Synthesis of novel 1,2,4-triazoles, triazolothiadiazines and triazolothiadiazoles as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.08.047

A series of new N-substituted-3-mercapto-1,2,4-triazoles (3a,b and 7a-d), triazolo[1,3,4]thiadiazines (5a,b) and triazolo[1,3,4]thiadiazoles (4a-d, 6 and 8a-d) have been synthesized starting from isonicotinic acid hydrazide. The structure of the newly synthesized compounds was confirmed on the basis of their spectral data and elemental analyses. All the compounds were screened for their in vitro anticancer activity against 6 human cancer cell lines and normal fibroblasts. Seven of the tested compounds (3a,b, 4c, 5a and 8b-d) exhibited significant cytotoxicity against most cell lines. Among these derivatives compound 4c exhibited equivalent cytotoxic effect to the standard CHS 828 against gastric cancer cell line (IC₅₀ = 25 nM). Normal fibroblast cells (WI38) were affected to a much lesser extent (IC₅₀ > 10,000 nM).

Full text of DOI:10.1016/j.ejmech.2014.08.047

European Journal of Medicinal Chemistry 86 (2014) 75e80
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 1,2,4-triazoles, triazolothiadiazines and
triazolothiadiazoles as potential anticancer agents
,
Mona M. Kamel a *, Nadia Y. Megally Abdo b
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, 11562 Cairo, Egypt
b Chemistry Department, Faculty of Education, Alexandria University, 21526 Alexandria, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new N-substituted-3-mercapto-1,2,4-triazoles (3a,b and 7aed), triazolo[1,3,4]thiadiazines
(5a,b) and triazolo[1,3,4]thiadiazoles (4aed, 6 and 8aed) have been synthesized starting from iso-
nicotinic acid hydrazide. The structure of the newly synthesized compounds was confirmed on the basis
of their spectral data and elemental analyses. All the compounds were screened for their in vitro anti-
cancer activity against 6 human cancer cell lines and normal fibroblasts. Seven of the tested compounds
(3a,b, 4c, 5a and 8bed) exhibited significant cytotoxicity against most cell lines. Among these derivatives
compound 4c exhibited equivalent cytotoxic effect to the standard CHS 828 against gastric cancer cell
Received 24 February 2014
Received in revised form
11 August 2014
Accepted 14 August 2014
Available online 15 August 2014
Keywords:
line (IC50
(IC50 > 10,000 nM).
¼
25 nM). Normal fibroblast cells (WI38) were affected to a much lesser extent
Isonicotinic acid hydrazide
Mercaptotriazoles
Triazolothiadiazoles
Triazolothiadiazines
Anticancer activity
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
preparative organic chemistry as useful intermediates for the
preparation of some triazolothiadiazoles and triazolothiadiazines.
The amino and mercapto groups are ready made nucleophilic
centers for the synthesis of condensed heterocyclic rings [13].
Meanwhile, the triazolothiadiazoles and triazolothiadiazines
exhibiting broad spectrum biological profile have matured into
indispensable heterocyclic scaffolds [14,15]. New analogs of tri-
azolothiadiazoles and triazolothiadiazines bearing different sub-
stituents on the 3 and 6 positions were found to possess significant
anticancer activity. Holla et al. [16] reported the synthesis of several
structurally modified triazolothiadiazines which were evaluated
for their anticancer activity against full panel of 60 cell lines derived
from seven cancer types. The screening results showed that the
compound bearing a 2-chloroaryloxy methyl group at C-3 and 4-
chlorobenzylidine at C-7 positions exhibited the highest activity
Cancer is a life threatening disease and remains a major health
problem around the globe. It is the second most occurring disease
after cardiovascular diseases. Thus, the development of potent and
effective novel antineoplastic drugs is one of the most intensely
persuaded goals of contemporary medicinal chemistry.
The chemistry of 1,2,4-triazoles and their fused heterocyclic
derivatives have received considerable attention owing to their
synthetic and effective biological importance. For example, a large
number of 1,2,4-triazoles have been incorporated into a wide va-
riety of therapeutically interesting drug candidates possessing
antimicrobial [1e5], anti-inflammatory [6], analgesic [7] and anti-
cancer activities [8,9]. Literature survey reveals that important
chemotherapeutics, such as Vorozole, Letrozole and Anastrozole
(Fig. 1) that consist of substituted 1,2,4-triazole ring, are currently
being used for the treatment of breast cancer [10]. Among these
heterocycles, the mercapto and thione substituted 1,2,4-triazole
ring systems have been well studied and so far a variety of bio-
logical activities have been reported for a large number of their
derivatives [11,12]. In addition to these important biological appli-
cations, mercapto 1,2,4-triazoles are also of great utility in
with GI50 < 10 mM against all cell lines. Subsequently, Poojary and
co-workers [17] prepared and screened a series of novel tri-
azolothiadiazoles and triazolothiadiazines for their anti-tumor ac-
tivity. The structure activity relationship indicated that compounds
containing chlorine atoms at various positions, especially the tri-
azolothiadiazoles bearing chlorophenyl groups at position 6, are
more active as compared to other derivatives. Moreover, Isloor et al.
[18] carried out the synthesis of 6-[3-(4-fluorophenyl)-1H-pyrazol-
4-yl]-3-[(2-naphthyloxy)methyl][1,2,4]triazolo[3,4-b][1,3,4]-thia-
diazole (FPNT). The anticancer activity along with possible mech-
anism of action of the triazolothiadiazole in HepG2 cells was
* Corresponding author.
E-mail addresses: mona_mounir50@hotmail.com, mona.kamel@pharma.cu.edu.
eg (M.M. Kamel).
http://dx.doi.org/10.1016/j.ejmech.2014.08.047
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

76
M.M. Kamel, N.Y. Megally Abdo / European Journal of Medicinal Chemistry 86 (2014) 75e80
Fig. 1. Chemical structures of breast cancer drugs.
explored using MTT assay, [3H] thymidine assay, flow cytometry
and chromatin condensation studies. It was concluded that FPNT
exhibited potent cytotoxic activity through induction of growth
inhibition followed by apoptosis in HepG2 cells.
Recently, Husain et al. [19] reported the in vitro anticancer ac-
tivity of a library of triazolothiadiazole and triazolothiadiazine
derivatives incorporating benzimidazole scaffold. The anticancer
screening results revealed that the triazolothiadiazole derivatives
carrying a benzimidazole moiety exhibited better anticancer ac-
tivity than their thiadiazine analogs.
isothiocyanates in pyridine for 30 min. Meanwhile, carrying the
latter reaction for 2e3 h till complete evolution of H2S (tested by
lead acetate paper) afforded the cyclized analogs 8aed. The
disappearance of the singlet signal at
d 5.8 ppm assignable to the
2NH groups of the thiourea moiety present in the 1H NMR spectra
of compounds 7aed confirmed formation of the latter cyclized
derivatives.
2.2. In vitro cytotoxicity
In a valuable attempt to explore the mode of action of tri-
azolothiadiazoles, Ibrahim [20] reported the synthesis of a new
series of 3,6-disubstituted triazolo[3,4-b]thiadiazole derivatives
which were designed to act as tyrosine kinase inhibitors. The
synthesized compounds were evaluated for their cytotoxic activity
against a panel of 60 human cancer cell lines by the National Cancer
Institute (NCI) and some of them demonstrated inhibitory effects
on the growth of a wide range of cancer cell lines generally at
10ꢀ5e10ꢀ7 M concentrations. However, the anti-tumor activity of
the synthesized compounds could not be interpreted in terms of
tyrosine kinase inactivation but more likely as a relatively broad
specificity for the ATP-binding domain of other kinases. The
biochemical assay of the synthesized compounds as CDK inhibitors
was determined and expressed as IC50. Some compounds showed
modest activity (IC50 < 10 mM).
The heterocyclic compounds, prepared in this study, were
evaluated according to standard protocols for their in vitro cyto-
toxicity against six human cancer cell lines including cells derived
from human gastric cancer (NUGC), human colon cancer (DLD1),
human liver cancer (HA22T and HEPG2), nasopharyngeal carci-
noma (HONE1), human breast cancer (MCF) and normal fibroblast
cells (WI38). For comparison purposes CHS 828, a pyridyl cyano-
guanidine, was used as a standard cytotoxic drug (Fig. 4) [21]. All of
IC50 values in nanomolar (nM) are listed in (Table 1) and the results
are represented graphically in (Figs. 2 and 3). Seven of the tested
compounds namely: 3a, 3b, 4c, 5a, 8b, 8c and 8d showed significant
cytotoxic effects with IC50 values <800 nM. Normal fibroblast cells
(WI38) were affected to a much lesser extent (IC50 > 10,000 nM).
The N-substituted-mercaptotriazole derivative 3a showed better
cytotoxic activity than 3b with IC50 in the range of 42e53 nM
against colon, gastric and liver cancer cell lines. Moreover, com-
pound 5a, a triazolothiadiazine derivative, possessed potential
cytotoxic activity against all the tested cancer cell lines except colon
cancer. The triazolothiadiazole derivatives 4c, 8b, 8c and 8d showed
remarkable cytotoxic activity against most cell lines. Among these
derivatives compound 4c exhibited equivalent cytotoxic effect to
the standard CHS 828 against gastric cancer cell line (IC50 ¼ 25 nM).
While some of the compounds were not the most potent, their
specific activity against particular cell lines makes that of interest
for further development as anticancer drugs.
In our search for new classes of potential anticancer agents, the
aforementioned findings promoted us to synthesize a series of N-
substituted-3-mercapto-[1,2,4]triazoles, 3,6-disubstituted-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazoles and 3,6-disubstituted-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazines. The newly synthesized compounds
were evaluated for their in vitro anticancer activity.
2. Results and discussion
2.1. Chemistry
The reaction sequence employed for the synthesis of the title
compounds is shown in Scheme 1. The reaction of isonicotinic acid
hydrazide with CS2 in methanolic KOH yielded the corresponding
dithiocarbazinate 1 which upon heating under reflux with hydra-
zine hydrate afforded the starting 3-mercaptotriazole 2. The N-
acylated derivatives 3a,b were obtained by the reaction of 2 with
acetyl chloride and p-nitrobenzoyl chloride respectively. On the
other hand, condensation of 2 with various aromatic carboxylic
acids in POCl3 yielded the corresponding 6-aryltriazolothiadiazoles
4aed. The triazolothiadiazine derivatives 5a,b were obtained from
the reaction of 2 with phenacyl bromides. Moreover, the reaction of
2 with acetic anhydride afforded the 6-methyltriazolothiadiazole 6.
The synthesis of thiourea derivatives 7aed was achieved via
3. Conclusion
The present research reports the successful synthesis, charac-
terization and anticancer activity of new 1,2,4-triazole derivatives
starting from isonicotinic acid hydrazide. Seven of the tested
compounds namely: the 3-mercaptotriazoles 3a, 3b; tri-
azolothidiazine derivative 5a and triazolothiadizoles 4c, 8b, 8c and
8d showed significant cytotoxic effects with IC50 values <800 nM.
Among these derivatives compound 4c exhibited equivalent cyto-
toxicity effect to the standard CHS 828 against gastric cancer cell
line (IC50 ¼ 25 nM). Normal fibroblast cells (WI38) were affected to
a much lesser extent (IC50 > 10,000 nM). The obtained results
suggest that these compounds may serve as lead chemical entities
heating under reflux
a mixture of 2 with various aryl

M.M. Kamel, N.Y. Megally Abdo / European Journal of Medicinal Chemistry 86 (2014) 75e80
77
Scheme 1. Synthesis of compounds: 3a,b; 4aed; 5a,b; 6; 7aed and 8aed.
for further modification in the search of new classes of potential
anticancer agents.
University, Egypt) using TMS as internal standard. Chemical shift
values are recorded in ppm on scale. Mass spectra were recorded
d
on a Hewlett Packard 5988 spectrometer (Microanalysis Center,
Cairo University, Egypt). Elemental analyses were carried out at the
Microanalysis Center, Cairo University, Egypt; found values were
within 0.35% of the theoretical ones. Progress of the reactions was
monitored using thin layer chromatography (TLC) sheets precoated
with UV fluorescent silica gel Merck 60F 254 and were visualized
using UV lamp.
4. Experimental
4.1. Chemistry
All melting points were determined on a Stuart apparatus and
the values given are uncorrected. IR spectra (KBr, cmꢀ1) were
determined on a Shimadzu IR 435 spectrophotometer (Faculty of
Pharmacy, Cairo University, Egypt). 1H NMR and 13C NMR spectra
were recorded on Varian Gemini 300 MHz (Microanalysis Center,
Cairo University, Egypt) and Bruker Ascend 400 MHz spectropho-
tometers (Microanalytical Unit, Faculty of Pharmacy, Cairo
4.1.1. General method for preparation of 4-amino-5-pyridin-4-yl-
4H-[1,2,4]triazole-3-thiol (2)
Isonicotinic acid hydrazide (11.37 g, 0.083 mol) was treated with
a solution of KOH (6.4 g, 0.125 mol) dissolved in methanol (50 mL)

78
M.M. Kamel, N.Y. Megally Abdo / European Journal of Medicinal Chemistry 86 (2014) 75e80
cold water and acidified by HCl. The obtained white solid was
filtered, washed with water, dried and crystallized from ethanol.
4.1.2.1. N-(3-Mercapto-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-yl)acet-
amide(3a). Yield: 60%; m.p.: 275e277 ꢁC; IR (kBr, cmꢀ1): 3248
(NH), 2966 (CH aliphatic), 2621 (SH), 1714 (C]O), 1608 (C]N); 1H
NMR(DMSO-d6):
d 2.07 (s, 3H, CH3), 3.38 (s, 1H, NH, D2O
exchangeable), 7.68 (s, 2H, Pyridine H-3 and H-5), 8.76 (s, 2H,
Pyridine H-2 and H-6), 11.53 (s, 1H, SH, D2O exchangeable); 13C
NMR (DMSO-d6):
d 20.8, 122.1, 133.4, 147.8, 150.6, 168.2, 170.8; MS:
m/z (%) 235 (Mþ, 41). Anal. Calcd. for C9H9N5OS: C, 45.95; H, 3.86; N,
29.77. Found: C, 45.87; H, 3.77; N, 29.61.
4.1.2.2. N-(3-Mercapto-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-yl)-4-
nitrobenzamide(3b). Yield: 68%; m.p.: 235e237 ꢁC; IR (kB, cmꢀ1):
3316 (NH), 3062 (AreH), 2551 (SH), 1691 (C]O), 1608 (C]N); 1H
Fig. 2. Cytotoxicity of 3a, 3b, 4c, 5a and CHS 828 against NUGC, gastric cancer; DLDI,
colon cancer; HA22T, liver cancer; HEPG2, liver cancer; HONEI, nasopharyngeal car-
cinoma; MCF, breast cancer.
NMR (DMSO-d6):
d 3.32 (s, 1H, NH, D2O exchangeable), 7.35e7.87
(m, 4H, AreH), 8.15e8.61 (m, 4H, Pyridine-H), 13.56 (s, 1H, SH, D2O
exchangeable); MS: m/z (%) 342 (Mþ, 21). Anal. Calcd. for
at 0e5 ꢁC with stirring. Carbon disulphide (9.6 g, 0.125 mol) was
added slowly and the reaction mixture was stirred over night at
room temperature. The solid product of potassium dithiocarbazi-
nate 1 was filtered, washed with chilled methanol and dried. It was
directly used in the next step without further purification.
C14H10N6O3S: C, 49.12; H, 2.94; N, 24.55. Found: C, 49.40; H, 2.87; N,
24.42.
4.1.3. General method for the preparation of compounds 4aed
An equimolar mixture (0.01 mol) of 4-amino-5-pyridin-4-yl-
4H-[1,2,4]triazole-3-thiol (2) and the appropriate aromatic acid in
POCl3 (10 mL) was refluxed for 8e10 h. The reaction mixture was
cooled to room temperature and gradually poured onto ice cold
water with stirring. The mixture was allowed to stand overnight
and the solid separated out was filtered, treated with dilute NaOH
solution and washed thoroughly with cold water. The solid ob-
tained was filtered, dried and crystallized from ethanol.
Hydrazine hydrate (9 g, 0.2 mol) was added to a solution of 1
(22 g, 0.1 mol) in water (20 mL) and the reaction mixture heated
under reflux till complete evolution of H2S. The reaction mixture
was poured on ice cold water and acidified with HCl. The yellow
precipitate was filtered, washed with cold water, dried and crys-
tallized from ethanol. Yield: 80%; m.p.: 240e242 ꢁC; IR (kBr, cmꢀ1):
3271 (NH2), 3159 (AreH), 2881 (CH aliphatic), 2561 (SH), 1608 (C]
N); 1H NMR (DMSO-d6):
d 5.87 (s, 2H, NH2, D2O exchangeable), 8.01
(d, 2H, J ¼ 6.0 Hz, Pyridine H-3 and H-5), 8.75 (d, 2H, J ¼ 6.0 Hz,
Pyridine H-2 and H-6), 14.12 (s, 1H, SH, D2O exchangeable).
4.1.3.1. 6-Phenyl-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zole (4a). Yield: 55%; m.p.: 216e218 ꢁC; IR (kBr, cmꢀ1): 3080
4.1.2. General method for the preparation of compounds 3a,b
A mixture of 4-amino-5-pyridin-4-yl-4H-[1,2,4]triazole-3-thiol
(2) (0.01 mol) and the appropriate acid chloride (0.01 mol) was
heated under reflux in DMF in presence of triethylamine (0.5 mL)
for 5 h. The reaction mixture was allowed to cool, poured onto ice
(AreH), 1633, 1600 (C]N). 1H NMR (DMSO-d6):
d 7.65e8.12 (m, 5H,
AreH), 8.46 (d, 2H, J ¼ 6 Hz, Pyridine H-3 and H-5), 8.96 (d, 2H,
J ¼ 6 Hz, Pyridine H-2 and H-6); MS: m/z (%) 279 (Mþ, 100). Anal.
Calcd. for C14H9N5S: C, 60.20; H, 3.25; N, 25.07. Found: C, 60.12; H,
3.07; N, 25.30.
Fig. 3. Cytotoxicity of 8a, 8b, 8c, 8d and CHS 828 against NUGC, gastric cancer; DLDI, colon cancer; HA22T, liver cancer; HEPG2, liver cancer; HONEI, nasopharyngeal carcinoma;
MCF, breast cancer.

M.M. Kamel, N.Y. Megally Abdo / European Journal of Medicinal Chemistry 86 (2014) 75e80
79
4.1.4.1. 6-Phenyl-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazine (5a). Yield: 87%; m.p.: 220e222 ꢁC; IR (kBr, cmꢀ1):
3039 (AreH), 1598 (C]N); 1H NMR (DMSO-d6):
d 4.48 (s, 2H, CH2),
7.36e8.02 (m, 5H, AreH), 8.04 (d, 2H, J ¼ 6 Hz, Pyridine H-3 and H-
5), 8.79 (d, 2H, J ¼ 6 Hz, Pyridine H-2 and H-6); 13C NMR (DMSO-
d6): d 156.5, 150.3, 149.6, 143.8, 133.2, 132.9, 132.1, 129.1, 127.7, 121.4,
22.8; MS: m/z (%) 293 (Mþ, 85). Anal. Calcd. for C15H11N5S: C, 61.42;
Fig. 4. Chemical structure of CHS 828.
H, 3.78; N, 23.87. Found: C, 61.21; H, 3.58; N, 23.92.
4.1.3.2. 6-(2-Chlorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b]
4.1.4.2. 6-(4-Bromophenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-
[1,3,4]thiadiazole (4b). Yield: 75%; m.p.: 235e237 ꢁC; IR (kBr,
b][1,3,4]thiadiazine (5b). Yield: 84%; m.p.: 283e285 ꢁC; IR (kBr,
cmꢀ1): 3080 (AreH), 1602 (C]N). 1H NMR (DMSO-d6):
d 7.04e7.53
cmꢀ1): 3078 (AreH),1600 (C]N). 1H NMR (DMSO-d6):
d 4.52 (s, 2H,
CH2), 7.68e8.06 (m, 4H, AreH), 8.39e8.73 (m, 4H, Pyridine-H); MS:
m/z (%) 372 (Mþ, 42). Anal. Calcd. for C15H10BrN5S: C, 48.40; H, 2.71;
N, 18.81. Found: C, 48.13; H, 2.71; N, 18.52.
(m, 4H, AreH), 8.14 (d, 2H, J ¼ 6 Hz, Pyridine H-3 and H-5), 8.81 (d,
2H, J ¼ 6 Hz, Pyridine H-2 and H-6); MS: m/z (%) 313 (Mþ, 51). Anal.
Calcd for C14H8ClN5S: C, 53.59; H, 2.57; N, 22.32. Found: C, 53.34; H,
2.76; N, 22.54.
4.1.5. General method for the preparation of 6-methyl-3-(pyridin-
4-yl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (6)
4.1.3.3. 6-(4-Chlorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (4c). Yield: 82%; m.p.: 278e280 ꢁC, IR (kBr, cmꢀ1):
A mixture of 4-amino-5-pyridin-4-yl-4H-[1,2,4]triazole-3-thiol
(2) (0.01 mol) and acetic anhydride (10 mL) was heated under
reflux for 7 h. The reaction mixture was left to cool, poured onto ice
cold water. The obtained solid, filtered, washed with water, dried
and crystallized from ethanol. Yield: 60%; m.p.: 246e248 ꢁC; IR
(kBr, cmꢀ1): 3043 (AreH), 2958 (CH aliphatic), 1604 (C]N); 1H
3022 (AreH), 1604 (C]N). 1H NMR (DMSO-d6):
d 7.36e8.15 (m, 4H,
AreH), 8.24 (d, 2H, J ¼ 5.8 Hz, Pyridine H-3 and H-5), 8.84 (d, 2H,
J ¼ 5.8 Hz, Pyridine H-2 and H-6); 13C NMR (DMSO-d6):
d 120.2,
128.8,129.7,131.4,134.0,134.3,143.4,148.6,151.3,167.7; MS: m/z (%)
313 (Mþ, 53). Anal. Calcd. for C14H8ClN5S: C, 53.59; H, 2.57; N, 22.32.
Found: C, 53.78; H, 2.32; N, 22.60.
NMR (DMSO-d6):
d
2.48 (s, 3H, CH3), 8.13 (d, 2H, J ¼ 5.9 Hz, Pyridine
H-3 and H-5), 8.80 (d, 2H, J ¼ 5.9 Hz, Pyridine H-2 and H-6); MS: m/
z (%) 217 (Mþ, 42). Anal. Calcd. for C9H7N5S: C, 49.76; H, 3.25; N,
32.24. Found: C, 49.56; H, 3.12; N, 31.95.
4.1.3.4. 6-(2,4-Dichlorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole (4d). Yield: 84%; m.p.: 215e217 ꢁC; IR (kBr,
cmꢀ1): 3039 (AreH), 1600 (C]N). 1H NMR (DMSO-d6):
d 7.73e8.20
(m, 3H, AreH), 8.22 (d, 2H, J ¼ 6 Hz, Pyridine H-3 and H-5), 8.83 (d,
2H, J ¼ 6 Hz, Pyridine H-2 and H-6); MS: m/z (%) 348 (Mþ, 11). Anal.
Calcd. for C14H7Cl2N5S: C, 48.29; H, 2.03; N, 20.11. Found: C, 48.50;
H, 2.18; N, 20.09.
4.1.6. General method for the preparation of compounds 7aed
An equimolar mixture (0.01 mol) of 4-amino-5-pyridin-4-yl-
4H-[1,2,4]triazole-3-thiol (2) and the appropriate aryl isothiocya-
nate was heated under reflux in pyridine (10 mL) for 30 min. The
reaction mixture was poured onto ice cold water. The obtained
solid was filtered, washed with water, dried and crystallized from
ethanol.
4.1.4. General method for the preparation of compounds 5a,b
A mixture of 4-amino-5-pyridin-4-yl-4H-[1,2,4]triazole-3-thiol
(2) (0.01 mol) and the appropriate phenacyl bromide (0.012 mol)
in absolute ethanol was heated under reflux for 6 h. The reaction
mixture was poured onto crushed ice and neutralized with Na2CO3.
The solid product obtained was filtered, washed with water, dried
and crystallized from ethanol.
4.1.6.1. 1-(4-Chlorophenyl)-3-(3-mercapto-5-(pyridin-4-yl)-4H-
1,2,4-triazol-4-yl)thiourea (7a). Yield: 90%; m.p.: 235e238 ꢁC; IR
(kBr, cmꢀ1): 3300 (NH), 3138 (AreH), 1608 (C]N), 1315 (C]S); 1H
NMR (DMSO-d6):
d 5.86 (s, 2H, NH, D2O exchangeable), 7.34e7.77
(m, 4H, AreH), 8.02 (d, 2H, J ¼ 6.1 Hz, Pyridine H-3 and H-5), 8.75
(d, 2H, J ¼ 6.1 Hz, Pyridine H-2 and H-6), 10.9 (s, 1H, SH, D2O
exchangeable); MS: m/z (%) 362 (Mþ, 35). Anal. Calcd. for
C14H11ClN6S2: C, 46.34; H, 3.06; N, 23.16. Found: C, 46.14; H, 3.20; N,
23.40.
Table 1
a
Cytotoxicity of IMT-samples against a variety of cancer cell lines [IC50 (nM)].
Compd.
Cytotoxicity (IC50 in nM)
NUGC
DLDI
HA22T
HEPG2
HONE1
MCF
WI38
3a
3b
4a
4b
4c
4d
5a
5b
6
7a
7b
7c
7d
8a
44
360
1280
3220
25
42
266
3360
2665
49
1188
2350
2377
3285
2248
2288
2152
3270
3263
128
166
1114
1265
1260
120
53
89
1177
2279
2426
1169
488
2102
1092
1538
2177
1624
3224
80
na
na
na
na
na
na
na
na
650
2189
na
na
1288
na
na
480
na
4.1.6.2. 1-(3-Mercapto-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-yl)-3-(4-
methoxyphenyl)thiourea (7b). Yield: 92%; m.p.: 200e202 ꢁC; IR
(kBr, cmꢀ1): 3215 (NH), 3020 (AreH), 1610 (C]N), 1340 (C]S); 1H
1365
1328
1228
2265
469
2289
2735
2022
1299
1230
2782
1980
333
NMR (DMSO-d6):
d 3.78 (s, 3H, OCH3), 5.86 (s, 2H, NH, D2O
2157
59
3068
122
1170
110
exchangeable), 6.88e7.56 (m, 4H, AreH), 8.02 (d, 2H, J ¼ 6.1 Hz,
Pyridine H-3 and H-5), 8.75 (d, 2H, J ¼ 6.1 Hz, Pyridine H-2 and H-
6), 9.42 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 359 (MþH, 7).
Anal. Calcd. for C15H14N6OS2: C, 50.26; H, 3.94; N, 23.45. Found: C,
49.98; H, 3.70; N, 23.28.
2489
2188
2120
1824
2766
3289
1289
56
2063
1723
2014
2371
2379
3196
3074
282
1784
1078
1220
2269
1289
1684
2265
2217
3283
2217
15
2273
219
3231
1672
2170
1592
1259
215
120
229
18
4.1.6.3. 1-(3-Mercapto-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-yl)-3-(p-
tolyl)thiourea (7c). Yield: 88%; m.p.: 210e212 ꢁC; IR (kBr, cmꢀ1):
3233 (NH), 3159 (AreH), 1608 (C]N), 1315 (C]S); 1H NMR (DMSO-
8b
8c
8d
CHS 828
540
136
25
1380
320
2315
277
782
2067
312
128
1245
d6):
d 1.91 (s, 3H, CH3), 5.84 (s, 2H, NH, D2O exchangeable),
na
6.83e7.79 (m, 4H, AreH), 8.02 (d, 2H, J ¼ 6 Hz, Pyridine H-3 and H-
5), 8.74 (d, 2H, J ¼ 6 Hz, Pyridine H-2 and H-6); MS: m/z (%) 342
(Mþ, 14). Anal. Calcd. for C15H14N6S2: C, 52.61; H, 4.12; N, 24.54.
Found: C, 52.43; H, 4.12; N, 24.72.
NUGC, gastric cancer, DLDI, colon cancer, HA22T, liver cancer, HEPG2, liver cancer
HEPG2, liver cancer; HONEI, nasopharyngeal carcinoma; MCF, breast cancer; WI38,
normal fibroblast cells.
a
The sample concentration produces a 50% reduction in cell growth.

80
M.M. Kamel, N.Y. Megally Abdo / European Journal of Medicinal Chemistry 86 (2014) 75e80
4.1.6.4. 1-(4-Bromophenyl)-3-(3-mercapto-5-(pyridin-4-yl)-4H-
1,2,4-triazol-4-yl)thiourea (7d). Yield: 94%; m.p: 290e292 ꢁC; IR
(kBr, cmꢀ1): 3300 (NH), 3134 (AreH), 1610 (C]N), 1313 (C]S); 1H
4.2. In vitro cytotoxic assay
4.2.1. Chemicals
NMR (DMSO-d6):
d
5.86 (s, 2H, NH, D2O exchangeable), 7.29e7.53
Fetal bovine serum (FBS) and L-glutamine, were purchased from
(m, 4H, AreH), 8.02 (d, 2H, J ¼ 6 Hz, Pyridine H-3 and H-5), 8.76 (d,
2H, J ¼ 6 Hz, Pyridine H-2 and H-6), 10.9 (s, 1H, SH, D2O
exchangeable); MS: m/z (%) 407 (Mþ, 29). Anal. Calcd. for
Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium was pur-
chased from Cambrex (New Jersey, USA). Dimethyl sulfoxide
(DMSO), doxorubicin, penicillin, streptomycin and sulforhodamine
B (SRB) were purchased from Sigma Chemical Co. (Saint Louis,
USA).
C
14H11BrN6S2: C, 41.28; H, 2.72; N, 20.63. Found: C, 40.96; H, 2.52;
N, 20.71.
4.1.7. General method for the preparation of compounds 8aed
An equimolar mixture (0.01 mol) of 4-amino-5-pyridin-4-yl-
4H-[1,2,4]triazole-3-thiol (2) and the appropriate aryl isothiocya-
nate was heated under reflux in pyridine (10 mL) for 2e3 h until the
complete evolution of H2S (detected by lead acetate paper). The
reaction mixture was poured onto ice cold water. The obtained
solid was filtered, washed with water, dried and crystallized from
ethanol.
4.2.2. Cell cultures
Cell cultures were obtained from the European Collection of cell
Cultures (ECACC, Salisbury, UK) and human gastric cancer (NUGC),
human colon cancer (DLD1), human liver cancer (HA22T and
HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma
(HONE1) and normal fibroblast cells (WI38) were kindly provided
by the National Cancer Institute (NCI, Cairo, Egypt). They were
grown as monolayer and routinely maintained in RPMI-1640 me-
dium supplemented with 5% heat inactivated FBS, 2 mM glutamine
and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL), at
37 ꢁC in a humidified atmosphere containing 5% CO2. Exponentially
growing cells were obtained by plating 1.5 ꢂ 105 cells/mL for the six
human cancer cell lines followed by 24 h of incubation. The effect of
the vehicle solvent (DMSO) on the growth of these cell lines was
evaluated in all the experiments by exposing untreated control cells
to the maximum concentration (0.5%) of DMSO used in each assay.
4.1.7.1. N-(4-Chlorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-6-amine (8a). Yield: 91%; m.p: >300 ꢁC; IR (kBr,
cmꢀ1): 3300 (NH), 3100 (AreH), 1643, 1612 (C]N); 1H NMR
(DMSO-d6):
d
7.50e7.65 (m, 4H, AreH), 8.13 (d, 2H, J ¼ 5.9 Hz,
Pyridine H-3 and H-5), 8.81 (d, 2H, J ¼ 5.9 Hz, Pyridine H-2 and H-
6), 10.89 (s, 1H, NH, D2O exchangeable); MS: m/z (%) 328 (Mþ, 17).
Anal. Calcd. for C14H9ClN6S: C, 51.14; H, 2.76; N, 25.56. Found: C,
51.32; H, 2.96; N, 25.40.
References
4.1.7.2. N-(4-Methoxyphenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-6-amine (8b). Yield: 87%; m.p.: 180e182 ꢁC; IR
(kBr, cmꢀ1): 3213 (NH), 3020 (AreH), 1630, 1610 (C]N); 1H NMR
[1] A.M. Isloor, B. Kalluraya, P. Shetty, Eur. J. Med. Chem. 44 (2009) 3784e3787.
[2] N. Demirbas, S.A. Karaoglu, A. Demirbas, K. Sanack, Eur. J. Med. Chem. 39
(2004) 793e804.
[3] O. Prakash, D.K. Aneja, K. Hussain, P. Lohan, P. Ranjan, S. Arora, C. Sharma,
K.R. Aneja, Eur. J. Med. Chem. 46 (2011) 5065e5073.
[4] S.N. Swamy, Basappa, B.S. Priya, B. Prabhuswamy, B.H. Doreswamy, J.S. Prasad,
K.S. Rangappa, Eur. J. Med. Chem. 41 (2006) 531e538.
[5] V.S. Palekar, A.J. Damle, S.R. Shukla, Eur. J. Med. Chem. 44 (2009) 5112e5116.
[6] M.F. Shehy, A. Abu-Hashem, E.M. El-Telbani, Eur. J. Med. Chem. 45 (2010)
1906e1911.
(DMSO-d6):
d 3.78 (s, 3H, OCH3), 6.88e7.55 (m, 4H, AreH), 8.14 (d,
2H, J ¼ 6 Hz, Pyridine H-3 and H-5), 8.81 (d, 2H, J ¼ 6 Hz, Pyridine H-
2 and H-6), 9.42 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-
d6): d 161.9, 156.5, 155.7, 150.6, 132.1, 126.0, 120.5, 119.1, 114.6, 113.6,
55.3; MS: m/z (%) 325 (MþH, 14). Anal. Calcd. for C15H12N6OS: C,
55.54; H, 3.73; N, 25.91. Found: C, 55.80; H, 3.91; N, 25.71.
[7] V. Mathew, J. Keshavayya, V.P. Vaidya, D. Giles, Eur. J. Med. Chem. 42 (2007)
823e840.
[8] B.S. Holla, K.N. Poojary, B.S. Rao, M.K. Shivananda, Eur. J. Med. Chem. 37 (2002)
511e517.
[9] V. Padmavathi, G.S. Reddy, A. Padmaja, P. Kondaiah, Ali-Shazia, Eur. J. Med.
Chem. 44 (2009) 2106e2112.
[10] M. Clemons, R.E. Coleman, S. Verma, Cancer Treat. Rev. 30 (2004) 324e332.
[11] M. Ashok, B.S. Holla, J. Pharmacol. Toxicol. 2 (3) (2007) 256e263.
[12] B.S. Holla, B. Veerendra, M.K. Shivananda, B. Poojary, Eur. J. Med. Chem. 38
(2003) 759e767.
[13] N. Demirbas, A. Demirbas, S.A. Karaoglu, E. Celik, Arkivoc I (2005) 75e91.
[14] V. Sumangala, B. Poojary, N. Chidananda, T. Arulmoli, S. Shenoy, Eur. J. Med.
Chem. 54 (2012) 59e64.
4.1.7.3. N-(4-Methylphenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-6-amine (8c). Yield: 94%; m.p.: 290e291 ꢁC; IR
(kBr, cmꢀ1): 3249 (NH), 2951 (AreH), 1608, 1589 (C]N); 1H NMR
(DMSO-d6): d 2.27 (s, 3H, CH3), 7.13e7.39 (m, 4H, AreH), 8.04 (d, 2H,
J ¼ 5.9 Hz, Pyridine H-3 and H-5), 8.76 (d, 2H, J ¼ 5.9 Hz, Pyridine H-
2 and H-6), 10.9 (s, 1H, NH, D2O exchangeable); MS: m/z (%) 308
(Mþ, 62). Anal. Calcd. C15H12N6S: C, 58.43; H, 3.92; N, 27.25. Found:
C, 55.34; H, 3.51; N, 27.42.
[15] I. Khan, A. Ibrar, N. Abbas, Eur. J. Med. Chem. 63 (2013) 854e868.
[16] B.S. Holla, B.S. Rao, B.K. Sarojini, P.M. Akberali, N.S. Kumari, Eur. J. Med. Chem.
41 (2006) 657e663.
[17] K.S. Bhat, B. Poojary, D.J. Prasad, P. Naik, B.S. Holla, Eur. J. Med. Chem. 44
(2009) 5066e5070.
[18] D. Sunil, A.M. Isloor, P. Shetty, K. Satyamoorthy, A.S.B. Prasad, Arab. J. Chem. 3
(2010) 211e217.
[19] A. Husain, M. Rashid, M. Shaharyar, A.A. Siddiqui, R. Mishra, Eur. J. Med. Chem.
62 (2013) 785e798.
4.1.7.4. N-(p-Bromophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-6-amine (8d). Yield: 89%; m.p: >300 ꢁC; IR (kBr,
cmꢀ1): 3300 (NH), 2918 (AreH), 1645, 1612 (C]N); 1H NMR
(DMSO-d6):
d
7.44e7.84 (m, 4H, AreH), 8.02 (d, 2H, J ¼ 6 Hz, Pyr-
idine H-3 and H-5), 8.71 (d, 2H, J ¼ 6 Hz, Pyridine H-2 and H-6),
10.95 (s, 1H, NH, D2O exchangeable); MS: m/z (%) 373 (Mþ, 63).
Anal. Calcd. for C14H9BrN6S: C, 45.05; H, 2.43; N, 22.52. Found: C,
45.14; H, 2.39; N, 22.38.
[20] D.A. Ibrahim, Eur. J. Med. Chem. 44 (2009) 2776e2781.
[21] P.J. Hjarnaa, E. Jonsson, S. Latini, S. Dhar, R. Larsson, E. Bramm, T. Skov,
L. Binderup, Cancer Res. 59 (22) (1999) 5751e5757.