Synthesis, crystal structure and biological evaluation of some novel 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and 1,2,4-triazolo[3,4-b]-1,3,4- thiadiazines

Article abstract of DOI:10.1016/j.ejmech.2014.03.046

Nitrogen-containing heterocycles are of particular interest and significant importance for the discovery of potent bioactive agents in pharmaceutical industry. The present study reports the synthesis of a library of new conjugated heterocycles including 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4- thiadiazoles (4a-g and 5a-e) and 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4- thiadiazines (6a-h), by cyclocondensation reaction of 4-amino-5-(pyridin-4-yl)- 4H-1,2,4-triazole-3-thiol 3 with various substituted aromatic acids and phenacyl bromides, respectively. The structures of newly synthesized compounds were characterized by elemental analysis, IR, ¹H and ¹³C NMR spectroscopy and in case of 4c by X-ray crystallographic analysis. Newly synthesized triazolothiadiazoles and thiadiazines were screened for acetyl- and butyryl-cholinesterases and alkaline phosphatase inhibition. Almost all of the compounds showed good to excellent activities against acetylcholinesterase more than the reference drugs. Compound 5d exhibited IC₅₀ value 0.77 ± 0.08 μM against acetylcholinesterase and 4a showed IC₅₀ 9.57 ± 1.42 μM against butyrylcholinesterase. Among all the tested compounds, 4a also proved as excellent inhibitor of alkaline phosphatase with IC₅₀ 0.92 ± 0.03 μM. These heteroaromatic hybrid structures were also tested for their anticancer activity against lung carcinoma (H157) and kidney fibroblast (BHK-21) cell lines and leishmanias. Variable cell growth inhibitory activities were obtained and many compounds exhibit potent %inhibition.

Full text of DOI:10.1016/j.ejmech.2014.03.046

European Journal of Medicinal Chemistry 78 (2014) 167e177
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, crystal structure and biological evaluation of some novel
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and 1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazines
Imtiaz Khan a, Sumera Zaib b, Aliya Ibrar a, Nasim Hasan Rama a, Jim Simpson c,
,d,
Jamshed Iqbal b
*
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
c Department of Chemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand
d Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitrogen-containing heterocycles are of particular interest and significant importance for the discovery
of potent bioactive agents in pharmaceutical industry. The present study reports the synthesis of a library
of new conjugated heterocycles including 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles (4a
eg and 5aee) and 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazines (6aeh), by cyclo-
condensation reaction of 4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol 3 with various substituted
aromatic acids and phenacyl bromides, respectively. The structures of newly synthesized compounds
were characterized by elemental analysis, IR, 1H and 13C NMR spectroscopy and in case of 4c by X-ray
crystallographic analysis. Newly synthesized triazolothiadiazoles and thiadiazines were screened for
acetyl- and butyryl-cholinesterases and alkaline phosphatase inhibition. Almost all of the compounds
showed good to excellent activities against acetylcholinesterase more than the reference drugs. Com-
Received 7 February 2014
Received in revised form
14 March 2014
Accepted 15 March 2014
Available online 19 March 2014
Keywords:
Heterocycles
Cyclocondensation
Alkaline phosphatase inhibition
Leishmanias
pound 5d exhibited IC50 value 0.77 ꢀ 0.08
9.57 ꢀ 1.42 M against butyrylcholinesterase. Among all the tested compounds, 4a also proved as
M. These heteroaromatic hybrid
mM against acetylcholinesterase and 4a showed IC50
Lung carcinoma
m
excellent inhibitor of alkaline phosphatase with IC50 0.92 ꢀ 0.03
m
structures were also tested for their anticancer activity against lung carcinoma (H157) and kidney
fibroblast (BHK-21) cell lines and leishmanias. Variable cell growth inhibitory activities were obtained
and many compounds exhibit potent %inhibition.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
therapy offers symptomatic improvements or delays in the pro-
gression of cognitive, behavioral and functional deficits. Recently,
Alzheimer’s disease (AD) with its associated loss of brain activity
is the most common cause of dementia among people aged 65
years and older. It shows the typical characteristic of a progressive
cognitive and functional decline in patients [1]. Degradation of
cholinergic neurons in the cerebral cortex and other areas of the
brain, with resulting deficits in cholinergic transmission and
acetylcholine (ACh) levels, is considered to be a primary cause of
cognitive decline [2]. Acetylcholinesterase inhibitor (AChEI)
preclinical studies indicate that AChEIs could prevent damage and
protect neurons from death [3].
AChEIs are known to inhibit AChE, which catalyzes the break-
down of ACh in the synaptic cleft, thus enhancing ACh. Two cho-
linesterases hydrolyze ACh in the human brain, AChE and
butyrylcholinesterase (BChE); current evidence indicates that AChE
activity declines with the progressive loss of cortical neurons in AD,
and at the same time BChE levels increase and may take over the
function to metabolize ACh at the synapse. Moreover, the rate of
cognitive decline has been shown to correlate with BChE levels in
the temporal cortex [4].
* Corresponding author. Department of Pharmaceutical Sciences, COMSATS
Institute of Information Technology, Abbottabad 22060, Pakistan.
E-mail addresses: drjamshed@ciit.net.pk, jamshediqb@gmail.com, jamshed.
iqbal@uni-bonn.de (J. Iqbal).
Alkaline phosphatase (ALP, E.C. 3.1.3.1.) is a non-specific
phosphomonoester hydrolase that catalyzes the hydrolysis and
transphosphorylation
of
a
wide
variety of
organic
http://dx.doi.org/10.1016/j.ejmech.2014.03.046
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

168
I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
monophosphates and regulates the functions of many biological
systems [5e7]. The most common occurrence of ALP in nature
suggests its involvement in fundamental biochemical processes,
however, there is no positive evidence regarding its physiological
functions. The possible roles of ALP have been suggested as hy-
drolysis of phosphoesters, phosphate transferase activity, protein
phosphatase activity, phosphate transport, modulation of organic
cation transport, and involvement in cell proliferation [8,9]. In
serum, the biological action of ALP is associated with metabolic
bone (Hypophosphatasia) and liver diseases and also is used as a
marker of osteoblastic differentiation [10]. Alkaline phosphatases
may potentially be employed as therapeutic agents and thera-
peutic targets and show several uses in clinical medicine and in
biotechnology.
Cell/tissue culture has been devised at the beginning of this
century [11] as a method for studying the behavior of animal cells,
free of systemic variations that may arise in the animals either
during normal homeostasis or under the stress of an experiment.
As the name implies the technique is elaborated first with un-
disaggregated fragments of tissue, with occasional mitosis in the
outgrowth [12]. On the other hand, the development of resistance
to chemotherapeutic agents is a common obstacle in the treatment
of different types of cancers [13].
Leishmaniasis is one of the world’s most neglected diseases,
largely affecting the poorest of the poor, mainly in developing
countries. Over 350 million people are considered at risk of con-
tracting leishmaniasis, and approximately 2 million new cases
occur yearly [14]. Leishmania donovani is the causative agent for
visceral leishmaniasis (VL), the most fatal form of the disease. The
choice of drugs available to treat leishmaniasis is limited [15];
current treatments provide limited efficacy and many are toxic at
therapeutic doses. In addition, most of the first line treatment
drugs have already lost their utility due to increasing multiple
drug resistance [16]. The current pipeline of anti-leishmanial
drugs is also severely depleted. Sustained efforts are needed to
enrich a new anti-leishmanial drug discovery pipeline, and this
endeavor relies on the availability of suitable in vitro screening
models.
2. Results and discussion
2.1. Chemistry
The target compounds were synthesized as outlined in Scheme
1. The starting material isonicotinohydrazide 1, on reaction with
carbon disulfide in methanolic potassium hydroxide yielded cor-
responding dithiocarbazinate 2 in good yield and was directly used
for the next step without further purification. The triazole 3 was
obtained by refluxing 2 with hydrazine hydrate (80%). Infra-red and
NMR data displayed by compound 3 was consistent as obtained
previously [27]. Reaction of 4-amino-5-(pyridin-4-yl)-4H-1,2,4-
triazole-3-thiol 3 with various aromatic carboxylic acids in phos-
phorous oxychloride under reflux conditions yielded, 1,2,4-triazolo
[3,4-b][1,3,4]thiadiazoles 4aeg and 5aee [25].
The appearance of new stretching bands and disappearance of
characteristic peaks for SH and NH2 indicated the formation of
conjugated scaffolds. Their proton NMR spectra also lacked signals
characteristic of SH and NH protons, and showed new peaks at
5.57e5.88 ppm (integration for two protons) attributed to OCH2
protons, respectively, establishing that COOH group of the aromatic
carboxylic acids reacted with SH and NH2 groups of the interme-
diate 3 to afford 1,2,4-triazolo[3,4-b][1,3,4]thiadiazole derivatives
4aeg and 5aee, respectively.
Furthermore,
4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-
thiol 3 was refluxed with various phenacyl bromides in absolute
ethanol to give 1,2,4-triazolo[3,4-b][1,3,4]thiadiazine derivatives
6aeh [28]. Infra-red spectra indicated the formation of condensed
heterocycles as they were devoid of characteristic stretching bands
attributable to SH and NH2 groups. In the proton NMR spectra, the
disappearance of SH and NH2 signals and appearance of new singlet
characteristic for methylene protons recorded in the range of 4.42e
4.67 ppm (integration for two protons) confirmed that the com-
pound 3 underwent ring closure to afford the 1,2,4-triazolo[3,4-b]
[1,3,4]thiadiazines. 13C NMR spectra of condensed heterocycles
showed the presence of carbon signals at appropriate chemical
shift values. The purity of all the newly synthesized compounds
was also confirmed by elemental analysis.
Compounds incorporating heterocyclic ring systems are of
significant importance and particular interest both in pharma-
ceutical as well as agrochemical industries. Indeed, with partic-
ular reference to the pharmaceutical industry, heterocyclic
scaffolds are especially prevalent with over 60% of the top
retailing drugs containing at least one heterocyclic nucleus as
part of the overall topography of the compound [17]. In addition,
these heterocyclic structures are commonly used as key tem-
plates for the design and development of more potent and spe-
cific biologically active agents for various biological targets. In
this regard, heteroaromatic hybrid structures like tri-
azolothiadiazoles and triazolothiadiazines have received consid-
erable attention from scientific community and are extensively
used as scaffolds with diverse spectra of biological activities
including potential anti-inflammatory and analgesic activities
[18], antitumor [19], CNS depressant [20], antibacterial, anti-
fungal [21], antiviral [22], anticancer [23], and antioxidant ac-
tivity [24]. Several triazolothiadiazoles have also been shown to
possess potent urease, alkaline phosphatase, acetylcholinesterase
and phosphodiesterase inhibitory activities [25].
In parallel, for a full structural elucidation, compound 4c was
confirmed by X-ray diffraction analysis. The crystal and instru-
mental parameters used in the unit cell determination, the data
collection, and structure refinement parameters are presented in
Table 1 while selected bond distances and angles in Table 2. The
structure comprises a central [1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zole unit substituted in the 3-position by a pyridine ring and with a
furan substituent on the C atom of the thiadiazole ring (Fig. 1). The
ꢀ
molecule is moderately planar with a rms deviation of only 0.123 A
from the best fit meanplane through all 19 non-hydrogen atoms. A
weak intramolecular C8eH8.N4 hydrogen bond supports the
planarity of the molecular structure. In keeping with this, the
dihedral angle between the triazole and thiadiazole rings of the
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole unit is 2.37(15)ꢁ while the
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole unit subtends angles of
12.80(11)ꢁ and 6.57(15)ꢁ to the pyridine and furan rings,
respectively.
The Cambridge database [29] reveals only one structure, ethyl 5-
(6-(furan-2-yl)-1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-3-yl)-2,6-
dimethylnicotinate with a furan substituent on the C atom of the
thiadiazole ring [30] and only one instance of a pyridine substituent
on the 3-position of the triazole ring in 6-(4-pyridyl)-3-(3,4,5-
trimethoxyphenyl)-1,2,4-triazolo[3,4-b][1,3,4]thiadiazole [31].
In the crystal structure intermolecular C5eH5.N hydrogen
So, in corollary of these fascinating findings and as part of a
programme aimed at finding a library of privileged heterocyclic
structures with potential bioactive profile [25,26], we have ratio-
nalized to target these novel heteroaromatic scaffolds. Further-
more, this paper describes the synthesis, characterization and
ꢀ
ꢀ
bonds together N2.S1 [2.782 (2) A] and N2.N2 [2.864(4) A]
contacts form separate inversion dimers that are linked by C12e
H12.S1 hydrogen bonds to form rows along c. The resulting rows
biological
activities
of
triazolothiadiazoles
and
triazolothiadiazines.

I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
169
Scheme 1. Synthetic pathway of title compounds 4aeg, 5aee and 6aeh.
are further stacked along the b axis to form a three dimensional
network (Fig. 2), with stacking interactions between two
value 9.57 ꢀ 1.42
m
M than donepezil (6.37 ꢀ 0.32
m
M). However,
p.p
other compounds were slightly less potent on the hBChE.
Among the newly synthesized analogs, the most potent com-
pound against EeAChE was 5d having strong electron-donating (e
OCH3) group at para position. However, any further substitution to
methoxy group reduces the activity as in the case of 6a (OCH3e
C6H4). If (eCH3) group is attached to ortho or para positions activity
was further decreased as observed in 5c and 6d, respectively.
Reduced activity was observed when (eNO2) group is attached at
meta position as in compound 6g. Substitution of various groups to
the phenyl group of 5aee compounds also affect inhibitory activity.
In case of 4aeg, different substituents are attached to triazolo[3,4-
b][1,3,4]thiadiazine and among all of them 4g showed excellent
inhibitory potential.
ꢀ
triazole rings [Cg.Cg ¼ 3.5298(14) A] and the triazole ring and the
ꢀ
pyridine ring [Cg.Cg ¼ 3.5583(14) A] further stabilizing the
structure.
2.2. Pharmacology
2.2.1. Cholinesterases inhibition
All of the synthesized molecules were screened for their in vitro
cholinesterase activity using spectrophotometric method (Table 3).
Neostigmine and Donepezil were used as reference drugs. Their
IC50 values for AChE are 14.2 ꢀ 1.01
respectively. Whereas for BChE, Neostigmine showed IC50 value of
26.1 ꢀ 2.03 M and Donepezil showed 6.37 ꢀ 0.32 M inhibition.
mM and 0.03 ꢀ 0.003
mM,
m
m
In general, all the tested compounds were also excellent in-
hibitors of butyrylcholinesterase. Among 4aeg compounds 4a was
found to be the most potent inhibitor of hBChE having 2-F,4-Cl-
The IC50 values suggested that most of designed compounds
exhibited potent and selective inhibitory activities in micromolar
range toward cholinesterases. The most potent compounds are 3,
4c, 4g, 5b, 5d and 6f on EeAChE, while in case of hBChE 4a, 5a, 6d,
6g and 6h are potent compounds. Our compounds have different
scaffold than neostigmine, therefore, they were more selective on
both enzymes and showed more selectivity as compared to refer-
ence compound. Compound 5d was many folds more active on
EeAChE than the neostigmine whereas showed comparable IC50
value to donepezil. Moreover, in case of hBChE 4a was found to be
3-folds more active than neostigmine and have slightly more IC50
C6H3 groups attached and showed IC50 value 9.57 ꢀ 1.42
mM. In all
of the 5aee compounds, 5a having hydroxyl group at ortho position
was most active in the series and showed IC50 value
18.01 ꢀ 3.58
mM. Among 6aeh compounds, 6g and 6h showed IC50
value 16.6 ꢀ 2.72 and 12.2 ꢀ 1.67
mM, respectively.
2.2.2. Alkaline phosphatase
Potassium dihydrogen phosphate with IC50 value of
2.41 ꢀ 0.03 M was used as standard drug for alkaline phosphatase
m

170
I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
Table 1
Crystal data and structure refinement for 4c.
Empirical formula
C12H7N5OS
Formula weight
Temperature
Wavelength
269.29
93(2) K
0.71073 A
ꢀ
Crystal system
Space group
Unit cell dimensions
Monoclinic
P 21/n
¼ 90ꢁ
ꢀ
a
a ¼ 11.2955(10) A,
¼ 97.136(4)ꢁ
ꢀ
b ¼ 4.9203(4) A,
b
¼ 90ꢁ
ꢀ
c ¼ 20.4714(18) A,
g
3
ꢀ
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta ¼ 23.95ꢁ
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
1128.93(17) A
4
1.584 mg/m3
0.285 mmꢂ1
552
0.43 ꢃ 0.18 ꢃ 0.09 mm3
Fig. 1. The structure of 4c showing the atom numbering with ellipsoids drawn at the
50% probability level.
3.64e23.95ꢁ
ꢂ12 ꢄ h ꢄ 12, ꢂ5 ꢄ k ꢄ 5, ꢂ23 ꢄ l ꢄ 19
7421
1752 [R(int) ¼ 0.0476]
2.2.3. Cytotoxic inhibition
99.7%
The anticancer drugs used in chemotherapy are systemic anti-
proliferative agents that preferentially kill those cells that are
dividing. Antiproliferative activity of newly synthesized tri-
azolothiadiazoles and thiadiazines was measured in vitro at four
Semi-empirical from equivalents
0.745 and 0.656
Full-matrix least-squares on F2
1752/0/172
1.048
different concentrations (100, 10, 1 and 0.1 mM) concentration by
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole
s
(I)]
R1 ¼ 0.0368, wR2 ¼ 0.0837
the cell growth inhibition against kidney fibroblast cell lines (BHK-
21) and lung carcinoma (H157) and then compared with the stan-
dard anticancer drug vincristine to get percent inhibition of each
compound.
R1 ¼ 0.0480, wR2 ¼ 0.0914
ꢀꢂ3
0.233 and ꢂ0.251 e A
Most of the compounds exhibited (>50%) activity for both cell
lines with slightly different capacity due to their structure diversity
in terms of attached functional groups with triazolothiadiazoles
and thiadiazines (Tables 5 and 6). Among all the compounds
screened, 5d showed the highest inhibition 74.0% of activity for
BHK-21 cells which is same as that of standard drug vincristine
(74.5%). The compounds 6a and 6d have also proved to be potent
inhibitors for cancer therapy when tested against BHK-21 cells
showed 73.3% and 72.6% inhibition, respectively. Compound 5d has
methoxy group attached at para position. Again in case of 6a,
OCH3eC6H4 group attached at meta position while for 6d, methyl
group attached at para position. Compounds 3, 4g and 6e showed
inhibition value less than 50%. Among the series, least active
compound was 4d with inhibition value 31.2% and has furanyl
group at meta position. When the same group was attached to
ortho position as in case of 4c, it showed 50.9% inhibition.
In case of H157, from the series of novel triazolothiadiazoles and
thiadiazines, compound 4a was the most potent compound with
inhibition assay. Compound 4a with IC50 value of 0.92 ꢀ 0.03
mM
emerged as the most active compound of the series. It has 2-F and
4-Cl-C6H3 as substituents to parent structure 3, whose activity is
4.43 ꢀ 0.14
mM, it means the attachment of substituent enhances
the activity 4-folds. Compound 4e with percent inhibition of 29.86%
was the least active compound of the series. Compound 6h and 4b
also showed potent inhibition of IC50 value 1.32 ꢀ 0.20
2.01 ꢀ 0.08 M, respectively, which was less than standard drug
used in the assay. All other compounds showed IC50 values ranging
M. Some of the compounds
mM, and
m
from 2.59 ꢀ 0.22
m
M to 9.83 ꢀ 0.11
m
were less active and their IC50 values were not determined and the
percent inhibition of 4e, 4f, 5a, 6a, 6b and 6d was reported in
Table 4.
Table 2
ꢁ
ꢀ
Selected bond lengths [A] and angles [ ] for 4c.
inhibition value 85.5% at 100
near to vincristine inhibition at 100
attached, whereas when 3-Cl,4-F-C6H3 group was substituent
attached to same ring as in case of 4b inhibition decreases to 53.7%.
Another potent inhibitor of H157 cells was 6c having 78.6%
m
M while 73.7% at 0.1
mM which was
ꢀ
Bond lengths [A]
C(1)-N(1)
C(1)-N(3)
N(1)-N(2)
N(2)-C(3)
C(3)-N(3)
C(3)-S(1)
S(1)-C(2)
mM. It has 2-F,4-Cl-C6H3 group
1.322(3)
1.379(3)
1.392(3)
1.319(3)
1.360(3)
1.725(2)
1.769(2)
1.309(3)
1.387(2)
1.458(3)
C(4)-C(5)
C(5)-C(6)
C(6)-N(5)
N(5)-C(7)
C(2)-C(9)
C(9)-C(10)
C(9)-O(1)
C(10)-C(11)
C(11)-C(12)
C(12)-O(1)
1.395(3)
1.376(3)
1.340(3)
1.336(3)
1.438(3)
1.357(3)
1.377(3)
1.428(3)
1.338(3)
1.368(3)
C(2)-N(4)
N(4)-N(3)
C(1)-C(4)
Bond angles [ꢁ]
N(1)-C(1)-N(3)
C(1)-N(1)-N(2)
C(3)-N(2)-N(1)
N(2)-C(3)-N(3)
N(2)-C(3)-S(1)
N(3)-C(3)-S(1)
C(3)-S(1)-C(2)
N(4)-C(2)-S(1)
107.96(19)
109.45(17)
105.49(18)
111.23(19)
138.40(18)
110.32(15)
86.90(11)
C(2)-N(4)-N(3)
C(3)-N(3)-C(1)
C(3)-N(3)-N(4)
C(1)-N(3)-N(4)
N(1)-C(1)-C(4)
N(3)-C(1)-C(4)
N(4)-C(2)-C(9)
C(9)-C(2)-S(1)
106.31(18)
105.85(17)
118.42(18)
135.60(19)
124.3(2)
127.7(2)
123.0(2)
118.90(17)
118.04(17)
Symmetry transformations used to generate equivalent atoms:
#1 ꢂ x þ 1; ꢂy; ꢂz þ 2
Fig. 2. Crystal packing of 4c viewed along the b axis.

I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
171
Table 3
Table 5
Acetyl- and butyryl-cholinesterase inhibition activities of synthesized compounds
(3, 4aeg, 5aee and 6aeh).
Anticancer activity of synthesized compounds (3, 4aeg, 5aee and 6aeh) against
kidney fibroblast (BHK-21) cell lines.
Entry
Ar/R
EeAChE
hBChE
Entry
Percent inhibition ꢀ SEM
100 10
IC50 ꢀ SEM (
m
M)a/%inhibitionb
m
M
m
M
1
mM
0.1 mM
3
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
6a
6b
6c
48.3 ꢀ 2.5
55.6 ꢀ 1.8
51.4 ꢀ 1.9
50.9 ꢀ 2.3
31.2 ꢀ 2.1
64.7 ꢀ 3.2
58.9 ꢀ 3.2
48.6 ꢀ 2.4
53.8 ꢀ 4.1
60.5 ꢀ 2.9
62.1 ꢀ 2.5
74.0 ꢀ 3.2
52.7 ꢀ 3.8
73.3 ꢀ 3.1
56.4 ꢀ 2.9
64.3 ꢀ 3.2
72.6 ꢀ 2.8
45.8 ꢀ 2.6
69.2 ꢀ 2.1
67.1 ꢀ 2.7
63.5 ꢀ 3.3
74.5 ꢀ 2.9
45.1 ꢀ 2.4
51.6 ꢀ 2.9
46.3 ꢀ 3.2
48.2 ꢀ 3.2
28.7 ꢀ 3.1
61.6 ꢀ 2.4
53.9 ꢀ 2.8
45.4 ꢀ 2.8
49.9 ꢀ 3.7
56.2 ꢀ 2.1
56.5 ꢀ 2.9
71.9 ꢀ 2.4
48.3 ꢀ 2.5
69.9 ꢀ 2.2
53.3 ꢀ 2.5
61.2 ꢀ 2.2
68.7 ꢀ 1.8
41.8 ꢀ 1.9
64.7 ꢀ 2.7
62.5 ꢀ 2.8
59.4 ꢀ 2.6
72.6 ꢀ 3.1
42.1 ꢀ 3.1
47.2 ꢀ 2.3
43.6 ꢀ 2.6
44.9 ꢀ 2.0
23.3 ꢀ 2.5
57.0 ꢀ 2.4
49.7 ꢀ 2.4
40.9 ꢀ 2.8
46.3 ꢀ 2.9
53.7 ꢀ 2.4
54.9 ꢀ 2.5
68.8 ꢀ 2.3
45.4 ꢀ 3.3
66.7 ꢀ 2.4
50.3 ꢀ 2.3
56.9 ꢀ 1.9
62.0 ꢀ 1.8
37.5 ꢀ 2.2
60.7 ꢀ 2.3
55.1 ꢀ 3.0
56.5 ꢀ 1.8
70.9 ꢀ 2.4
39.6 ꢀ 2.7
44.4 ꢀ 2.3
40.9 ꢀ 3.1
41.0 ꢀ 3.2
20.3 ꢀ 3.1
54.6 ꢀ 3.0
46.7 ꢀ 2.7
38.2 ꢀ 2.6
43.8 ꢀ 2.8
51.6 ꢀ 1.9
51.2 ꢀ 2.4
63.3 ꢀ 2.6
43.8 ꢀ 3.1
52.4 ꢀ 3.3
46.2 ꢀ 3.2
54.6 ꢀ 2.5
53.1 ꢀ 2.7
32.7 ꢀ 2.8
53.3 ꢀ 2.7
48.2 ꢀ 2.2
52.8 ꢀ 3.1
69.8 ꢀ 1.9
3
13.46 ꢀ 0.41a
49.58 ꢀ 0.47a
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
6a
6b
6c
6d
2-F,4-Cl-C6H3
3-Cl,4-F-C6H3
2-furanyl
3-furanyl
2-CH3-3-furanyl
2-Cl-4,5-diF-C6H2
4-OC2H5eC6H4eCH2
2-OH
4-OH
2-CH3
4-OCH3
4-F
3-OCH3-C6H4
3-Cl-C6H4
3-F-C6H4
4-CH3-C6H4
biphenyl
Naphthyl
3-NO2-C6H4
3,4-diCl-C6H3
e
21.26 ꢀ 0.12a
30.19 ꢀ 2.37a
12.4 ꢀ 0.08a
34.63b
9.57 ꢀ 1.42a
14.4b
61.55 ꢀ 0.31a
36.2 ꢀ 3.06a
41.2b
51.18 ꢀ 0.06a
47.20b
18.21b
3.48 ꢀ 1.58a
54.05 ꢀ 0.17a
15.64 ꢀ 0.08a
41.48 ꢀ 0.02a
0.77 ꢀ 0.08a
36.84 ꢀ 0.19a
23.8 ꢀ 0.94a
22.74 ꢀ 0.16a
18.6 ꢀ 0.32a
46.09b
32.1 ꢀ 1.08a
18.01 ꢀ 3.58a
39.15 ꢀ 0.09a
51.34 ꢀ 0.09a
37.92 ꢀ 0.16a
54.09 ꢀ 0.21a
28.90b
6d
6e
6f
6g
39.41 ꢀ 0.55a
38.66b
26.07 ꢀ 0.23a
42.6 ꢀ 3.09a
52.76 ꢀ 0.16a
16.6 ꢀ 2.72a
12.2 ꢀ 1.67a
26.1 ꢀ 2.03a
6.37 ꢀ 0.32a
6h
6e
6f
6g
6h
16.11 ꢀ 0.23a
13.66 ꢀ 0.31a
28.03 ꢀ 0.05a
45.99b
Vincristine
anticancer drugs with reduced toxicity and improved therapeutic
indices. The present studies revealed some of the antiproliferative
properties of newly synthesized triazolothiadiazoles and thiadia-
zines with some most promising results against kidney fibroblast
cells as well as lungs carcinoma.
Neostigmine
Donepezil
14.2 ꢀ 1.01a
e
0.03 ꢀ 0.003a
a
IC50
.
b
%age inhibition.
inhibition. Among all the tested compounds 5e was least active
with 32.0% inhibition and 4f and 5a also exhibit less than 50%
activity.
Despite several decades of intensive research, the long-term
outlook for patients with aggressive cancer remains discouraging,
and there is a need for innovative approaches toward the design of
2.2.4. Antileishmanial activity
The antileishmanial activity of novel triazolothiadiazoles and
thiadiazines was measured by MTT method and %inhibition of all
the compounds at different concentrations was reported in Table 7.
Amphotericin B was used as standard drug for anti-leishmanial
activity.
The results of this research shows the antileishmanial activity of
synthesized compounds against Leishmania major in vitro. Among
Table 4
Alkaline phosphatase inhibition activities of synthesized compounds (3, 4aeg, 5aee
and 6aeh).
Entry
Ar/R
IC50 ꢀ SEM (
m
M)a/%inhibitionb
Table 6
Anticancer activity of synthesized compounds (3, 4aeg, 5aee and 6aeh) against
lung carcinoma (H157) cell lines.
3
4.43 ꢀ 0.14a
Entry
Percent inhibition ꢀ SEM
100 10
m
M
m
M
1
mM
0.1 mM
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
6a
2-F,4-Cl-C6H3
3-Cl,4-F-C6H3
2-furanyl
0.92 ꢀ 0.03a
2.01 ꢀ 0.08a
3.24 ꢀ 0.11a
7.71 ꢀ 0.14a
29.86b
3
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
6a
6b
6c
51.5 ꢀ 2.1
85.5 ꢀ 2.8
53.7 ꢀ 2.7
67.9 ꢀ 2.7
64.3 ꢀ 2.3
58.4 ꢀ 3.1
49.2 ꢀ 3.0
70.5 ꢀ 2.9
41.8 ꢀ 2.4
63.7 ꢀ 2.5
59.2 ꢀ 2.4
68.9 ꢀ 2.3
32.0 ꢀ 3.2
54.4 ꢀ 2.1
67.4 ꢀ 2.2
78.6 ꢀ 2.4
63.8 ꢀ 2.5
56.9 ꢀ 2.3
68.2 ꢀ 2.5
69.4 ꢀ 2.1
72.1 ꢀ 2.6
74.5 ꢀ 2.9
47.4 ꢀ 2.4
80.9 ꢀ 2.3
50.9 ꢀ 2.1
63.8 ꢀ 2.8
61.6 ꢀ 2.9
55.1 ꢀ 2.6
45.2 ꢀ 2.7
67.4 ꢀ 2.4
37.3 ꢀ 2.5
60.9 ꢀ 3.1
56.8 ꢀ 3.8
65.6 ꢀ 3.1
28.5 ꢀ 1.9
51.1 ꢀ 1.8
63.8 ꢀ 2.3
75.6 ꢀ 3.2
57.3 ꢀ 3.1
62.3 ꢀ 3.0
63.7 ꢀ 2.7
65.5 ꢀ 2.4
67.4 ꢀ 2.1
72.6 ꢀ 3.1
43.4 ꢀ 2.5
77.8 ꢀ 2.6
46.6 ꢀ 2.3
60.9 ꢀ 2.5
57.7 ꢀ 2.7
51.9 ꢀ 2.1
41.4 ꢀ 1.3
63.8 ꢀ 2.4
35.9 ꢀ 2.7
56.3 ꢀ 2.5
53.2 ꢀ 2.5
61.7 ꢀ 2.2
25.6 ꢀ 3.1
47.5 ꢀ 2.6
60.8 ꢀ 2.5
72.3 ꢀ 2.9
53.2 ꢀ 2.1
47.8 ꢀ 2.7
59.9 ꢀ 2.6
62.1 ꢀ 2.1
63.5 ꢀ 2.8
70.9 ꢀ 2.4
40.4 ꢀ 2.7
73.7 ꢀ 2.4
43.6 ꢀ 3.1
56.0 ꢀ 2.3
54.8 ꢀ 2.4
47.1 ꢀ 2.3
36.4 ꢀ 2.8
60.2 ꢀ 2.1
31.8 ꢀ 2.4
52.6 ꢀ 2.6
50.5 ꢀ 2.9
57.2 ꢀ 3.0
21.3 ꢀ 2.1
43.1 ꢀ 2.4
57.4 ꢀ 3.5
69.6 ꢀ 2.1
50.3 ꢀ 2.7
44.5 ꢀ 3.3
55.2 ꢀ 1.8
57.9 ꢀ 3.2
59.4 ꢀ 2.5
69.8 ꢀ 1.9
3-furanyl
2-CH3-3-furanyl
2-Cl-4,5-diF-C6H2
4-OC2H5eC6H4eCH2
2-OH
4-OH
2-CH3
31.62b
6.17 ꢀ 0.17a
46.04b
5.61 ꢀ 0.05a
3.08 ꢀ 0.16a
3.72 ꢀ 0.03a
2.59 ꢀ 0.22a
38.47b
4-OCH3
4-F
3-OCH3-C6H4
3-Cl-C6H4
3-F-C6H4
4-CH3-C6H4
biphenyl
6b
6c
6d
6e
47.98b
9.83 ꢀ 0.11a
35.74b
3.84 ꢀ 0.09a
4.45 ꢀ 0.13a
3.59 ꢀ 0.11a
1.32 ꢀ 0.20a
2.41 ꢀ 0.03a
6f
naphthyl
6d
6e
6f
6g
6g
6h
KH2PO4
3-NO2-C6H4
3,4-diCl-C6H3
e
6h
a
IC50
.
Vincristine
b
%age inhibition.

172
I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
Table 7
Product spots were visualized under UV light at 254 and 365 nm.
Melting points were recorded on a Stuart melting point apparatus
(SMP3) and are uncorrected. Infra-red (IR) spectra were recorded
on FTS 3000 MX, Bio-Rad Merlin (Excalibur model) spectropho-
tometer. 1H NMR spectra were recorded on a Bruker Avance
Antileishmanial activity of synthesized compounds (3, 4aeg, 5aee and 6aeh).
Entry
Percent inhibition ꢀ SEM
100 10
m
M
mM
1
m
M
0.1 mM
3
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
6a
6b
6c
39.5 ꢀ 2.5
52.5 ꢀ 2.1
56.7 ꢀ 2.4
81.2 ꢀ 2.8
54.6 ꢀ 2.6
34.9 ꢀ 2.4
40.3 ꢀ 2.5
66.8 ꢀ 2.3
37.2 ꢀ 2.7
48.0 ꢀ 2.1
71.4 ꢀ 2.6
60.5 ꢀ 2.3
53.8 ꢀ 2.1
38.6 ꢀ 3.1
41.8 ꢀ 2.5
52.3 ꢀ 2.2
43.9 ꢀ 2.4
61.2 ꢀ 2.3
55.6 ꢀ 2.3
63.1 ꢀ 2.7
59.8 ꢀ 2.4
79.8 ꢀ 1.8
36.7 ꢀ 1.9
49.5 ꢀ 2.5
54.3 ꢀ 2.1
77.2 ꢀ 1.9
51.5 ꢀ 2.3
30.8 ꢀ 2.0
37.9 ꢀ 2.7
63.0 ꢀ 2.2
34.6 ꢀ 2.8
45.3 ꢀ 3.2
68.9 ꢀ 2.8
57.2 ꢀ 2.1
51.4 ꢀ 2.5
35.2 ꢀ 2.6
38.3 ꢀ 2.4
49.6 ꢀ 2.1
39.8 ꢀ 2.0
58.9 ꢀ 1.9
52.9 ꢀ 2.7
59.6 ꢀ 1.8
56.4 ꢀ 1.4
76.3 ꢀ 1.4
33.6 ꢀ 2.3
46.2 ꢀ 1.7
51.6 ꢀ 1.6
75.8 ꢀ 1.7
48.5 ꢀ 1.3
27.3 ꢀ 1.8
35.4 ꢀ 2.6
60.8 ꢀ 2.7
31.2 ꢀ 3.1
41.9 ꢀ 3.3
65.5 ꢀ 2.6
52.2 ꢀ 2.4
49.1 ꢀ 2.6
32.3 ꢀ 3.0
34.6 ꢀ 1.9
46.1 ꢀ 1.7
37.7 ꢀ 2.6
55.9 ꢀ 2.9
49.0 ꢀ 2.5
57.5 ꢀ 2.1
52.3 ꢀ 1.8
74.8 ꢀ 2.7
30.9 ꢀ 2.5
42.5 ꢀ 2.0
47.7 ꢀ 3.1
71.5 ꢀ 2.4
45.2 ꢀ 2.4
25.1 ꢀ 2.6
31.3 ꢀ 2.3
56.4 ꢀ 2.2
27.8 ꢀ 3.0
37.2 ꢀ 3.1
62.1 ꢀ 1.3
49.6 ꢀ 1.9
46.7 ꢀ 2.7
29.3 ꢀ 2.5
31.7 ꢀ 2.4
44.9 ꢀ 2.4
34.5 ꢀ 2.3
52.3 ꢀ 2.2
47.28 ꢀ 2.4
54.6 ꢀ 2.7
49.3 ꢀ 2.1
69.9 ꢀ 2.3
(300 MHz) spectrometer. Chemical shifts (d) are quoted in parts per
million (ppm) downfield of tetramethylsilane, using residual pro-
tonated solvent as internal standard (DMSO-d6 at 2.50 ppm). Ab-
breviations used in the description of resonances are: s (singlet),
d (doublet), t (triplet), q, (quartet), m (multiplet). Proton-decoupled
13C NMR spectra were recorded on a Bruker Avance (75 MHz)
spectrometer using deuterated solvent as internal standard
(DMSO-d6 at 39.52 ppm). The elemental analysis was performed on
Leco CHNS-932 Elemental Analyzer, Leco Corporation (USA).
4.2. Preparation of 4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-
thiol (3)
6d
6e
6f
6g
Isonicotinohydrazide 1 (1 mmol) was stirred with a solution of
potassium hydroxide (1.5 mmol) dissolved in methanol (10 mL) at
0e5 ꢁC. Then, to this stirred solution carbon disulfide (1.5 mmol)
was added slowly and the reaction mixture was left overnight at
room temperature. The solid product of potassium dithiocarbazi-
nate 2 was filtered, washed with chilled methanol and dried. It was
directly used for next step without purification.
6h
Amphotericin B
all the compounds screened in the series, 4c showed excellent in-
hibition of 81.2% whereas Amphotericin B showed 79.8% inhibition.
Compound 4c has furanyl group at ortho position while 4d has
furanyl group at meta position but inhibitory activity decreases
from 81.2% to 54.6% when substitution goes from ortho to meta
position. Another potent compound in the series was 5c having
71.4% inhibition against Leishmania major. It has methyl group
attached as substituent at ortho position, while 6d has 43.9% in-
hibition although it contains methyl substituent but at para posi-
tion. Least active compound among the series was 4e having 34.9%
inhibition. Compounds which showed less than 50% inhibition in-
cludes 3, 4f, 5a, 5b, 6a, 6b and 6d. The results indicate that these
compounds may be used as therapeutic agent to treat
leishmaniasis.
The above potassium dithiocarbazinate 2 was taken in water
(8 mL) and hydrazine hydrate (2 mmol) and refluxed for 4e5 h.
During progress of the reaction, the reaction mixture turned to
green with evolution of hydrogen sulfide and finally it became
homogeneous. It was then diluted with little cold water and acid-
ified with conc. hydrochloric acid. The white precipitated solid was
filtered, washed with cold water and recrystallized from aqueous
methanol. 4-Amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol
3
displayed spectroscopic data consistent with those observed pre-
viously [27].
4.3. Preparation of 1,2,4-triazolo[3,4-b][1,3,4]thiadiazoles (4aeg)
and (5aee)
A
mixture of 4-amino-5-(pyridin-3-yl)-4H-[1,2,4]triazole-3-
3. Conclusion
thiol 3 (1 mmol) and substituted aromatic acids (1.1 mmol) in
POCl3 (5 mL) was refluxed for 6e7 h. The reaction mixture was
slowly poured into crushed ice with stirring and neutralized with
sodium bicarbonate. Solid material was filtered, washed with cold
water, dried, and recrystallized from ethanol to afford 1,2,4-triazolo
[3,4-b][1,3,4]thiadiazoles 4aeg and 5aee [25].
The synthesis and biological evaluation of newly synthesized
triazolothiadiazoles and thiadiazines derivatives have been re-
ported. The cholinesterase inhibition studies revealed that all
compounds were potential inhibitors of the investigated enzymes
and can be used for treatment of Alzheimer’s disease. The study of
their anticholinesterase activities might lead to a novel family of
potent anti-AD compounds in near future. Some of the investigated
compounds were more potent on alkaline phosphatase, better than
the reference drug and this study will prompt the design and
screening of more such derivatives as small molecule probes for the
study of the pathophysiological role of ALP. Due to synthetic and
biological versatility of tested compounds, they may be used as
therapeutic agents having antiproliferative and antileishmanial
activities as well.
4.3.1. 6-(4-Chloro-2-fluorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazole (4a)
Off white solid (76%): m.p 222e223 ꢁC; Rf: 0.70 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3097 (AreH), 1607 (C]N), 1574,
1524 (C]C), 1216 (CeF), 1085 (CeCl); 1H NMR (300 MHz, DMSO-
d6):
8.63 (m, 1H, AreH), 7.90e7.86 (m, 1H, AreH), 7.70e7.66 (m, 1H, Are
H); 13C NMR (75 MHz, DMSO-d6):
170.86, 163.65, 159.76, 156.42,
d
9.46 (d, 2H, J ¼ 6.0 Hz, AreH), 8.78e8.76 (m, 2H, AreH), 8.66e
d
150.43, 142.38, 132.85, 132.25, 131.65, 131.12, 124.63, 124.03, 122.40,
116.36, 116.07. Analysis Calcd. for C14H7ClFN5S: C, 50.68; H, 2.13; N,
21.11; S, 9.67. Found: C, 50.54; H, 2.18; N, 21.01; S, 9.52.
4. Experimental
4.1. General
4.3.2. 6-(3-Chloro-4-fluorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazole (4b)
Unless otherwise noted, all materials were obtained from
commercial suppliers (Aldrich and Merck companies) and used
without further purification. Thin layer chromatography (TLC) was
performed on Merck DF-Alufoilien 60F254 0.2 mm precoated plates.
Yellow solid (78%): m.p 258e259 ꢁC; Rf: 0.70 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3033 (AreH), 1603 (C]N), 1557,
1519 (C]C), 1201 (CeF), 1075 (CeCl); 1H NMR (300 MHz, DMSO-
d6):
d
8.80 (d, 2H, J ¼ 6.0 Hz, AreH), 8.14 (d, 2H, J ¼ 6.0 Hz, AreH),

I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
173
8.11e8.04 (m, 1H, AreH), 7.62e7.57 (m, 2H, AreH); 13C NMR
(75 MHz, DMSO-d6): 169.14, 157.78, 151.44, 148.67, 146.45, 144.39,
137.65, 132.65, 132.11, 130.39, 128.40, 127.90, 126.71, 125.47, 114.12.
Analysis Calcd. for C14H7ClFN5S: C, 50.68; H, 2.13; N, 21.11; S, 9.67.
Found: C, 50.75; H, 1.98; N, 21.16; S, 9.54.
4.3.8. 2-((3-(Pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-
d
yl)methoxy)phenol (5a)
Off white solid (76%): m.p 213e214 ꢁC; Rf: 0.65 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3112 (OH), 3006 (AreH), 1607 (C]
N), 1574, 1524 (C]C); 1H NMR (300 MHz, DMSO-d6):
d 8.73 (d, 2H,
J ¼ 6.0 Hz, AreH), 8.02e8.00 (m, 2H, AreH), 7.48e7.45 (m, 4H, Are
4.3.3. 6-(Furan-2-yl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (4c)
H), 5.88 (s, 2H, OCH2), 5.83 (s, 1H, OH); 13C NMR (75 MHz, DMSO-
d6):
124.04, 123.64, 119.28, 118.91, 70.80. Analysis Calcd. for
15H11N5O2S: C, 55.38; H, 3.41; N, 21.53; S, 9.86. Found: C, 55.27; H,
d 173.01, 167.71, 151.53, 149.82, 148.98, 148.20, 148.15, 136.05,
Off white solid (75%): m.p 250e251 ꢁC; Rf: 0.68 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3092 (AreH), 1602 (C]N), 1592,
C
1558 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
8.84 (d, 2H, J ¼ 6.0 Hz,
AreH), 8.18 (d, 2H, J ¼ 6.3 Hz, AreH), 7.67e7.61 (m, 2H, AreH),
6.90e6.88 (m, 1H, AreH); 13C NMR (75 MHz, DMSO-d6):
157.81,
3.22; N, 21.61; S, 9.70.
d
4.3.9. 4-((3-(Pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-
yl)methoxy)phenol (5b)
151.20, 148.47, 147.54, 146.97, 133.71, 124.49, 119.83, 116.11, 113.97.
Analysis Calcd. for C12H7N5OS: C, 53.52; H, 2.62; N, 26.01; S, 11.91.
Found: C, 53.38; H, 2.69; N, 26.11; S, 11.76.
Off white solid (72%): m.p 198e199 ꢁC; Rf: 0.62 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3112 (OH), 3004 (AreH), 1605 (C]
N), 1554, 1504 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
8.81 (d, 2H,
J ¼ 6.0 Hz, AreH), 8.14e8.11 (m, 2H, AreH), 7.45e7.42 (m, 4H, Are
H), 5.59 (s, 3H, OH, OCH2); 13C NMR (75 MHz, DMSO-d6):
173.01,
4.3.4. 6-(Furan-3-yl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (4d)
d
Off white solid (79%): m.p 262e263 ꢁC; Rf: 0.73 (chloroform :
169.27, 155.33, 151.51, 133.69, 130.12, 124.69, 123.64, 121.65, 116.38,
65.85. Analysis Calcd. for C15H11N5O2S: C, 55.38; H, 3.41; N, 21.53; S,
9.86. Found: C, 55.47; H, 3.26; N, 21.59; S, 9.74.
methanol, 9:1); IR (neat, cmꢂ1): 3043 (AreH), 1601 (C]N), 1579,
1526 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
9.02 (d, 2H, J ¼ 6.0 Hz,
AreH), 8.81 (d, 2H, J ¼ 6.0 Hz, AreH), 7.66e7.58 (m, 2H, AreH),
6.90e6.88 (m, 1H, AreH); 13C NMR (75 MHz, DMSO-d6):
157.43,
d
4.3.10. 3-(Pyridin-4-yl)-6-(o-tolyloxymethyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (5c)
154.56, 151.59, 147.95, 147.60, 132.38, 125.43, 122.72, 115.90, 115.13.
Analysis Calcd. for C12H7N5OS: C, 53.52; H, 2.62; N, 26.01; S, 11.91.
Found: C, 53.34; H, 2.49; N, 25.88; S, 11.78.
Brown solid (75%): m.p 189e190 ꢁC; Rf: 0.63 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3054 (AreH), 1602 (C]N), 1556,
1529 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
8.83 (d, 2H, J ¼ 6.0 Hz,
AreH), 8.15 (d, 2H, J ¼ 6.0 Hz, AreH), 7.24e7.13 (m, 3H, AreH),
6.98e6.93 (m, 1H, AreH), 5.65 (s, 2H, OCH2), 2.27 (s, 3H, CH3); 13
NMR (75 MHz, DMSO-d6): 170.06, 155.96, 155.61, 151.21, 143.88,
4.3.5. 6-(2-Methylfuran-3-yl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole (4e)
C
Brown solid (77%): m.p 245e246 ꢁC; Rf: 0.69 (chloroform :
d
methanol, 9:1); IR (neat, cmꢂ1): 3036 (AreH), 1597 (C]N), 1557,
132.78, 131.31, 127.64, 126.60, 122.35, 119.83, 112.74, 65.40, 16.37.
Analysis Calcd. for C16H13N5OS: C, 59.43; H, 4.05; N, 21.66; S, 9.92.
Found: C, 59.33; H, 4.16; N, 21.54; S, 9.81.
1534 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
8.87 (d, 2H, J ¼ 6.0 Hz,
AreH), 8.24 (d, 2H, J ¼ 6.0 Hz, AreH), 7.67e7.62 (m, 1H, AreH), 7.04
(d, 1H, J ¼ 6.8 Hz, AreH), 2.68 (s, 3H, CH3); 13C NMR (75 MHz,
DMSO-d6):
d
161.19, 157.85, 154.74, 148.59, 147.95, 146.97, 134.31,
4.3.11. 6-((4-Methoxyphenoxy)methyl)-3-(pyridin-4-yl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadia-zole (5d)
125.87, 124.72, 119.76, 14.38. Analysis Calcd. for C13H9N5OS: C,
55.11; H, 3.20; N, 24.72; S, 11.32. Found: C, 55.03; H, 3.12; N, 24.55;
S, 11.21.
Brown solid (76%): m.p 199e200 ꢁC; Rf: 0.66 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3039 (AreH), 1604 (C]N), 1547,
1524 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
8.81 (d, 2H, J ¼ 6.0 Hz,
4.3.6. 6-(2-Chloro-4,5-difluorophenyl)-3-(pyridin-4-yl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadia-zole (4f)
AreH), 8.13e8.11 (m, 2H, AreH), 7.10e7.05 (m, 2H, AreH), 6.92e
6.89 (m, 2H, AreH), 5.57 (s, 2H, OCH2), 3.70 (s, 3H, OCH3); 13C NMR
Brown solid (74%): m.p 230e231 ꢁC; Rf: 0.66 (chloroform:
methanol, 9:1); IR (neat, cmꢂ1): 3007 (AreH), 1603 (C]N), 1558,
1509 (C]C) 1179 (CeF), 1022 (CeCl); 1H NMR (300 MHz, DMSO-
(75 MHz, DMSO-d6):
132.75, 119.81, 116.79, 115.24, 66.02, 55.86. Analysis Calcd. for
16H13N5O2S: C, 56.63; H, 3.86; N, 20.64; S, 9.45. Found: C, 56.47; H,
d 169.89, 156.02, 154.96, 151.44, 151.19, 143.86,
C
d6):
d
8.83 (d, 2H, J ¼ 6.0 Hz, AreH), 8.41e8.35 (m, 1H, AreH), 8.24
4.01; N, 20.49; S, 9.29.
(d, 2H, J ¼ 6.0 Hz, AreH), 8.14e8.08 (m, 1H, AreH); 13C NMR
(75 MHz, DMSO-d6):
d
170.89, 163.55, 159.74, 156.82, 151.26, 144.09,
4.3.12. 6-((4-Fluorophenoxy)methyl)-3-(pyridin-4-yl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (5e)
132.65, 132.23, 131.24, 130.86, 126.89, 126.24, 121.98, 121.27, 120.99,
119.99. Analysis Calcd. for C14H6ClF2N5S: C, 48.08; H, 1.73; N, 20.02;
S, 9.17. Found: C, 47.95; H, 1.56; N, 19.87; S, 9.10.
Off white solid (79%): m.p 192e193 ꢁC; Rf: 0.68 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3081 (AreH), 1604 (C]N), 1541,
1505 (C]C), 1203 (CeF); 1H NMR (300 MHz, DMSO-d6):
d
8.81 (d,
2H, J ¼ 6.0 Hz, AreH), 8.13e8.11 (m, 2H, AreH), 7.23e7.16 (m, 4H,
AreH), 5.63 (s, 2H, OCH2); 13C NMR (75 MHz, DMSO-d6):
169.31,
4.3.7. 6-(4-Ethoxybenzyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (4g)
d
Brown solid (77%): m.p 206e208 ꢁC; Rf: 0.67 (chloroform:
159.43, 156.27, 156.04, 153.84, 153.81, 151.18, 143.88, 132.74, 119.81,
117.24,117.13,116.79,116.48, 65.96. Analysis Calcd. for C15H10FN5OS:
C, 55.04; H, 3.08; N, 21.39; S, 9.80. Found: C, 54.93; H, 3.17; N, 21.46;
S, 9.63.
methanol, 9:1); IR (neat, cmꢂ1): 3032 (AreH), 1613 (C]N), 1578,
1512 (C]C); 1H NMR (300 MHz, DMSO-d6):
d 9.37 (d, 1H, 1.5 Hz,
AreH), 8.74e8.73 (m, 1H, AreH), 8.55e8.51 (m, 1H, AreH), 7.67e
7.63 (m, 1H, AreH), 7.36 (d, 2H, J ¼ 8.7 Hz, AreH), 6.93 (d, 2H,
J ¼ 8.7 Hz, AreH), 4.45 (s, 2H, CH2), 4.01 (q, 2H, J ¼ 7.2 Hz, OCH2),
4.4. Preparation of 1,2,4-triazolo[3,4-b][1,3,4]-thiadiazines (6aeh)
1.31 (t, 3H, J ¼ 6.9 Hz, CH3); 13C NMR (75 MHz, DMSO-d6):
d 172.40,
158.55, 155.68, 151.42, 147.00, 143.50, 133.64, 130.95, 127.40, 124.64,
122.43, 115.26, 63.50, 36.86, 15.08. Analysis Calcd. for C17H15N5OS:
C, 60.52; H, 4.48; N, 20.76; S, 9.50. Found: C, 60.44; H, 4.31; N,
20.65; S, 9.37.
A mixture of 4-amino-5-(pyridin-4-yl)-4H-[1,2,4]triazole-3-
thiol 5 (1 mmol) and substituted phenacyl bromides (1.2 mmol)
was refluxed in absolute ethanol (10 mL) for 6e7 h. The reaction
mass was poured into crushed ice and neutralized with sodium

174
I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
bicarbonate. Solid product obtained was filtered, washed with
water, dried and recrystallized from ethanol to afford conjugated
products 6aeh [28].
4.4.6. 6-(Naphthalen-1-yl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine (6f)
Light yellow solid (71%): m.p 256e257 ꢁC; Rf: 0.54 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3036 (AreH), 2969, 2850 (CH2),
1631 (C]N), 1596, 1538 (C]C); 1H NMR (300 MHz, DMSO-d6):
4.4.1. 6-(3-Methoxyphenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine (6a)
d
9.04 (d, 2H, J ¼ 6.0 Hz, AreH), 8.74e8.72 (m, 1H, AreH), 8.49 (d,
Light yellow solid (72%): m.p 249e250 ꢁC; Rf: 0.56 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3009 (AreH), 2887, 2843 (CH2),
1632 (C]N), 1603, 1513 (C]C); 1H NMR (300 MHz, DMSO-d6):
2H, J ¼ 6.0 Hz, AreH), 8.19 (d, 1H, J ¼ 7.8 Hz, AreH), 8.10e8.03 (m,
3H, AreH), 7.71e7.66 (m, 2H, AreH), 4.67 (s, 2H, SCH2); 13C NMR
(75 MHz, DMSO-d6): d 156.55, 148.97, 148.00, 147.55, 146.77, 146.56,
d
9.02 (d, 2H, J ¼ 6.0 Hz, AreH), 8.44 (d, 2H, J ¼ 6.3 Hz, AreH), 8.08
145.58, 140.45, 140.89, 137.76, 134,29, 131.91, 131.45, 130.83, 122.49,
115.08, 21.60. Analysis Calcd. for C19H13N5S: C, 66.45; H, 3.82; N,
20.39; S, 9.34. Found: C, 66.32; H, 3.61; N, 20.46; S, 9.23.
(d, 1H, J ¼ 8.7 Hz, AreH), 8.01e7.86 (m, 2H, AreH), 7.15 (s, 1H, Are
H), 4.48 (s, 2H, SCH2), 3.87 (s, 3H, OCH3); 13C NMR (75 MHz, DMSO-
d6):
125.43, 124.72, 123.47, 115.13, 56.13, 23.18. Analysis Calcd. for
16H13N5OS: C, 59.43; H, 4.05; N, 21.66; S, 9.92. Found: C, 59.63; H,
d 163.14, 157.05, 148.59, 145.95, 138.69, 132.54, 130.39, 127.12,
4.4.7. 6-(3-Nitrophenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazine (6g)
C
4.11; N, 21.44; S, 9.78.
Light pink solid (73%): m.p 262e263 ꢁC; Rf: 0.57 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3043 (AreH), 2984, 2925 (CH2),
1638 (C]N), 1610, 1513 (C]C), 1445, 1349 (CeNO2); 1H NMR
4.4.2. 6-(3-Chlorophenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine (6b)
(300 MHz, DMSO-d6):
J ¼ 6.0 Hz, AreH), 8.23 (s, 1H, J ¼ 8.7 Hz, AreH), 8.04e7.83 (m, 3H,
AreH), 4.54 (s, 2H, SCH2); 13C NMR (75 MHz, DMSO-d6):
156.51,
d
9.17 (d, 2H, J ¼ 6.0 Hz, AreH), 8.55 (d, 2H,
Light yellow solid (75%): m.p 290e291 ꢁC; Rf: 0.51 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3035 (AreH), 2857, 2842 (CH2),
1633 (C]N), 1603, 1509 (C]C), 1109 (CeCl); 1H NMR (300 MHz,
d
148.87, 148.07, 147.55, 146.77, 140.45, 139.67, 137.12, 135.24, 131.45,
130.83, 125.43, 21.54. Analysis Calcd. for C15H10N6O2S: C, 53.25; H,
2.98; N, 24.84; S, 9.48. Found: C, 53.29; H, 2.84; N, 24.69; S, 9.41.
DMSO-d6):
AreH), 8.17 (d, 1H, J ¼ 8.4 Hz, AreH), 7.91e7.82 (m, 3H, AreH), 4.44
(s, 2H, SCH2); 13C NMR (75 MHz, DMSO-d6):
157.10, 148.85, 145.74,
d
9.02 (d, 2H, J ¼ 6.3 Hz, AreH), 8.43 (d, 2H, J ¼ 6.3 Hz,
d
138.29, 137.41, 134.19, 132.28, 129.62, 129.07, 127.75, 127.37, 123.48,
23.41. Analysis Calcd. for C15H10ClN5S: C, 54.96; H, 3.07; N, 21.37; S,
9.78. Found: C, 54.81; H, 2.96; N, 21.24; S, 9.85.
4.4.8. 6-(3,4-Dichlorophenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine (6h)
Light orange solid (79%): m.p 286e287 ꢁC; Rf: 0.54 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3056 (AreH), 2953, 2886 (CH2),
1630 (C]N), 1603, 1511 (C]C), 1026 (CeCl); 1H NMR (300 MHz,
4.4.3. 6-(3-Fluorophenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine (6c)
DMSO-d6):
AreH), 7.79 (d, 1H, J ¼ 1.5 Hz, AreH), 7.54e7.41 (m, 2H, AreH), 4.44
(s, 2H, SCH2); 13C NMR (75 MHz, DMSO-d6):
157.11, 148.96, 147.55,
d
9.02 (d, 2H, J ¼ 6.0 Hz, AreH), 8.43 (d, 2H, J ¼ 6.6 Hz,
Light yellow solid (78%): m.p 230e231 ꢁC; Rf: 0.50 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3042 (AreH), 2915, 2851 (CH2),
1631 (C]N), 1597, 1532 (C]C), 1230 (CeF); 1H NMR (300 MHz,
d
146.77, 144.12, 137.24, 135.72, 133.58, 131.91, 128.54, 126.19, 122.48,
21.45. Analysis Calcd. for C15H9Cl2N5S: C, 49.74; H, 2.50; N, 19.33; S,
8.85. Found: C, 49.58; H, 2.34; N, 19.21; S, 8.90.
DMSO-d6):
AreH), 8.16 (d, 1H, J ¼ 8.4 Hz, AreH), 7.77e7.63 (m, 3H, AreH), 4.50
(s, 2H, SCH2); 13C NMR (75 MHz, DMSO-d6):
157.10, 148.85,146.25,
d
8.96 (d, 2H, J ¼ 6.0 Hz, AreH), 8.32 (d, 2H, J ¼ 6.3 Hz,
d
145.74, 144.21, 139.12, 138.29, 132.28, 129.62, 129.07, 128.86, 127.75,
127.37, 123.48, 123.10, 23.41. Analysis Calcd. for C15H10FN5S: C,
57.87; H, 3.24; N, 22.49; S, 10.30. Found: C, 57.69; H, 3.05; N, 22.56;
S, 10.12.
5. Pharmacological protocols
5.1. In vitro inhibitory studies on EeAChE and hBChE
Ellman’s method was used to evaluate the inhibitory activities of
synthesized compounds against EeAChE and hBChE [32]. The
compounds were first dissolved in DMSO (end concentration of
DMSO was 1% in the assay) and studied at a 0.5 mM final concen-
tration for initial screening. The compounds exhibiting more than
50% inhibition were further tested by making eight to ten serial
dilutions in assay buffer (50 mM TriseHCl, 0.1 M NaCl and 0.02 M
4.4.4. 3-(Pyridin-4-yl)-6-p-tolyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazine (6d)
Light yellow solid (74%): m.p 281e282 ꢁC; Rf: 0.53 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3038 (AreH), 2917, 2865 (CH2),
1631 (C]N), 1603, 1510 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
9.01 (d, 2H, J ¼ 6.0 Hz, AreH), 8.39 (d, 2H, J ¼ 6.6 Hz, AreH), 8.05
(d, 2H, J ¼ 8.4 Hz, AreH), 7.88 (d, 2H, J ¼ 8.7 Hz, AreH), 4.42 (s, 2H,
SCH2), 3.1 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
157.14,
MgCl2, pH 8.0). Composition of the reaction mixture was: 20
mL
d
assay buffer, 10 L of test compound and 10 L of enzymes (0.5 and
m
m
148.85, 145.74, 139.48, 138.54, 132.28, 129.72, 129.07, 127.75, 123.48,
23.48, 21.53. Analysis Calcd. for C16H13N5S: C, 62.52; H, 4.26; N,
22.78; S, 10.43. Found: C, 62.41; H, 4.11; N, 22.65; S, 10.29.
3.4 U/mg of EeAChE or hBChE, respectively). A 10 min pre-
incubation of the reaction mixture at 25 ꢁC was carried out. At
the end of the pre-incubation period, 10
mL of 1 mM acetylth-
iocholine iodide or butyrylthiocholine chloride were added to the
respective EeAChE or hBChE enzyme solution to start the enzy-
matic reactions. The mixtures were incubated for 15 min at 25 ꢁC.
The change in the absorbance was measured at 405 nm to deter-
mine the amount of the product using a microplate reader (Bio-Tek
ELx800Ô). Neostigmine and donepezil were used as standard in-
hibitors. Assays were performed with a blank containing all of the
components except enzyme or substrate in order to account for a
non-enzymatic reaction. Each concentration was analyzed in trip-
licate. Enzyme dilution buffer consisted of 50 mM TriseHCl buffer
containing 0.1% (w/v) bovine serum albumin (BSA) and pH 8.0. The
linear regression parameters were determined for each curve and
4.4.5. 6-(4-Biphenyl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazine (6e)
Light yellow solid (75%): m.p 177e178 ꢁC; Rf: 0.59 (chloroform :
methanol, 9:1); IR (neat, cmꢂ1): 3041 (AreH), 2942, 2871 (CH2),
1632 (C]N), 1603, 1517 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
8.99 (d, 2H, J ¼ 6.3 Hz, AreH), 8.38 (d, 2H, J ¼ 6.6 Hz, AreH), 8.25e
8.14 (m, 4H, AreH), 7.56e7.43 (m, 5H, AreH), 4.52 (s, 2H, SCH2); 13
NMR (75 MHz, DMSO-d6): 156.89, 148.97, 148.00, 145.54, 140.89,
C
d
137.92, 132.04, 131.33, 131.08, 130.05, 124.92, 123.40, 116.97, 116.68,
21.49. Analysis Calcd. for C21H15N5S: C, 68.27; H, 4.09; N, 18.96; S,
8.68. Found: C, 68.10; H, 4.17; N, 18.82; S, 8.56.

I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
175
the IC50 values were measured. The computer program GraphPad
Prism 5.0 (San Diego, CA, USA) was used to analyze the data.
cold TCA solution at 4 ꢁC for 1 h. The plates were washed 5 times
with PBS and air dried. Fixed cells were further treated with 0.4% w/
v sulforhodamine B dye prepared in 1% acetic acid solution and left
at room temperature for 30 min. After that the plates were rinsed
with 1% acetic acid solution and allowed to dry. In order to solu-
bilize the dye, the dried plates were treated with 10 mM Tris base
solution for 10 min at room temperature. Absorbance was
measured at 490 nm subtracting the background measurement at
630 nm [36].
5.2. Activity against alkaline phosphatase
Newly synthesized triazolothiadiazoles and triazolothiadiazines
were tested against calf intestinal alkaline phosphatase (CIAP). The
compounds were initially tested against these enzymes at 10 mM
concentration to further make a doseeresponse curve of potential
inhibitors of synthesized compounds by using previously described
method by J. Iqbal [33]. The solution was made in assay buffer (pH
9.5) comprises of Tris-hydrochloride (50 mM), MgCl2 (5 mM), ZnCl2
(0.1 mM) and glycerol 50% in the solution. The solution of enzyme
substrate p-nitrophenyl phosphate (p-NPP) and test compounds
was also prepared in the same buffer without glycerol. The assay
5.4. Antileishmanial activity
5.4.1. Parasite and culture
Leishmania major promastigotes were cultured at 25 ꢀ 1 ꢁC to
logarithmic phase in D-MEM/F-12 medium (Gibco BRL) without
phenol red, supplemented by 10% heat inactivated fetal bovine
was started in 96 wells plate by adding 70
test compound (from 100 M compound stock solution), 10
optimized enzyme (0.5 U/mL) and then incubated at 37 ꢁC for
10 min. To initialize the reaction a 10 L of substrate (5 mM p-NPP)
mL of assay buffer,10
mL of
m
m
L of
serum (FBS), 100 IU/mL penicillin and 100
mg/mL streptomycin,
then washed 3 times with phosphate-buffered saline (PBS) by
centrifugation at 1500 rpm for 10 min at room temperature and
resuspended at a concentration of 2.5 ꢃ 106 parasites/mL in
medium.
m
was added into each well and again incubated for additional
30 min. The change in absorbance of released p-nitrophenolate was
monitored at 405 nm, using a 96-well microplate reader (Bio-Tek
ELx 800TM, Instruments, Inc. USA). The percent inhibition of each
compound was calculated by comparing the results with the con-
trol wells having no inhibitor at all. The compounds which showed
ꢅ50% inhibition were selected for further determination of dosee
response curves against all compounds. For this purpose 7e9 serial
dilutions of each compound spanning three orders of magnitude
were prepared in enzyme assay buffer and their dose response
curves were obtained by adopting the same methods used for
initial screening. All experiments were repeated three times in
triplicate. Results reported are mean of three independent experi-
ments (ꢀSEM) and expressed as percent inhibitions calculated by
the formula;
5.4.2. Antileishmanial activity assays (MTT assay)
The antileishmanial activity of the compounds was evaluated
in vitro against the promastigote forms of Leishmania major using
an MTT (3-(4,5-dimethylthiazol- 2yl)-2,5-diphenyltetrazolium
bromide)-based microassay as a marker of cell viability. The MTT
assay used was based on that originally described by Mosmann
(1983) [37] modified by Niks and Otto (1990) [38]. A stock solution
of MTT (Sigma Chemical Co., St. Louis, Mo.) was prepared by dis-
solving in phosphate-buffered saline (PBS) at 5 mg/mL and storing
in the dark at 4 ꢁC for up to 2 weeks before use. For the anti-
leishmanial activity assays, 100 mL/well of the culture which con-
tained 2.5 ꢃ 106 cells/mL promastigotes was seeded in 96-well flat-
bottom plates. Then, 10
mL/well from various concentrations of
Inhibitionð%Þ ¼ ½100 ꢂ ðabs of test comp=abs of controlÞ
ꢃ 100ꢆ
compounds were added to triplicate wells and plates were incu-
bated for 72 h at 25 ꢀ 1 ꢁC. The first well of 96 wells was as a blank
well which only contained of 100 mL culture medium without any
IC50 values of selected compounds exhibiting >50% activity at
0.5 mM were calculated after dilutions using computer program
GraphPad, San Diego, California, USA.
compound, drug or parasite. Amphotericin B was used as standard
drug. At the end of incubation, 10 mL of MTT was added to each well
and plates were incubated for 3 h at 25 ꢀ 1 ꢁC. Enzyme reaction was
then stopped by the addition of 100 mL of 50% isopropanol and 10%
5.3. Cell lines and cultures
sodium dodecyl sulfate. The plates were incubated for an additional
30 min. under agitation at room temperature. Relative optical
density (OD) was then measured at a wavelength of 570 nm using a
96-well microplate reader (Bio-Tek ELx 800TM, Instruments, Inc.
USA). The background absorbance of plates was measured at
690 nm and subtract from 570 nm measurement. The absorbance of
the formazan produced by the action of mitochondrial de-
hydrogenases of metabolically active cells is shown to correlate
with the number of viable cells. All experiments were repeated at
least three times. Results reported are mean of three independent
experiments (ꢀSEM) and expressed as percent inhibitions calcu-
lated by the formula;
Lung carcinoma (H157), (ATCC CRL-5802) and kidney fibroblast
(BHK-21), (ATCC CCL-10) cell lines were kept in RPMI-1640 [having
heat-inactivated fetal bovine serum (10%) glutamine (2 mM), Py-
ruvate (1 mM), 100 U/mL penicillin and 100 mg/mL streptomycin] in
T-75 cm2 sterile tissue culture flasks in a 5% CO2 incubator at 37 ꢁC
[34]. 6-Well plates were used for growing H157 and BHK-21 cells by
inoculating 104 cells per 100
mL per well. For experiments, both cell
lines were grown in 96-well plates by inoculating 104 and
5 ꢃ 104 cells/100
mL/well respectively, and plates were incubated at
37 ꢁC in a 5% CO2 incubator. Within 24 h, a uniform monolayer was
formed which was used for experiments.
Inhibitionð%Þ ¼ ½100 ꢂ ðabs of test comp=abs of controlÞ
ꢃ 100ꢆ
5.3.1. Cytotoxicity analysis by sulforhodamine B (SRB) assays
To perform cytotoxicity assay with H157 and BHK-21 cells, a
previously described method by Skehan et al. [35] was adopted
with some modifications. Briefly, cells were cultured in different 96
well plates for 24 h. The compounds in different concentrations
5.5. X-ray structure determination
(100, 10, 1 and 0.1
and blank wells were also prepared contain standard drug (VCN)
and culture media with cells respectively. The plates were then
m
M) were inoculated in test wells while control
Diffraction data for synthesized compound 4c were collected on
ꢀ
a Bruker APEXII CCD diffractometer using Mo-K
a
(l
¼ 0.71073 A) at
w90 K. Each crystal was mounted on a mylar loop in a thin film of
Paratone N oil. A full sphere of data was collected using ø and u-
incubated for 48 h. After that cells were fixed with 50
mL of 50% ice

176
I. Khan et al. / European Journal of Medicinal Chemistry 78 (2014) 167e177
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q
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We thank the University of Otago for purchase of the diffrac-
tometer. This work was financially supported by COMSTECHe
TWAS, German-Pakistani Research Collaboration Programme and
Higher Education Commission (HEC) of Pakistan.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.046.
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