Synthesis, antimicrobial and cytotoxic activities of novel 4-trifluoromethyl-(1,2,3)-thiadiazolo-5-carboxylic acid hydrazide Schiff's bases

Article abstract of DOI:10.1007/s00044-012-0168-x

A series of novel 1,2,3-thiadiazolo-5-carboxylic acid hydrazide Schiff's bases 4a-4r were prepared starting from ethyl-4,4,4-trifluoroacetoacetate and ethyl carbazate in four steps. All the compounds were screened for antibacterial activity against various bacterial strains at 150 μg/ml concentration and found no activity. Similarly, all the compounds were screened for antifungal activity against various fungal strains at 100 and 150 μg/ml concentrations. Compounds 4a, 4m, and 4q found to show moderate activity against Candida albicans. Further, compounds were evaluated for cytotoxic activity against breast carcinoma cells MDA-MB 231 (aggressive), MCF-7 (non-aggressive) using doxorubicin as standard. Compound 4n was found to show 25.39 % cell viability against MDA-MB 231 and 63.60 % cell viability against MCF-7 cells.

Full text of DOI:10.1007/s00044-012-0168-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1747–1755
DOI 10.1007/s00044-012-0168-x
ORIGINAL RESEARCH
Synthesis, antimicrobial and cytotoxic activities of novel
4-trifluoromethyl-(1,2,3)-thiadiazolo-5-carboxylic acid hydrazide
Schiff’s bases
•
P. Sambasiva Rao C. Kurumurthy B. Veeraswamy
•
•
•
G. Santhosh kumar B. Narsaiah K. Pranay Kumar
•
•
•
U. S. N. Murthy Santosh Karnewar Srigiridhar Kotamraju
•
Received: 15 November 2011 / Accepted: 28 June 2012 / Published online: 18 July 2012
Ó Springer Science+Business Media, LLC 2012
Abstract
A
series of novel 1,2,3-thiadiazolo-5-car-
Introduction
boxylic acid hydrazide Schiff’s bases 4a–4r were prepared
starting from ethyl-4,4,4-trifluoroacetoacetate and ethyl
carbazate in four steps. All the compounds were screened
for antibacterial activity against various bacterial strains at
150 lg/ml concentration and found no activity. Similarly,
all the compounds were screened for antifungal activity
against various fungal strains at 100 and 150 lg/ml con-
centrations. Compounds 4a, 4m, and 4q found to show
moderate activity against Candida albicans. Further,
compounds were evaluated for cytotoxic activity against
breast carcinoma cells MDA-MB 231 (aggressive), MCF-7
(non-aggressive) using doxorubicin as standard. Compound
4n was found to show 25.39 % cell viability against MDA-
MB 231 and 63.60 % cell viability against MCF-7 cells.
The thiadiazole nucleus present in many biologically active
compounds and more specifically 1,2,3-thiadiazole deriv-
atives known to have antihelminthic (Sarett and Brown,
1962, 1967, 1969), insecticide (McConnell and Coover,
1963), antibacterial activities (Pain and Slack, 1965) and
some of the derivatives was found as necroptosis inhibitors
(Teng et al., 2007). The 1,2,3-thiadiazole ring system is
prepared mainly in five ways: (i) cyclization of hydrazones
with thionyl chloride (Hurd–Mori synthesis) (Hurd and
Mori, 1955), (ii) cycloaddition of diazoalkanes with C=S
bond (Pechmann synthesis) (Pechmann and Nold, 1896),
(iii) hetero-cyclization of a-diazo thiocarbonyl compounds
(Wolff synthesis) (Wolff, 1904), (iv) ring transformation of
other sulfur-containing heterocyclic compounds (Kurzer,
1973), and (v) elaboration of preformed 1,2,3-thiadiazole
Keywords 1,2,3-Thiadiazole ꢀ Schiff’s bases ꢀ
Hurd–Mori synthesis ꢀ Antibacterial activity ꢀ
Antifungal activity ꢀ Cytotoxic activity
´
(Thomas, 1984; Bakulev and Mokrushin, 1986; L’abbe
et al., 1996; Morzherin et al., 2003). Recent reports claim
that the 1,2,3-thiadiazole derivatives are identified as novel
class of potent HIV-1 non-nucleoside reverse transcriptase
inhibitors (NNRTIs) (Peng et al., 2008, 2009). In addition,
it was found that the fluorine (Hertel et al., 1988) or tri-
fluoromethyl (Filler et al., 1993; Chae et al., 2004) group at
a strategic position of an organic molecule dramatically
alters the properties of molecule in terms of lipid solubility,
oxidative thermal stability, permeability, oral bioavail-
ability thereby enhancement of transport mechanism.
Based on the importance of 1,2,3-thiadiazole nucleus and
in continuation of our efforts (Manichandrika et al., 2008,
2010; Sirisha et al., 2010; Kurumurthy et al., 2011) on
synthesis of trifluoromethyl-substituted compounds, we
have synthesized a series of novel 4-trifluoromethyl-1,2,3-
thiadiazole-5-carboxylic acid hydrazide Schiff’s bases in
four steps starting from ethyl 4,4,4-trifluoroacetoacetate
P. Sambasiva Rao ꢀ C. Kurumurthy ꢀ B. Veeraswamy ꢀ
G. Santhosh kumar ꢀ B. Narsaiah (&)
Fluoroorganic Division, Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500607, India
e-mail: narsaiah_1984@yahoo.co.in
K. Pranay Kumar ꢀ U. S. N. Murthy
Biology Division, Indian Institute of Chemical Technology,
Tarnaka, Hyderabad 500607, India
S. Karnewar ꢀ S. Kotamraju
Chemical Biology Division, Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500607, India
123

1748
Med Chem Res (2013) 22:1747–1755
and ethyl carbazate. All the final compounds screened for
antibacterial, antifungal, and cytotoxic activities against
breast carcinoma cells MDA-MB 231 (aggressive) and
MCF-7 (non-aggressive) and reporting here for the first
time.
Table 1 Physical properties of compounds 4a–4r
S. no. Compounds R
R0
Yield (%) mp (°C)
1
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
H
H
C6H5
98
96
95
92
98
223–225
228–230
244–245
223–225
203–205
250–256
178–180
260–263
219–221
246–248
229–231
203–209
218–220
208–211
186–188
175–176
186–188
191–194
2
4-F–C6H4
4-Cl–C6H4
4-Br–C6H4
4-NO2–C6H4
3
4
5
Chemistry
6
4-N(Me)2–C6H4 90
7
4g
4h
4i
4-OH–C6H4
4-OMe–C6H4
Furan-2-yl
98
94
96
A mixture of ethyl 4,4,4-trifluoroacetoacetate and ethyl
carbazate in ethanol was heated at reflux for 6 h to obtain
3-(ethoxy carbonyl hydrazino)-4,4,4-trifluorobutyric acid
ethylester 1. The product 1 was further treated with thionyl
chloride and heated at 60 °C for 8 h, by Hurd–Mori synthesis
obtained 4-trifluoromethyl-(1,2,3)-thiadiazolo-5-carboxylic
acid ethylester 2. The compound 2 was refluxed in hydrazine
hydrate to form 4-trifluoromethyl-(1,2,3)-thiadiazole-5-
carboxylic acid hydrazide 3. The hydrazide 3 was further
condensed with different substituted benzaldehydes and
ketones in ethanol and obtained 4-trifluoromethyl-(1,2,3)-
thiadiazolo-5-carboxylic acid hydrazide Schiff’s bases 4 in
high yields. The details of reactions outlined in Scheme 1
and products are tabulated in Table 1.
8
9
10
11
12
13
14
15
16
17
18
4j
Thiophene-2-yl 95
4k
4l
CH3 C6H5
92
92
91
88
95
CH3 4-NO2–C6H4
CH3 2,4-Br2–C6H3
CH3 Pyridine-2-yl
CH3 CH3
(RR0=)–CH2(CH2)2CH2– 96
(RR0=)–CH2(CH2)3CH2– 95
(RR0=)–CH2(CH2)4CH2– 91
4m
4n
4o
4p
4q
4r
species of Gram-positive and Gram-negative bacteria. The
details of compounds and their activity profile against
various microorganisms are tabulated in Table 2.
Antimicrobial activity
In vitro antibacterial assays
In vitro antifungal activity assays
Compounds 4a–4r were dissolved in acetone and screened
for in vitro antibacterial activity against Gram-positive
(Bacillus subtilis, Staphylococcus aureus, Staphylococcus
epidermidis) and Gram-negative (Pseudomonas aerugin-
osa, Escherichia coli, Klebsiella pneumoniae) bacteria.
The MIC values of the compounds were compared with
those obtained with penicillin and streptomycin. However,
no compounds showed significant activity against all
The in vitro antifungal activity of compounds 4a–4r were
screened against the fungal strains, viz., Candida albicans
(MTCC 227), Saccharomyces cerevisiae (MTCC 36) and
filamentous fungal cultures like Aspergillus niger (MTCC
1344), Candida rugosa (NCIM 3462) by agar cup diffusion
method. The strains were obtained from the Institute of
Microbial Technology, Chandigarh. Compounds 4a and
4m showed moderate activity against C. albicans at 100
Scheme 1 Preparation of
compounds 4a–4r. Reagents
O
CF3
COOC2H5
F3C
and conditions: (i) Ethanol,
reflux, 6 h; (ii) SOCl2, 60 °C,
8 h; (iii) hydrazine hydrate,
reflux, 3 h; (iv) (RR0) C=O,
ethanol, reflux, 1–2 h.
F3C
N
N
i
ii
COOC2H5
COOC2H5
COOC2H5
+
N
S
N
H
1
H2N-NH-COOC2H5
2
iii
CF3
CF3
NH-NH2
O
N
N
iv
HN N C(RR')
N
N
S
S
O
3
4a-r
123

Med Chem Res (2013) 22:1747–1755
1749
Table 2 Antibacterial activity of compounds 4a–4r
Compounds MIC (lg/ml)
Table 4 Cytotoxic activity of compounds 4a–4q against MDA-MB
231 and MCF-7 cells treated for 48 h by MTT assay at 10 lM
concentration
B.
S.
S.
E.
P.
subtilis aureus epidermidis coli aeruginosa pneumoniae
K.
S. no.
Compounds
Percentage of cell viability
MDA-MB 231
MCF-7
4a
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
150 150
12.5 12.5
6.25 1.562
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
6.25
3.125
4b
4c
1
Control (DMSO)
100.00
101.55
102.43
87.27
89.53
91.93
94.52
94.73
25.39
84.20
78.56
22.85
100.00
101.95
99.53
2
4a
4d
4e
3
4b
4
4h
101.30
99.98
4f
5
4i
4g
6
4j
102.15
115.02
122.72
63.60
4h
4i
7
4k
8
4m
4j
9
4n
4k
4l
10
11
12
4p
97.94
4m
4n
4o
4q
97.15
Doxorubicin (5 lM)
43.13
4p
4q
4r
obtained with 50 lg/ml of standard amphotericin. The
details of the results have been tabulated in Table 3.
Penicillin
1.562 1.562 3.125
6.25 3.125
In vitro cytotoxic activity
Streptomycin 6.25
and 150 lg/ml, whereas 4q showed moderate activity
against C. albicans at 150 lg/ml. The inhibitory zone
diameters of the compounds are compared with those
The compounds 4a–4b, 4h–4k, 4m–4n, and 4p–4q were
screened against breast carcinoma cells MDA-MB 231
(aggressive) and MCF-7 (non-aggressive) using
Table 3 Antifungal activity of
compounds 4a–4r
Compounds
Zone of inhibition
C. albicans
C. rugosa
S. cerevisiae
A. niger
100 lg
150 lg
100 lg
150 lg
100 lg
150 lg
100 lg
150 lg
4a
10
0
12
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4b
4c
0
0
4d
0
0
4e
0
0
4f
0
0
4g
0
0
4h
0
0
4i
0
0
4j
0
0
4k
0
0
4l
0
0
4m
11
0
15
0
4n
4o
0
0
4p
0
0
4q
0
7
4r
0
0
Amphotericin B
23.5
21
22
25
123

1750
Med Chem Res (2013) 22:1747–1755
Table 5 % of cell viability of 4n at different concentrations against
MDA-MB 231 cells
Concentration (lM)
Percentage of cell viability
10
5
25.39
34.81
41.74
1
Table 6 IC50 (lg/ml) values of compound 4n and doxorubicin
Fig. 3 Percentage of cell viability of 4n at different concentrations
against MDA-MB 231 cells
Compound
IC50
MDA-MB 231
MCF-7
4n
2.74
1.81
6.01
4.12
activity with increase in concentration. The data are out-
lined in Table 5 and IC50 values are calculated and com-
pared with standard doxorubicin in Table 6. All other
compounds showed moderate cell viability, however,
compounds 4k and 4m showed proliferation of cells rather
than viability against MCF-7 cell lines. Further work is in
progress to identify the lead compound. The details are
outlined in Table 4 and Figs. 1, 2.
Doxorubicin
Conclusion
A series of novel trifluoromethyl-substituted thiadiazole
carboxylic acid hydrazide Schiff’s bases were prepared in
four steps and screened for antimicrobial and cytotoxic
activities. The compounds 4a, 4m, and 4q showed prom-
ising antifungal activity and compound 4n showed prom-
ising percentage cell viability (Fig. 3).
Fig. 1 Cytotoxic activity of compounds 4a–4q against MDA-MB
231 breast carcinoma cells at 10 lM concentration
Experimental
Melting points were recorded on Cassia-Siamia (VMP-
AM) melting point apparatus and are uncorrected. IR
spectra were recorded on a Perkin-Elmer FT-IR 240-C
spectrophotometer using KBr optics. 1H NMR spectra were
recorded on Bruker AV 300 MHz in CDCl3 and DMSO-d6
using TMS as internal standard. 13C NMR spectra were
recorded on Bruker AV 75 MHz in CDCl3 and DMSO-d6.
Electron impact (EI) and chemical ionization mass spectra
were recorded on a VG 7070 H instrument at 70 eV. All
the reactions were monitored by thin layer chromatography
(TLC) on precoated silica gel 60 F254 (mesh); spots were
visualized with UV light. Merck silica gel (60–120 mesh)
was used for column chromatography. CHN analysis was
recorded on a Vario EL analyser. Six bacterial test
organisms and four fungal strains were selected for
screening and obtained from the Institute of Microbial
Technology, Chandigarh. The breast carcinoma cells
Fig. 2 Cytotoxic activity of compounds 4a–4q against MCF-7 breast
carcinoma at 10 lM concentration
doxorubicin as standard, in order to assess the growth
inhibitory/cytotoxic effects. Compound 4n was found to
show promising cell viability against both the cell lines and
is attributed to the presence of thiadiazole and pyridine ring
in single molecule. Thus, compound 4n was further eval-
uated at different concentrations and found to be increased
123

Med Chem Res (2013) 22:1747–1755
1751
Preparation of 4-(trifluoromethyl)-1,2,3-thiadiazole-5-
MDA-MB 231 (aggressive) and MCF-7 (non-aggressive)
were used for cytotoxic effects.
carbohydrazide (3)
Procedure
Preparation of ethyl 2-(4-ethoxy-1,1,1-trifluoro-
4-oxobutan-2-ylidene) hydrazinecarboxylate (1)
The 4-trifluoromethyl-(1,2,3)-thiadiazol-5-carboxylic acid
ethylester 2 (1.0 g, 4.5 mmol) and hydrazine hydrate
(0.5 ml, 10 mmol) in ethanol (10 mL) was refluxed for 3 h.
On cooling, yellow solid was separated, filtered, dried, and
recrystallised to obtain pure compound 3. Yellow solid,
Yield: 72 %; mp 180 °C; IR (KBr, cm-1): 3311, 3369
Procedure
A
mixture of ethyl-4,4,4-trifluoroacetoacetate (1.0 g,
5.4 mmol) and ethyl carbazate (0.56 g, 5.4 mmol) in 10 ml
ethanol was heated at reflux for 6 h. After completion of
reaction, the solvent was removed under vacuum and the
crude residue was purified by passing through a column
packed with silica gel using petroleum ether/EtOAc (6:4)
as eluents. White solid, Yield: 92 %, mp 111–113 °C; IR
(KBr, cm-1): 3227 (–NH), 1747 (–C=O), 1619 (C=N),
1
(–NHNH2), 1705 (–C=O), 1515 (C=C), 1359 (–C–F); H
NMR (DMSO-d6, 300 MHz): 10.60 (br., 1H, NH),
4.32(br., 2H, NH2); 13C NMR (DMSO-d6, 75 MHz): 167.8,
148.1, 146.8, 118.6; MS (EI, 70 eV): m/z (%) : 212 (M?,
100), 181 (M?-NHNH2, 65), 143 (M?-CF3, 32); Anal.
Calcd. for C4H3F3N4OS: C, 22.65; H, 1.43; N, 26.41 %.
Found: C, 22.66; H, 1.44; N, 26.39 %.
1
1363 (–C–F); H NMR (DMSO-d6, 300 MHz): d 1.20 (t,
J = 6.6 Hz, 3H, CH3), 1.40 (t, J = 6.7 Hz, 3H, CH3), 3.61
(s, 2H, –CH2–), 4.22 (q, J = 6.6 Hz, 2H, OCH2), 4.29 (q,
J = 6.7 Hz, 2H, OCH2), 10.71 (s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): 171.2, 159.1, 155.1, 118.6, 59.2,
58.2, 56.6, 13.6, 13.3; MS (EI, 70 eV): m/z (%) : 270(M?,
100), 201 (M?-CF3, 28); Anal. Calcd. for C9H13F3N2O4:
C, 40.01; H, 4.85; N, 10.37 %. Found: C, 40.03; H, 4.84;
N, 10.35 %.
Preparation of N0-(arylmethylidene)-4-
(trifluoromethyl)-1,2,3-thiadiazole-5-carbohydrazide
(4a–4r)
General procedure
A mixture of 4-trifluoromethyl-(1,2,3)-thiadiazol-5-car-
boxylic acid hydrazide 3 (0.2 g, 1 mmol) and aryl aldehyde
(1 mmol) in ethanol (5 ml) was refluxed on a water bath
for 2 h. The solvent was removed and the residue was
purified by column chromatography using petroleum ether/
EtOAc (8:2) as eluents.
Preparation of ethyl 4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carboxylate (2)
Procedure
The 3-(ethoxycarbonyl hydrazino)-4,4,4-trifluorobutyric
acid ethyl ester 1 (1.0 g, 4 mmol) was cooled at 0 °C, and
SOCl2 (2.0 ml) was added dropwise with stirring during
10 min, and the temperature of reaction mixture was raised
to 60 °C and stirring was continued for 8 h. The reaction
mixture was cooled to room temperature and excess SOCl2
was removed under vacuum. The residue was treated with
cold water and neutralized with aqueous NaHCO3. The
product was extracted thrice with ethyl acetate, and com-
bined extracts were washed with water and dried over
anhydrous sodium sulfate. The solvent was removed under
vacuum and the crude product was purified by passing
through a column packed with silica gel using petroleum
ether/EtOAc (6:4) as eluents. Yellow oil, Yield: 48 %; IR
(KBr, cm-1): 1725 (–C=O), 1519 (C=C), 1361 (–C–F); 1H
NMR (CDCl3, 300 MHz): 1.41 (t, J = 6.6 Hz, 3H, CH3),
4.33(q, J = 6.6 Hz, 2H, OCH2); 13C NMR (DMSO-d6,
75 MHz): 167.2, 148.4, 146.9, 118.3, 59.4, 15.6; MS (EI,
70 eV): m/z (%): 226 (M?, 100), 181 (M?-OC2H5, 49),
157 (M?-CF3, 31); Anal. Calcd. for C6H5F3N2O2S: C,
31.86; H, 2.23; N, 12.39 %. Found: C, 31.88; H, 2.21; N,
12.37 %.
N0-(phenylmethylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4a)
Yellow solid, Yield: 98 %, mp 223–225 °C; IR (KBr,
cm-1): 3429 (–NH), 1689 (–C=O), 1621 (C=N), 1369 (–C–
F); 1H NMR (DMSO-d6, 300 MHz): d 7.41 (m, 3H, Ar–H),
7.59 (t, J = 8.3 Hz, 2H, Ar–H), 8.21 (s, 1H, –CH=N),
12.70 (s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz): 170.8,
154.8, 147.6, 146.8, 131.2, 130.6, 129.6, 128.6, 128.4,
128.3, 119.6; MS (EI, 70 eV): m/z (%): 300 (M?, 100),
231(M?-CF3, 31); Anal. Calcd. for C11H7F3N4OS: C,
44.00; H, 2.35; N, 18.66 %. Found: C, 44.02; H, 2.34; N,
18.68 %.
N0-((4-fluorophenyl)methylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4b)
Pale yellow solid, Yield: 96 %, mp 228–230 °C; IR (KBr,
cm-1): 3420(–NH),1689(–C=O),1624(C=N),1371(–C–F);
123

1752
Med Chem Res (2013) 22:1747–1755
1H NMR (DMSO-d6, 300 MHz): d 7.30 (d, J = 8.4 Hz, 2H,
Ar–H), 7.61 (d, J = 8.4 Hz, 2H, Ar–H), 8.39 (s, 1H,
–CH=N–), 12.79 (s, 1H, NH); 13C NMR (DMSO-d6,
75 MHz): 169.8, 158.8, 148.6, 146.9, 146.8, 132.2, 131.6,
122.6, 124.6, 127.4, 119.6; MS (EI, 70 eV): m/z (%): 318
(M?, 100), 249 (M?-CF3, 35); Anal. Calcd. for
C11H6F4N4OS: C, 41.51; H, 1.90; N, 17.60 %. Found: C,
41.49; H, 1.89; N, 17.62 %.
N0-((4-(dimethylamino)phenyl)methylidene)-4-
(trifluoromethyl)-1,2,3-thiadiazole-5-carbohydrazide (4f)
Red solid, Yield: 90 %, mp 250–256 °C; IR (KBr, cm-1):
3389 (–NH), 1669 (–C=O), 1619 (C=N), 1369 (–C–F); H
1
NMR (DMSO-d6, 300 MHz): d 3.02 (s, 6H, CH3), 7.11 (d,
J = 8.6 Hz, 2H, Ar–H), 7.29 (d, J = 8.6 Hz, 2H, Ar–H),
8.11 (s, 1H, –CH=N–), 12.66 (s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): 170.4, 154.7, 150.6, 146.9, 146.8,
137.2, 131.6, 131.4, 116.2, 116.1, 118.9, 41.6, 41.5; MS
(EI, 70 eV): m/z (%): 343(M?, 100), 274(M?-CF3, 29);
Anal. Calcd. for C13H12F3N5OS: C, 45.48; H, 3.52; N,
20.40 %. Found: C, 45.51; H, 3.53; N, 20.38 %.
N0-((4-chlorophenyl)methylidene)-4-(trifluoromethyl)-
1,2,3-thiadiazole-5-carbohydrazide (4c)
Pale yellow solid, Yield: 95 %, mp 244–245 °C; IR (KBr,
cm-1): 3418 (–NH), 1686 (–C=O), 1625 (C=N), 1368 (–C–
N0-((4-hydroxyphenyl)methylidene)-4-(trifluoromethyl)-
1,2,3-thiadiazole-5-carbohydrazide (4g)
F); 1H NMR (DMSO–d6, 300 MHz):
d 7.31 (d,
J = 8.3 Hz, 2H, Ar–H), 7.62 (d, J = 8.3 Hz, 2H, Ar–H),
8.18 (s, 1H, –CH=N–), 12.71(s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): 170.2, 154.8, 148.6, 146.9, 136.8,
132.2, 131.6, 129.6, 129.2, 127.4, 119.6; MS (EI, 70 eV):
m/z (%): 334(M?, 100), 265(M?-CF3, 38); Anal. Calcd.
for C11H6ClF3N4OS: C, 39.47; H, 1.81; N, 16.74 %.
Found: C, 39.49; H, 1.82; N, 16.71 %.
Yellow solid, Yield: 98 %, mp 178–180 °C; IR (KBr,
cm-1): 3420 (–OH), 3386 (–NH), 1683 (–C=O), 1632
1
(C=N), 1358 (–C–F); H NMR (DMSO-d6, 300 MHz): d
6.91 (d, J = 8.1 Hz, 2H, Ar–H), 7.52 (d, J = 8.1 Hz, 2H,
Ar–H), 8.32 (s, 1H, –CH=N), 9.81 (s, 1H, –OH), 12.15 (s,
1H, NH); 13C NMR (DMSO-d6, 75 MHz): 170.1, 159.8,
154.6, 146.3, 146.2, 130.2, 130.1, 128.6, 118.8, 128.7,
118.6; MS (EI, 70 eV): m/z (%): 316(M?, 100), 247(M?
-CF3, 41); Anal. Calcd. for C11H7F3N4O2S: C, 41.78; H,
2.23; N, 17.72 %. Found: C, 40.77; H, 2.22; N, 17.74 %.
N0-((4-bromophenyl)methylidene)-4-(trifluoromethyl)-
1,2,3-thiadiazole-5-carbohydrazide (4d)
N0-((4-methoxyphenyl)methylidene)-4-(trifluoromethyl)-
1,2,3-thiadiazole-5-carbohydrazide (4h)
Pale yellow solid, Yield: 92 %, mp 223–225 °C; IR (KBr,
cm-1): 3420 (–NH), 1680 (–C=O), 1629 (C=N), 1365 (–C–
F); 1H NMR (DMSO-d6, 300 MHz): d 7.29 (d, J = 8.3 Hz,
2H, Ar–H), 7.60 (d, J = 8.3 Hz, 2H, Ar–H), 8.31 (s, 1H, –
CH=N–), 12.77 (s, 1H, NH); 13C NMR (DMSO-d6,
75 MHz): 170.1, 154.7, 148.6, 146.9, 132.8, 132.2, 131.6,
129.6, 129.2, 125.4, 119.2; MS (EI, 70 eV): m/z (%):
379(M?, 100), 310(M?-CF3, 28); Anal. Calcd. for
C11H6BrF3N4OS: C, 34.85; H, 1.60; N, 14.78 %. Found: C,
34.87; H, 1.61; N, 14.77 %.
Yellow solid, Yield: 94 %, mp 260–263 °C; IR (KBr,
cm-1): 3389 (–NH), 1686 (–C=O), 1629 (C=N), 1354 (–C–
1
F); H NMR (DMSO-d6, 300 MHz): d 3.90 (s, 3H, CH3),
6.91 (d, J = 8.1 Hz, 2H, Ar–H), 7.62 (d, J = 8.1 Hz, 2H,
Ar–H), 8.10 (s, 1H, –CH=N), 12.71 (s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): 170.8, 158.8, 155.6, 147.1, 146.8,
131.2, 131.1, 128.8, 128.7, 118.7, 118.6, 56.8; MS (EI,
70 eV): m/z (%): 330(M?, 100), 261(M?-CF3, 38); Anal.
Calcd. for C12H9F3N4O2S: C, 43.64; H, 2.75; N, 16.96 %.
Found: C, 43.66; H, 2.76; N, 16.94 %.
N0-((4-nitrophenyl)methylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4e)
N0-(furan-2-ylmethylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4i)
Brown solid, Yield: 98 %, mp 203–205 °C; IR (KBr,
cm-1): 3429 (–NH), 1689 (–C=O), 1621 (C=N), 1525
1
Yellow solid, Yield: 96 %, mp 219–221 °C; IR (KBr,
cm-1): 3360 (–NH), 1690 (–C=O), 1615 (C=N), 1349 (–C–
F); 1H NMR (DMSO-d6, 300 MHz): d 6.51 (d, J = 8.3 Hz,
1H, Ar–H), 6.80 (m, 1H, Ar–H), 7.10 (d, J = 8.2 Hz, 1H,
Ar–H), 8.1 (s, 1H, –CH=N–), 12.59 (s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): 170.1, 154.8, 146.6, 146.2, 143.6,
143.5, 114.9, 110.7, 110.6; MS (EI, 70 eV): m/z (%):
290(M?, 100), 221(M?-CF3, 42); Anal. Calcd. for
(–NO2), 1367 (–C–F); H NMR (DMSO-d6, 300 MHz): d
7.41 (d, J = 8.5 Hz, 2H, Ar–H), 7.59 (d, J = 8.5 Hz, 2H,
Ar–H), 8.21 (s, 1H, –CH=N–), 12.70 (s, 1H, NH); 13C
NMR (DMSO-d6, 75 MHz): 170.4, 154.8, 150.6, 146.9,
146.8, 137.2, 131.6, 129.6, 123.2, 123.1, 118.4; MS (EI,
70 eV): m/z (%): 345(M?, 100), 276(M?-CF3, 32); Anal.
Calcd. for C11H6F3N5O3S: C, 38.27; H, 1.75; N, 20.28 %.
Found: C, 38.29; H, 1.74; N, 20.29 %.
123

Med Chem Res (2013) 22:1747–1755
1753
C9H5F3N4O2S: C, 37.25; H, 1.74; N, 19.30 %. Found: C,
37.26; H, 1.73; N, 19.32 %.
(DMSO-d6, 75 MHz): 170.8, 155.8, 146.4, 146.2, 136.6,
136.2, 134.5, 134.1, 122.8, 122.6, 115.9, 12.6; MS (EI,
70 eV): m/z (%): 472(M?, 100), 403(M?-CF3, 42); Anal.
Calcd. for C12H7Br2F3N4OS: C, 30.53; H, 1.49; N,
11.87 %. Found: C, 30.56; H, 1.48; N, 11.88 %.
N0-(thiophene-2-ylmethylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4j)
Yellow solid, Yield: 95 %, mp 246–248 °C; IR (KBr,
cm-1): 3396 (–NH), 1696 (–C=O), 1621 (C=N), 1364 (–C–
F); 1H NMR (DMSO-d6, 300 MHz): d 7.11 (d, J = 7.9 Hz,
1H, Ar–H), 7.40 (m, 1H, Ar–H), 7.51 (d, J = 8.1 Hz, 1H,
Ar–H), 8.30 (s, 1H, –CH=N–), 12.61 (s, 1H, NH); 13C
NMR (DMSO-d6, 75 MHz): 169.6, 154.1, 145.6, 145.3,
125.5, 125.4, 123.5, 123.4, 115.6; MS (EI, 70 eV): m/
z (%): 306 (M?, 100), 237 (M?-CF3, 46); Anal. Calcd. for
C9H5F3N4OS2: C, 35.29; H, 1.65; N, 18.29 %. Found: C,
35.31; H, 1.66; N, 18.27 %.
N0-(1-(pyridine-2-yl)ethylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4n)
White solid, Yield: 88 %, mp 208–211 °C; IR (KBr,
cm-1): 3930(–NH), 1659 (–C=O), 1632 (C=N), 1359 (–C–
1
F); H NMR (DMSO-d6, 300 MHz): d 2.59 (s, 3H, CH3),
7.40 (d, J = 7.9 Hz, 1H, Ar–H), 7.80 (m, 2H, Ar–H), 8.61
(d, J = 8.1 Hz, 1H, Ar–H), 12.71 (s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): 170.3, 155.8, 152.3, 148.9, 146,7,
146.5, 136.8, 128.9, 125.1, 116.1, 11.9; MS (EI, 70 eV): m/
z (%): 315(M?, 100), 246(M?-CF3, 29); Anal. Calcd. for
C11H8F3N5OS: C, 41.91; H, 2.56; N, 22.21 %. Found: C,
41.92; H, 2.55; N, 22.23 %.
N0-(1-phenylethylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4k)
Pale yellow solid, Yield: 92 %, mp 229–231 °C; IR (KBr,
N0-(1-methylethylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4o)
cm-1): 3390(–NH), 1696 (–C=O), 1626 (C=N), 1361 (–C–
1
F); H NMR (DMSO-d6, 300 MHz): d 2.41 (s, 3H, CH3),
7.42 (m, 3H, Ar–H), 7.71 (d, J = 8.4 Hz, 2H, Ar–H), 12.60
(s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz): 170.2, 155.8,
146.4, 146.2, 131.8, 131.1, 129.8, 129.7, 128.8, 128.6,
114.7, 12.8; MS (EI, 70 eV): m/z (%): 314(M?, 100),
245(M?-CF3, 45); Anal. Calcd. for C12H9F3N4OS: C,
45.86; H, 2.89; N, 17.83 %. Found: C, 46.84; H, 2.90; N,
17.84 %.
White solid, Yield: 95 %, mp 186–188 °C; IR (KBr,
cm-1): 3410 (–NH), 1696 (–C=O), 1626 (C=N), 1369 (–C–
1
F); H NMR (DMSO-d6, 300 MHz): d 2.21 (s, 6H, CH3),
11.21 (s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz): 170.2,
155.9, 146.8, 146.6, 115.6, 16.8, 12.4; MS (EI, 70 eV): m/
z (%): 252 (M?, 100), 183(M?-CF3, 34); Anal. Calcd. for
C7H7F3N4OS: C, 33.33; H, 2.80; N, 22.21 %. Found: C,
33.35; H, 2.79; N, 22.23 %.
N0-(1-(4-nitrophenyl)ethylidene)-4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide (4l)
N0,N00-pentane-1,5-diylidenebis(4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide) (4p)
Yellow solid, Yield: 92 %, mp 203–209 °C; IR (KBr,
cm-1): 3410(–NH), 1686 (–C=O), 1624 (C=N), 1513
White solid, Yield: 96 %, mp 175–176 °C; IR (KBr,
cm-1): 3390(–NH), 1696 (–C=O), 1619 (C=N), 1361 (–C–
F); 1H NMR (DMSO-d6, 300 MHz): d 1.91 (m, 4H, CH2–),
2.40 (m, 4H, –CH2–), 11.71 (s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): 170.8, 155.3, 146.9, 146.7, 115.6,
35.4, 31.2, 31.1, 30.8; MS (EI, 70 eV): m/z (%): 278(M?,
100), 209(M?-CF3, 38); Anal. Calcd. for C9H9F3N4OS: C,
38.85; H, 3.26; N, 20.14 %. Found: C, 38.84; H, 3.27; N,
20.16 %.
1
(–NO2), 1361 (–C–F); H NMR (DMSO-d6, 300 MHz): d
2.51 (s, 3H, CH3), 7.42 (d, J = 8.6 Hz, 2H, Ar–H), 7.61 (d,
J = 8.6 Hz, 2H, Ar–H), 12.79 (s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): 170.6, 154.2, 150.7, 146.6, 146.2,
131.1, 129.8, 129.7, 128.8, 128.6, 115.1, 12.1; MS (EI,
70 eV): m/z (%): 359 (M?, 100), 290 (M?-CF3, 38); Anal.
Calcd. for C12H8F3N5O3S: C, 40.12; H, 2.24; N, 19.49 %.
Found: C, 40.14; H, 2.23; N, 19.47 %.
N0-(1-(2,4-dibromophenyl)ethylidene)-4-(trifluoromethyl)-
1,2,3-thiadiazole-5-carbohydrazide (4m)
N0,N00-hexane-1,6-diylidenebis(4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide) (4q)
Yellow solid, Yield: 91 %, mp 218–220 °C; IR (KBr,
White solid, Yield: 95 %, mp 186–188 °C; IR (KBr,
cm-1): 3390 (–NH), 1696 (–C=O), 1617 (C=N), 1356 (–C–
F); 1H NMR (DMSO-d6, 300 MHz): d 1.70 (m, 2H, CH2–),
1.95 (m, 4H, –CH2–), 2.34 (m, 4H, –CH2–), 11.81 (s, 1H,
NH); 13C NMR (DMSO-d6, 75 MHz): 170.3, 155.6, 146.2,
cm-1): 3410(–NH), 1696 (–C=O), 1619 (C=N), 1358 (–C–
1
F); H NMR (DMSO-d6, 300 MHz): d 2.55 (s, 3H, CH3),
7.52 (d, J = 8.1 Hz, 1H, Ar–H), 7.59 (d, J = 8.1 Hz, 1H,
Ar–H), 7.66 (s, 1H, Ar–H), 12.68 (s,1H, NH); 13C NMR
123

1754
Med Chem Res (2013) 22:1747–1755
146.1, 115.7, 33.4, 32.3, 26.9, 26.2, 26.1; MS (EI, 70 eV):
m/z (%): 292(M?, 100), 223(M?-CF3, 31); Anal. Calcd.
for C10H11F3N4OS: C, 41.09; H, 3.79; N, 19.17 %. Found:
C, 41.08; H, 3.78; N, 19.19 %.
(30, 100 lg/ml) of test solutions were added. Controls were
maintained with acetone and amphotericin B (50 lg/ml).
The treated and the controls were kept at 28 °C for 48 h.
Inhibition zones were measured and the diameter was
calculated in millimeter.
N0,N00-heptane-1,7-diylidenebis(4-(trifluoromethyl)-1,2,3-
thiadiazole-5-carbohydrazide) (4r)
Cytotoxic activity
White solid, Yield: 91 %, mp 191–194 °C; IR (KBr,
cm-1): 3380 (–NH), 1689 (–C=O), 1611 (C=N), 1352 (–C–
F); 1H NMR (DMSO-d6, 300 MHz): d 1.74 (m, 4H, CH2–),
1.92 (m, 4H, –CH2–), 2.44 (m, 4H, –CH2–), 11.83 (s, 1H,
NH); 13C NMR (DMSO-d6, 75 MHz): 170.4, 155.9, 146.3,
146.1, 115.9, 29.9, 29.6, 29.5, 25.4, 24.7, 24.6; MS (EI,
70 eV): m/z (%): 306(M?, 100), 237(M?-CF3, 28); Anal.
Calcd. for C10H10F3N4OS: C, 43.13; H, 4.28; N, 18.29 %.
Found: C, 43.15; H, 4.27; N, 18.31 %.
Procedure
The breast carcinoma cells MDA-MB 231 (aggressive) and
MCF-7 (non-aggressive) from ATCC were grown in Dul-
becco’s modified Eagle’s medium (DMEM) with 10 %
FBS supplemented with antibiotics and treated with test
compounds at 10 lM concentration and standard doxoru-
bicin at 5 lM concentration for a period of 48 h and
cytotoxicity was measured by standard MTT assay. At the
end of the treatments, medium was removed and cells were
washed with Dulbecco’s phosphate buffered saline (DPBS)
and 10 ll of 5 mg/ml MTT solution in 200 ll of culture
medium was added and incubated for 1 h at 37 °C. Cells
were solubilized with 200 ll of DMSO and absorbance
was measured at 562 nm in a spectrophotometer. The
absorbance of the wells containing treated cells was com-
pared with that of the wells in which the drug is omitted
(control).
Antibacterial activity
Procedure
Five bacterial test organisms such as B. subtilis (MTCC
441), S. aureus (MTCC 96), S. epidermidis (MTCC 435),
E. coli (MTCC 443) P. aeruginosa (MTCC 741), and K.
pneumoniae were selected and obtained from the Institute
of Microbial Technology, Chandigarh. Cultures of test
organisms were maintained on nutrient agar slants and
were subcultured in Petri dishes prior to testing. The media
used were nutrient agar, nutrient broth procured from
Himedia Laboratories, Mumbai. The minimum inhibitory
concentration was determined by broth dilution method.
Acknowledgments Authors are thankful to Dr. J.S.Yadav, Director,
IICT for his constant encouragement and authors (P. Sambasiva Rao,
C. Kurumurthy, B. Veeraswamy, and G. Santhosh kumar) are
thankful to CSIR for providing financial assistance in the form of
Senior Research Fellowship.
Antifungal activity
References
Procedure
Bakulev VA, Mokrushin VS (1986) The chemistry of 1,2,3-thiadiaz-
oles. Khim Geterotsikl Soedin 1011
Chae J, Konno T, Ishihara T, Yamanaka H (2004) A facile synthesis
of various fluorine containing indole derivatives via palladium
catalyzed annulations of internal alkynes. Chem Lett 33:314–315
Filler R, Kobayashi Y, Yagupolskii LM, (1993) Organofluorine
compounds in medicinal chemistry and biomedical applications,
studies in organic chemistry, vol 48. Elsevier, Amsterdam, p 362
Hertel LW, Kroin JS, Misner JW, Tustin JM (1988) Synthesis of
2-deoxy-2,2-difluoro-D-ribose and 2-deoxy-2,2-difluoro-D-ribo-
furanosyl nucleoside. J Org Chem 53:2406–2409
Antifungal activity was studied by agar cup diffusion
method. The readymade potato dextrose agar (PDA)
medium (Himedia, 39 g) was suspended in distilled water
(1000 ml) and heated until it gets dissolved completely.
The medium and Petri dishes were autoclaved at pressure
of 15 lb/inch for 20 min. Agar cup bioassay was employed
for testing antifungal activity. The medium was poured into
sterile Petri dishes under aseptic conditions in a laminar
flow chamber. When the medium in the plates solidified,
0.5 ml of (week-old) culture of test organism was inocu-
lated and uniformly spread over the agar surface with a
sterile L-shaped rod. Solutions were prepared by dissolving
the compound in acetone and different concentrations (30,
100 lg/ml) were made. After inoculation, cups were
scooped out with 6-mm sterile cork borer and the lids of the
dishes were replaced. To each cup, different concentrations
Hurd CD, Mori RI (1955) On acyl hydrazones and 1,2,3-thiadiazoles.
J Am Chem Soc 77:5359
Kurumurthy C, Sambasiva Rao P, Veera Swamy B, Santhosh Kumar
G, Shanthan Rao P, Narsaiah B, Velatooru LR, Pamanji R,
Venkateswara Rao J (2011) Synthesis of novel alkyltriazole
tagged pyrido[2,3-d]pyrimidine derivatives and their anticancer
activity. Eur J Med Chem 46:3462–3468
Kurzer F (1973) Chemistry of organic compounds with sulfur,
selenium. Tellurium 1:444
L’abb’e G, D’hooge B, Dehaen W (1996) Thiadiazoles as a
convenient source for the study of molecular rearrangements,
123

Med Chem Res (2013) 22:1747–1755
1755
single bond/no bond resonance and dendrimer synthesis. Mol-
ecules 1:190
Peng Z, Xinyong L, Zhenyu L, Zengjun F, Zhong L, Defeng W
(2009) Novel 1,2,3-thiadiazole derivatives as HIV-1 NNRTIs
with improved potency: synthesis and preliminary SAR studies.
Bioorg Med Chem 17:5920–5927
Sarett LH. and Brown HD (1962) Certain benzimadazoles carrying
thiazolyl and isothiazolyl substituents in the second position.
U.S. patent 3,017,415, Antihelmentic compositions containing
benzimidazole derivatives. 3,357,884 (1967) and 5-(or 6)-halo(or
amino) benzazoles and methods for preparation 3,478,046
(1969)
Sirisha B, Narsaiah B, Yakaiah T, Gayatri G, Sastry GN, Prasad MR,
Rao AR (2010) Synthesis and theoretical studies on energetics of
novel N- and O-perfluoroalkyl triazole tagged thienopyrimi-
dines—their potential as adenosine receptor ligands. Eur J Med
Chem 45:1739–1745
Manichandrika P, Yakaiah T, Rao AR, Narsaiah B, Reddy NC,
Sridhar V, Rao JV (2008) Synthesis of novel 4,6-disubstituted
quinazoline derivatives, their anti-inflammatory and anti-cancer
activity (cytotoxic) against U937 leukemia cell lines. Eur J Med
Chem 43:846–852
Manichandrika P, Yakaiah T, Gayatri G, Pranay Kumar K, Narsaiah
B, Murthy USN, RaghuRam Rao A (2010) Click chemistry:
studies on the synthesis of novel fluorous tagged triazol-4-yl
substituted quinazoline derivatives and their biological evalua-
tion—theoretical and experimental validation. Eur J Med Chem
45:78–84
McConnell RL, Coover HW (1963). Organo phosphorous derivatives
of 1,2,3-thiadiazoles. U.S. patent 3,072,669
Morzherin YuYu, Glukhareva TV, Bakulev VA (2003) Rearrange-
ments and transformations of 1,2,3-thiadiazoles in organic
synthesis (Review). Chem Heterocycl Compd 39:2003
Pain DL, Slack R (1965) Congeners of pyridine-4-carboxyhydrazides,
Part-II: derivatives of 1,2,3-thiadiazoles. J Chem Soc 5166
Pechmann H, Nold A (1896) Reactions of 1,2,3-thiadiazoles. Chem
Ber 28:2588
Teng X, Keys H, Jeevanandam A, Porco JA Jr, Degterev A, Yuan J,
Cuny GD (2007) Structure–activity relationship study of
[1,2,3]thiadiazole necroptosis inhibitors. Bio Org Med Chem
Lett 17(24):6836–6840
Thomas EW (1984) Thiadiazoles and their benzo derivatives. In:
Comprehensive heterocyclic chemistry, vol 6. Pergamon Press,
New York, p 447
Peng Z, Xinyong L, Yuan C, Yan W, Christophe P, Erik DC (2008)
1,2,3-Thiadiazole thioacetanilides as a novel class of potent
HIV-1 non-nucleoside reverse transcriptase inhibitors. Bioorg
Med Chem Lett 18:5368–5371
Wolff L (1904) Hetero cyclization of a-diazo thio carbonyl
compounds. Liebigs Ann Chem 1:333
123