle associated
with a conjugation of p and p electrons and distinct regions of
positive and negative charges leading to highly polarizable
derivatives. This distinctive characteristic allows mesoionic
compounds to effectively cross-cellular membranes and
interact with biological molecules in unique ways. The good
liposolubility of the sulphur atom in the heterocycle might
also have a positive effect on the biological activity and
The order of cytotoxic activity was electron-withdraw-
ing group on phenyl [ electron-donating group on
phenyl [ phenyl.
We can conclude that electron-releasing group on phe-
nyl ring is responsible for less activity.
123

Med Chem Res
pharmacokinetic properties of thiadiazole-containing com-
pounds. The thiadiazole ring possesses similar chemical
properties to the pyrimidine ring and can be considered a
bioisostere. Given that the pyrimidine structure is found in
nucleobases, components of nucleotides and the building
blocks of DNA and RNA, it seems likely that thiadiazole
could readily interact with DNA and RNA, potentially
explaining the broad and often potent activity. Furthermore,
this activity against DNA suggests that thiadiazoles deriva-
tives could potentially be used for chemical intervention at
the gene level. Compounds containing thiadiazole with high
potency have been reported here, and some of them dis-
played excellent activities against a range of tumour cells.
The ability of thiadiazoles to target DNA could explain their
potential anticancer activity as uncontrolled DNA replica-
tion/cell division is a hallmark of neoplastic diseases. Fur-
thermore, the heteroatoms of the thiadiazole are able to form
interactions, such as hydrogen bonds, with biological targets
that include key kinases that participate in tumorigenesis,
such as CA IX and XII.
Carlo Erba 1106 analyzer. The maximum percentage dif-
ferences between calculated and found values for each
element were within the error and amounted to 0.4 %.
The completion of reaction and the purity of the obtained
compounds were checked by TLC on aluminium oxide 60
F254 plates (Merck Co., Whitehouse Station, NJ, USA), in
a CHCl3/C2H5OH (3:1, v/v) solvent system. The spots
were developed in iodine chamber and visualized under
ultra violet lamp (k = 254 nm).
General procedure
N-[(5-Amino-1,3,4-thiadiazol-2-yl)sulfonyl]benzamide (4);
5-[(4-acetamido)benzene sulphonamido]-1,3,4-thiadiazol-
2-(N-benzoyl)sulphonamide (6); and 5-[(4-amino)benzene
sulphonamido]-1,3,4-thiadiazol-2-(N-benzoyl)sulphona-
mide (7) were synthesized, and their physicochemical and
spectral data were reported previously (Chhajed et al.,
2007, 2013). The synthesis outline is depicted in Scheme 1.
The sulfonyl group of sulphonamides is similar to the
carbonate ion and can competitively inhibit CAs. Com-
pounds containing a thiadiazole, a benzene bioisostere,
should also possess high inhibitory activity when bonded
with a sulphamide group. From lead compound, acetazol-
amide, some of the most potent compounds were synthe-
sized and evaluated several sulphonamides as inhibitors of
in vitro cancer cell growth compared with selective hCA
IX inhibitor, indisulam. The affinity of 1,3,4-thiadiazole for
hCA increases significantly when substituted with sulph-
onamides connected with Schiff base. These results indi-
cate that the thiadiazole ring has receptor-binding ability in
the context of hCA IX inhibition and in the prevention of
cancer associated with CA.
N-{[5-(Benzylidenamino)-1,3,4-thiadiazol-2-yl]sulphonyl}
benzamide (9a)
Offwhitecrystals (EtOH) (this compound was prepared by
refuxing 5-amino-1,3,4-thiadiazol-2-[N-(benzoyl)]sulph-
onamide (2.74 g, 0.01 mol) (4a) and benzaldehyde (8a)
(1.06 g, 0.01 mol) in ethanol (20 mL) using 2–3 drops of
sulphuric acid as catalyst, for 12 h. Pour it with thin stream
into crushed ice. It was obtained as yellowish coloured solid
and recrystallized by ethanol); yield: 63 %; Mp:
185–187 °C; UV (MeOH) kmax (log e) 287 nm; Rf = 0.62
(CHCl3/EtOH, 3/1); FT-IR (KBr): vmax 3,625.1, 3,037.4,
1,693.4, 1,678.7, 1,624.32, 1,598.4, 1,557.7, 1,517–1,530.9,
1,369.6, 1,290.5, 907.25, 764.44, 756.54, 694.91 cm-1; 1H-
NMR (DMSO, 400 MHz): d = 1.257 (1H, s, –CH–), 2.134
(6H, m, CH–C6H5), 2.590 (6H, m, CO–C6H5), 3.965 (1H, s,
CH=N), 4.18 (1H, s, N–H), 7.664–7.685 ppm (10H, m, Ar–
H); 13C-NMR ([D]6DMSO, 75 MHz): d = 171.46 (C,
amide), 168.56 (C2, thiadiazole), 166.67 (C5, thiadiazole),
160.68 (C, imine), 137.78 (C1, Ar0–C-imine), 136.05 (C1,
Ar–C-amide), 134.24 (C4, CH–Ar0), 132.52 (C3, CH–Ar),
131.71 (C3, CH–Ar0), 130.39 (C5, CH–Ar), 129.29 (C2, CH–
Ar0), 129.15 (C6, CH–Ar0), 128.84 (C2, CH–Ar), 128.42 (C6,
CH–Ar), 127.34 (C5, CH–Ar0); EIMS m/z [M]? 370.9 (100);
Anal. Calcd. for C16H12N4O3S2: C, 51.60; H, 3.25; N, 15.04;
S, 17.22. Found: C, 51.61; H, 3.24; N, 15.05; S, 17.22.
Experimental section
Synthetic study
Melting points were determined in one-end-open capillary
tubes on a Thermonik Precision melting point apparatus
(C-PMP-2, Mumbai, India) and presented without any
corrections. The IR spectra (m, cm-1) were recorded in KBr
~
tablets using Shimadzu FT-IR 8400s spectrophotometer.
1H nuclear magnetic resonance (1H-NMR) spectra were
recorded for the compounds on Varian EM-390 apparatus
by using TMS as an internal standard. 13C-NMR spectra
were recorded for the compounds on Bruker Avance II 400
NMR Spectrometer apparatus using TMS as an internal
standard, and chemical shifts are reported in ppm (d-scale).
Elemental analysis of the obtained compounds was
performed for C, H, N, S using Elemental Vario EL III
N-({5-[(4-Chlorobenzylidene)amino]-1,3,4-thiadiazol-2-
yl}sulfonyl)benzamide (9b)
Yield: 64.2 %: Mp: 212–214 °C; kmax (log e) 305 nm;
Rf = 0.65 (CHCl3/EtOH, 3/1); FT-IR (KBr): vmax 3,465.3,
3,417.47, 3,148.51, 1,673.2–1,668.7, 1,624.32–1,598.4,
123

Med Chem Res
1,545.9, 1,538.1–1,527.4, 1,368.9–1,358.8, 1,169.9, 968.07,
848–826.5, 764.43–674.43, 764.43 cm-1; 1H-NMR (DMSO,
400 MHz): d = 1.359 (1H, s, –CH–), 2.342 (6H, m, CH–
C6H5), 2.678 (6H, m, CO–C6H5), 3.623 (1H, s, CH=N), 4.41
(1H, s, N–H), 7.462–8.104 (10H, m, Ar–H) 8.24- 8.362 ppm
(1H, s, C(=O)N–H); 13C–NMR ([D]6DMSO, 75 MHz):
d = 170.64 (C, amide), 168.41 (C5, thiadiazole), 166.58
(C2, thiadiazole), 161.68 (C, imine), 136.24 (C4, Cl–C–Ar0),
134.16 (C1, Ar–C-amide), 133.78(C1, Ar0–C-imine), 130.25
(C4, CH–Ar), 129.15 (C3, CH–Ar0), 129.29 (C5, CH–Ar0),
129.02 (C3, CH–Ar), 128.97 (C5, CH–Ar), 128.84 (C2, CH–
Ar0), 128.42 (C6, CH–Ar0), 127.34 (C2, CH–Ar), 127.29
(C6, CH–Ar); EIMS m/z [M]? 412.9 (100); Anal. calcd. for
C16H11N4O3S2Cl: C, 47.23; H, 2.73; N, 13.77; S, 15.76.
Found: C, 47.24; H, 2.72; N, 13.75; S, 15.77.
C–Ar0-OCH3), 163.51 (C, imine), 162.85 (C2, thiadiazole),
162.34 (C5, thiadiazole), 134.29(C1, CH–Ar), 134.01 (C4,
CH–Ar), 130.49 (C6, CH–Ar0), 130.11 (C2, CH–Ar0), 128.94
(C3, CH–Ar), 128.22 (C5, CH–Ar), 128.11 (C1, CH–Ar0),
127.42 (C2, CH–Ar), 127.16 (C6, CH–Ar), 114.33 (C5, CH–
Ar0), 114.08 (C3, CH–Ar0), 69.41 (C, OCH3) ppm; EIMS m/z
[M]? 403.9 (100); Anal. calcd. for C17H14N4O4S2: C, 50.74;
H, 3.51; N, 13.92; S, 15.93. Found: C, 50.72; H, 3.52; N,
13.96; S, 15.94.
N-({5-[(4-Hydroxybenzylidene)amino]-1,3,4-thiadiazol-2-
yl}sulfonyl)benzamide (9e)
Yield: 68.2 %; Mp: 178–180 °C; UV (MeOH) kmax (log e)
375 nm; Rf = 0.59 (CHCl3/EtOH, 3/1); FT-IR (KBr): vmax
3,769–3,719.8, 3,671.56–3,523.8, 2,884.5, 1,713.8, 1,673.7–
1,665.4, 1,599.9–1,549, 1,454.6–1,424.2, 1,317.8, 1,292–
1,174.8, 1,174.8–1,052.1, 931.21–921.7, 786.79–762.6,
N-({5-[(2-Methoxybenzylidene)amino]-1,3,4-thiadiazol-2-
yl}sulfonyl)benzamide (9c)
761.6–725.58 cm-1 1H-NMR (400 MHz, DMSO): d =
;
Yield: 62.8 %; Mp: 201–203 °C; UV (MeOH) kmax (log e)
315 nm; Rf = 0.57 (CHCl3/EtOH, 3/1); FT-IR (KBr): vmax
3,625.4, 3,048.7, 2,915.3–2,903.2, 1,692.8, 1,681.1–1,665.4,
1,599.9–1,536.5, 1,426.5, 1,347.1, 1,290, 1,143.2–1,129.4,
930.13–923.7, 762.6–713.1, 762.6 cm-1 (thiadiazole C–N
stretching); 1H-NMR (DMSO, 400 MHz): d = 1.352 (1H, s,
–CH–), 3.134 (1H, s, CH–C6H5), 3.417–3.487 (3H, m,
–OCH3), 6.364 (1H, s, Ar0–H3,5), 6.84–7.16 (3H,
J = 7.2 Hz, t, Ar–H3,4,5), 8.285 (2H, J = 2.4 Hz, d, Ar–
3.569 (1H, s, CH=N), 4.684 (1H, s, –OH), 6.547–8. 623 (9H,
m, Ar–H), 8.31 ppm (1H, s, C(=O)N–H); 13C-NMR
([D]6DMSO, 75 MHz): d = 169.43 (C, imine), 167.11(C,
amide), 161.32 (C4, C–Ar0–OH), 161.02 (C2, thiadiazole),
160.98 (C5, thiadiazole), 134.52 (C1, CH–Ar), 131.17 (C4,
CH–Ar), 130.62 (C6, CH–Ar0), 130.26 (C2, CH–Ar0), 128.82
(C3, CH–Ar), 128.29 (C5, CH–Ar), 127.34 (C1, CH–Ar0),
127.55 (C2, CH–Ar), 127.21 (C6, CH–Ar), 114.83 (C5, CH–
Ar0), 114.12 (C3, CH–Ar0), ppm; EIMS m/z [M]? 386.6
(100); Anal. calcd. for C16H12N4O4S2: C, 49.48; H, 3.11; N,
14.42; S, 16.51. Found: C, 49.50; H, 3.12; N, 14.40; S, 16.51.
H
2,6), 8.58 ppm (1H, s, N–H); 13C-NMR ([D]6DMSO,
75 MHz): d = 168.21(C, amide), 164.03 (C2, C–Ar0–
OCH3), 163.77(C, imine), 162.32 (C2, thiadiazole), 162.28
(C5, thiadiazole), 134.25(C1, CH–Ar), 132.22 (C4, CH–Ar),
130.76 (C4, CH–Ar0), 130.32 (C6, CH–Ar0), 128.66 (C3, CH–
Ar), 128.45 (C5, CH–Ar), 128.23 (C1, CH–Ar0), 127.55 (C2,
CH–Ar), 127.46 (C6, CH–Ar), 120.84 (C3, CH–Ar0), 120.44
(C5, CH–Ar0), 62.32 (C, aliphatic, OCH3) ppm; EIMS m/z
[M]? 404.6 (100); Anal. calcd. for C17H14N4O4S2: C, 50.74;
H, 3.51; N, 13.92; S, 15.93. Found: C, 50.74; H, 3.52; N,
13.95; S, 15.92.
N-({5-[(2-Hydroxybenzylidene)amino]-1,3,4-thiadiazol-2-
yl}sulfonyl)benzamide (9f)
Yield: 64.6 %; Mp: 220–222 °C; UV (MeOH) kmax (log e)
478 nm; Rf = 0.64 (CHCl3/EtOH, 3/1); FT-IR (KBr): vmax
3,489.1, 3,261.43, 2,948.5–2,884.5, 1,731.22–1,635.4,
1,614.217–1,589, 1,436.06–1,505.64, 1,330.70, 1,232.41–
1,093.86, 1,093.86, 974.20–841.7, 822.2–780.44, 761.6–
725.58 cm-1; 1H-NMR (400 MHz, DMSO): d = 3.582 (1H,
s, CH = N), 4.237 (1H, s, –OH), 6.413–8.548(9H, m, Ar–H),
8.41 ppm (1H, s, C(=O)N–H); 13C-NMR ([D]6DMSO,
75 MHz): d = 166.14 (C, imine), 165.26 (C, amide), 164.21
(C, C2–Ar0–OH), 160.72 (C5, thiadiazole), 160.19 (C2, thia-
diazole), 134.82 (C1, CH–Ar), 132.77 (C4, CH–Ar0), 131.38
(C4, CH–Ar), 130.15 (C6, CH–Ar0), 128.81 (C3, CH–Ar),
128.49 (C5, CH–Ar), 128.09 (C5, CH–Ar0), 127.40 (C2, CH–
Ar), 127.12 (C6, CH–Ar), 114.52 (C1, CH–Ar0), 114.33 (C3,
CH–Ar0), ppm; EIMS m/z [M]? 389.4 (100); Anal. calcd. for
C16H12N4O4S2: C, 49.48; H, 3.11; N, 14.42; S, 16.51. Found:
C, 49.47; H, 3.12; N, 14.43; S, 16.52.
N-({5-[(4-Methoxybenzylidene)amino]-1,3,4-thiadiazol-2-
yl}sulfonyl)benzamide (9d)
Yield: 65.3 %; Mp: 215–217 °C; kmax (log e) 287 nm;
Rf = 0.45 (CHCl3/EtOH, 3/1); FT-IR (KBr): vmax 3,659.8–
3,625.4, 2,915.3–2,903.2, 2,884.5, 1,692.8, 1,681.1–1,665.4,
1,599.9–1,536.5, 1,426.5, 1,347.1, 1,290–1,274.4, 1,143.2–
1,013.4, 930.13–923.7, 786.79–762.6, 762.6 cm-1;1H-NMR
(DMSO, 400 MHz): d = 3.721(3H, s, –OCH3), 6.463(2H, s,
Ar0–H3,5), 7.331–7.62 (5H, J = 3.0 Hz, d, Ar–H), 8.125 (3H,
s, Ar–H2,6), 8.24 ppm (1H, s, C(=O)N–H); 13C-NMR
([D]6DMSO, 75 MHz): d = 170.34 (C, amide), 165.29 (C4,
123

Med Chem Res
(9H, m, Ar–H), 8.645 ppm (1H, s, C(=O)N–H); 13C-NMR
([D]6DMSO, 75 MHz): d = 168.27 (C, imine), 165.61 (C,
amide), 162.23 (C5, thiadiazole), 162.18 (C2, thiadiazole),
154.32 (C3, C–Ar0–NO2), 135.71 (C6, CH–Ar0), 134.67
(C1, CH–Ar0), 134.46 (C1, CH–Ar), 132.49 (C4, CH–Ar),
129.37 (C5, CH–Ar0), 128.35 (C3, CH–Ar), 128.22 (C5,
CH–Ar), 126.13 (C4, CH–Ar0), 117.11 (C2, CH–Ar0),
116.37 (C2, CH–Ar), 116.16 (C6, CH–Ar) ppm; EIMS m/z
[M]? 416.9 (100); Anal. calcd. for C16H11N5O5S2: C,
46.04; H, 2.66; N, 16.78; S, 15.36. Found: C, 46.05; H,
2.68; N, 16.80; S, 15.36.
N-({5-[(4-Hydroxy-3-methoxy benzylidene)amino]-1,3,4-
thiadiazol-2-yl}sulfonyl)benzamide (9g)
Yield: 64.2 %; Mp: 252–254 °C; UV (MeOH) kmax (log e)
268 nm; Rf = 0.67 (CHCl3/EtOH, 3/1); FT-IR (KBr): vmax
3,537.42, 3,371.43, 2,927.5–2,853.4, 1,692.8–1,681.1,
1,665.4–1,599.9, 1,536.05–1,426.5, 1,347.1–1,290, 1,274.4–
1,182.6, 1,013.4, 930.13–923.7, 844.17–762.6, 762.6–
713.1 cm-1; 1H-NMR (400 MHz, DMSO): d = 3.069 (3H,
s, –OCH3), 3.659 (1H, s, CH=N), 4.428 (1H, s, –OH),
6.126–8.262 (8H, m, Ar–H), 8.523 ppm (1H, s, C(=O)N–H);
13C-NMR ([D]6DMSO, 75 MHz): d = 170.43 (C, imine),
167.67(C, amide), 165.09 (C5, thiadiazole), 164.18 (C2,
thiadiazole), 154.32 (C3, C–Ar0–OCH3), 145.13 (C4, C–Ar0–
OH), 135.14 (C1, CH–Ar), 134.02 (C4, CH–Ar), 128.83 (C3,
CH–Ar), 128.41 (C5, CH–Ar), 127.34 (C1, CH–Ar0), 127.21
(C2, CH–Ar), 121.62 (C6, CH–Ar0), 117.61 (C6, CH–Ar),
117.26 (C5, CH–Ar0), 114.31 (C2, CH–Ar0), 65.17 (C, Ar–
OCH3), ppm; EIMS m/z [M]? 420.1 (100); Anal. calcd. for
C17H14N4O5S2: C, 48.80; H, 3.37; N, 13.39; S, 15.33. Found:
C, 48.78; H, 3.38; N, 13.41; S, 15.34.
N-({5-[(Furan-2-ylmethylidene)amino]-1,3,4-thiadiazol-2-
yl}sulfonyl)benzamide (9j)
Brownish crystals (EtOH) (this compound was prepared by
refuxing 5-amino-1,3,4-thiadiazol-2-[N-(benzoyl)]sulph-
onamide (2.74 g, 0.01 mol) (4a) and Furfuldehyde (8j)
(0.96 g, 0.01 mol) in ethanol (20 mL) using 2–3 drops of
sulphuric acid as catalyst, for 7 h. Pour it with thin stream
into crushed ice. It was obtained as dark brown coloured solid
and recrystallized by ethanol); Yield: 53.04 %; Mp:
261–263 °C; UV (MeOH) kmax (log e) 412 nm; Rf = 0.69
(CHCl3/EtOH, 3/1); FT-IR (KBr): vmax 3,634.9, 3,581.22,
3,054.2, 1,635.34, 1,622.4–1,595.9, 1,432.4, 1,254.31–
N-[(5-{[4-(Dimethylamino)benzylidene]amino}-1,3,4-
thiadiazol-2-yl)sulfonyl]benzamide (9h)
1,197.7, 824.3–776.9, 741.3–711.4 cm-1
;
1H-NMR
Yield: 67.7 %; Mp: 236–238 °C; UV (MeOH) kmax (log e)
305 nm; Rf = 0.42 (CHCl3/EtOH, 3/1); FT-IR (KBr): vmax
3,652.4, 3,532.12, 3,114.7, 2,985.3–2,896.4, 1,614.2–
1,591.4, 1,413.1, 1,238.52–1,174.7, 804.2–783.6, 743.9–
719.2 cm-1; 1H-NMR (400 MHz, DMSO): d = 2.547 (6H,
s, –NCH3), 3.956 (1H, s, CH=N), 4.114 (1H, s, N–H),
6.466–7.824 (9H, m, Ar–H), 8.511 ppm (1H, s, C(=O)N–H);
13C-NMR ([D]6DMSO, 75 MHz): d = 169.42 (C, imine),
165.21 (C, amide), 162.15 (C2, thiadiazole), 162.11 (C5,
thiadiazole), 154.32 (C4, C–Ar0–N(CH3)2), 134.63 (C1, CH–
Ar), 132.46 (C4, CH–Ar), 132.23 (C2, CH–Ar0), 132.18 (C3,
CH–Ar), 131.65 (C6, CH–Ar0), 128.12 (C2, CH–Ar), 128.03
(C6, CH–Ar), 127.37 (C1, CH–Ar0), 127.11 (C3, CH–Ar0),
117.52 (C5, CH–Ar), 117.11 (C5, CH–Ar0), 52.84 (C, Ar–
NCH3, Aliphatic), 52.47 (C, Ar–NCH3, Aliphatic) ppm;
EIMS m/z [M]? 415.7 (100); Anal. calcd. for
C19H18N4O3S2: C, 55.06; H, 4.38; N, 13.52; S, 15.47. Found:
C, 55.07; H, 4.38; N, 13.53; S, 15.46.
(400 MHz, DMSO): d = 2.547 (6H, s, –NCH3), 4.116 (1H,
s, CH=N), 6.724–7.211 (3H, m, furfuryl-H), 7.446–7.918
(5H, m, Ar–H), 8.426 ppm (1H, s, C(=O)N–H); 13C-NMR
([D]6DMSO, 75 MHz): d = 148.22 (C, imine), 167.19 (C,
amide), 154.32 (C2, C-furfuryl), 152.13 (C2, thiadiazole),
150.84 (C5, thiadiazole), 135.71 (C5, CH-furfuryl), 134.63
(C1, CH–Ar), 132.46 (C4, CH–Ar), 128.12 (C3, CH–Ar),
128.03 (C5, CH–Ar), 117.11 (C3, CH-furfuryl), 111.24 (C2,
CH–Ar), 111.06 (C6, CH–Ar), 106.10 (C4, CH-furfuryl)
ppm; EIMS m/z [M]? 364.3 (100); Anal. calcd. for
C14H10N4O4S2: C, 46.40; H, 2.78; N, 15.46; S, 17.70. Found:
C, 46.42; H, 2.79; N, 15.45; S, 17.39.
Pharmacological evaluation
Antioxidant and free radical scavenging activity
Total antioxidant activity The ability of the test sample to
scavenge 2,20-azinobis-(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTSꢀ?) radical cation was compared with 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) stan-
dard (Chang et al., 2007; Erel, 2004; Re et al., 1999). The
ABTSꢀ? radical cation was pregenerated by mixing ABTS
stock solution (7 mM) with potassium persulphate (2.
45 mM) (final concentration) and incubating for 12–16 h
in the dark at room temperature until the reaction was
complete and the absorbance was stable. The absorbance
N-({5-[(3-Nitrobenzylidene)amino]-1,3,4-thiadiazol-2-
yl}sulfonyl)benzamide (9i)
Yield: 61.3 %; Mp: 258–260 °C; UV (MeOH) kmax (log e)
352 nm; Rf = 0.51 (CHCl3/EtOH, 3/1); FT-IR (KBr): vmax
3,537.9–3,427.2, 3,128.2–3,022.3, 3,075–3,007.4, 2,341.6–
2,331.1, 1,445.8, 1,456.8–1,531.7, 827, 1,022.8–1,078.2,
713.1–619.5 cm-1
;
1H-NMR (400 MHz, DMSO): d =
3.239 (1H, s, CH=N), 4.751 (1H, s, –OH), 6.872–8.421
123

Med Chem Res
of the ABTSꢀ? solution was equilibrated to 0.70 ( 0.02)
by diluting with water at room temperature, then 1 mL of
solution was mixed with 10 lL of the test sample (0.05–
10 mg/mL), and the absorbance was measured at 734 nm
after 6 min. All experiments were repeated three times.
The percentage inhibition of absorbance was calculated
and plotted as a function of the concentration of standard
and sample to determine the trolox equivalent antioxidant
concentration (TEAC). To calculate the TEAC, the gra-
dient of the plot for the sample was divided by the gra-
dient of the plot for trolox. The IC50 inhibitory
concentration (nM/mL) values of tested compounds are
depicted in Table 1. The ABTSꢀ? radical scavenging
activity of the samples was expressed as
test tubes containing methanol (3 mL), DPPH reagent
(2 mg/mL) (50 lL) was added. The initial absorbance was
measured. To these test tubes, methanolic solution of dif-
ferent test solutions (1 mg/mL) were added (10–50 lL).
Ascorbic acid (0.5 mg/mL) was also added in the con-
centration of 10, 20, 30, 40, 50 and 100 lL. After 20 min,
absorbance was recorded at 516 nm. The experiment was
performed in triplicate. The percentage reduction in
absorbance was calculated from the initial and final
absorbance of each solution (Dhar and Taploo, 1982). The
IC50 (nM/mL) values are shown in Table 1.
Superoxide anion radical scavenging effect Measurement
of superoxide anion scavenging activity of the synthesized
compound was taken based on the method described by
Nishimiki et al. (1972) and slightly modified. About 1 mL
of nitroblue tetrazolium (NBT) solution (156 lM NBT in
100 mM phosphate buffer, pH 7.4), NADH solution
(1 mL) (reduced form of b-nicotinamide adenine dinucle-
otide) (468 lM in 100 mM phosphate buffer, pH 7.4) and
sample solution (0.1 mL) of compounds (10, 20, 30, 40, 50
and 100 lg) in distilled water were mixed and the reaction
started by adding phenazine methosulphate (PMS) solution
(100 lL) (60 lM PMS in 100 mM phosphate buffer, pH 7.
4). The reaction mixture was incubated at 25 °C for 5 min,
and the absorbance at 560 nm was measured against blank
samples. Catechin was used as reference compound. All
the experiments were performed in triplicate, and the
results were averaged. The percentage of inhibition was
determined by comparing the results of control and test
samples. The IC50 (nM/mL) value are depicted in Table 1.
S % ¼ ½ðAcontrolꢁAsampleÞ=Acontrolꢂ ꢃ 100
where Acontrol is the absorbance of the blank control
(ABTSꢀ? solution without test sample), and Asample is the
absorbance of the test sample.
Lipid peroxidation inhibitory activity Egg lecithin (3 mg/
mL phosphate buffer, pH 7.4) was sonicated in an ultra-
sonic sonicator for 10 min to ensure proper liposome for-
mation. Test samples or standard, ascorbic acid (100 lL)
of different concentrations (10, 20, 30, 40 50 and 100 lg/
mL) was added to liposome mixture (1 mL); the control
was without test sample. Lipid peroxidation was induced
by adding ferric chloride (10 lL, 400 mM) and L-ascorbic
acid (10 lL, 200 mM). After incubation for 1 h at 37 °C,
the reaction was stopped by adding hydrochloric acid
(2 mL, 0.25 N) containing trichloroacetic acid (150 mg/
mL), thiobarbituric acid (3.75 mg/mL) and butylated
hydroxy anisole (0.50 mg/mL). The reaction mixture was
subsequently boiled for 15 min, cooled and centrifuged at
1,000 rpm for 15 min, and the absorbance of the super-
natant was measured at 532 nm (Duh and Yen, 1997). The
IC50 values of all tested compounds are reported in
Table 1. The % inhibition at different concentrations was
calculated by the following formula
Nitric oxide radical scavenging effect Nitric oxide gen-
erated from sodium nitroprusside in aqueous solution at
physiological pH interacts with oxygen to produce nitrite
ions, which were measured by the Griess reaction (Marcocci
et al., 1994; Green et al., 1982). Scavenger of nitric oxide
competes with oxygen leading to reduced production of nitric
oxide (Mondal et al., 2006). The reaction mixture (3 mL)
containing sodium nitroprusside (10 mM) in phosphate-
buffered saline (PBS) and the compounds in different con-
centrations (10, 20, 30, 40, 50 and 100 lg) were incubated at
25 °C for 150 min. At every 30-min interval, the incubated
sample (0.5 mL) was removed and Griess reagent (1 %
sulphanilamide, 0.1 % naphthylethylene diamine dihydro-
chloride in 2 % H3PO4) (0.5 mL) was added. The absorbance
of the chromophore formed was measured at 546 nm. All the
analyses were performed in triplicate, and the results were
averaged. The percentage inhibition of nitric oxide generated
was measured by comparing the absorbance values of control
and test. Curcumin was used as a reference compound. The
IC50 (nM/mL) values are reported in Table 1.
% Inhibition ¼ ½1 ꢁ ðVt=VcÞꢂ ꢃ 100
where Vt = mean absorption of test compound, Vc = mean
absorption of control.
The IC50 (nM/mL) value was derived from the % inhi-
bition at different concentrations.
DPPH radical scavenging activity Compounds of SC
series were evaluated for their in vitro free radical scav-
enging activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH)
assay method (Blois, 1958; Shishoo et al., 1999; Chhajed
et al., 2007). To determine the free radical scavenging
activity, a method based on the reduction of a methanolic
solution of the coloured DPPH radical was used. To a set of
123

Med Chem Res
In vitro antimitotic activity by Allium cepa (onion)
meristem root model
number of cells scored and expressed as percentage
using following formula as per standard procedures.
Number of dividing cells
Mitotic index ¼
ꢃ 100
Small bulbs (1.5–2.0 cm in diameter) of the common onion,
A. cepa (2n = 16), were purchased from vendor at a local
market. Prior to initiating the test, the outer scales of the
bulbs and the dry bottom plate were removed without
destroying the root primordia. The roots of A. cepa were
grown in distilled water in Erlenmeyer flasks (200 mL
capacity) under laboratory conditions (dark 24 °C). For each
synthesized compound sample, after reaching a length of
3 cm ( 0.5 cm), a series of six bulbs were placed in distilled
water (pH 7.3) for 48 h and then onion roots were treated
with the synthesized compound at 1 mg/mL concentrations
of each tested compound. The test tubes were kept in an
incubator at 22 1 °C, and the test samples were changed
daily at the same time. Several of the newly formed root tips
were then cut from each bulb and examined for any visible
morphological abnormalities. The bulbs with satisfactory
root lengths (2–2.5 cm) were used in the study, while those
with exceptionally long or short roots were discarded (on
average 2–3 bulbs). Therefore, individual sets of five bulbs
were used for each extract sample.
Total number of cells
(ii) Chromatin aberrations (stickiness, breaks and polar
deviation) were used as end points for the
determination of cytogenetic effects, and micronuclei
(MNC) were scored in interphase cells per 1,000 cells
(% MNC) (Freshney, 2000).
(iii) The most frequent abnormalities are shown in
microphotographs. After 72 h of exposure to the test
samples, the root lengths were measured and used as
an index of general toxicity. The results for mitotic
index and root length are expressed as percentage of
the negative and positive controls. Visible morpho-
logical modifications, such as changes in root
consistency and colour as well as the presence of
swelling (c-tumours), hooks or twists in the roots,
were also observed.
In vitro cytotoxicity activity by MTT assay method
Distilled water (pH 7.3) was used as a negative control,
and EMS (2 9 10-2 M) used as a positive control
mutagen (Fiskesjo, 1993, 1997). After 24 h of exposure,
several root tips were removed from the bulbs, fixed in
3:1 (v/v) ethanol (90 %)/glacial acetic acid (45 %) and
stored overnight at 4 °C. The next day, they were placed
in 70 % (v/v) aqueous alcohol and refrigerated until used.
Allium roots were softened by digesting with HCl and
rinsed the roots in water. After removing the water from
the third rinse, the roots were covered with the orcein
acetate stain. The roots were incubated in the stain for
12 min. During this time, the very tip of the root begins
to turn red as the DNA stains the numerous small actively
dividing cells at the tip. A root was transferred to the
centre of a clean microscope slide, and a drop of water
was added. Using a razor blade most of the unstained part
of the root was cut off and discarded. The root tip was
covered with a cover slip and then carefully pushed down
on the cover slide with the wooden end of a dissecting
probe. Care should be taken to push hard, but do not twist
or push the cover slide sideways. The root tip should
spread out to a diameter about 0.5–1 cm. Five slides were
prepared per bulb.
Cell line and culture medium The cancer cell line cul-
tures of HEK 293 (epidermal kidney cell line), BT474
(breast cancer cell line) and NCI-H226 (lung cancer) were
obtained from Pasteur Institute of India, Coonoor, India,
and were cultured in RPMI-1640 and 10 % heat-activated
New born calf serum with antibiotics [penicillin (1,000 I.
U./mL), streptomycin (100 lg/mL) and amphotericin B
(25 lg/mL)]. The cells were maintained at 37 °C in a
humidified atmosphere with 5 % CO2 and were subcul-
tured twice a week.
Determination of cytotoxicity by microculture tetrazolium
(MTT) assay The monolayer cell culture (100 lL) was
trypsinized, and the cell count was adjusted to 3.
0 9 105 cells/mL using medium containing 10 % new
born calf serum. To each well of the 96-well microtitre
plate, the diluted cell suspension (approximately 10,000
cells) (0.1 mL) was added and kept for 24 h in incubator at
37 °C in 5 % CO2 atmosphere for cell monolayer forma-
tion. After 24 h, when a partial monolayer was formed at
the bottom of the well, the supernatant was flicked off, the
monolayer was washed once, and different drugs, i.e.
synthesized compounds (100 lL), were added to the cells
in microtitre plates. The plates were then incubated at
37 °C for 3 days in 5 % CO2 atmosphere, and microscopic
examination was carried out and observations recorded
every 24 h. After 72 h, the sample solution in the wells was
flicked off; MTT dye (50 mL) was added to each well;
plates were gently shaken and incubated for 4 h at 37 °C in
Determination of cytotoxicity and genotoxicity The fol-
lowing parameters were used for the determination of
cytotoxicity and genotoxicity:
(i) the mitotic index (MI) was calculated as the ratio
between the number of mitotic cells and the total
123

Med Chem Res
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Acknowledgments The authors are highly thankful to Director,
Sophisticated Analytical Instrumentation Facility (SAIF), Panjab
University, Chandigarh; Department of Chemistry, Pune University,
Pune, and Director, Sophisticated Analytical Instrumental Laboratory
(SAIL), School of Pharmaceutical Sciences, Rajiv Gandhi Proud-
yogiki Viswavidyalaya, Bhopal for providing for providing the nec-
essary spectral analysis facilities to carry out this research work.
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