Synthesis, acetylcholinesterase and alkaline phosphatase inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole derivatives

Article abstract of DOI:10.1071/CH12134

A new series of 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones (6as) and 2,5-disubstituted-1,3,4-thiadiazoles (7ah) was synthesized by intramolecular dehydrocyclization of various 1,4-disubstituted thiosemicarbazide derivatives (5as) by refluxing in 4N aqueous sodium hydroxide and by overnight stirring with polyphosphoric acid, respectively. The structures of these compounds were characterized by IR, 1H and 13C NMR, elemental analysis and mass spectroscopic studies. All the synthesized compounds were screened for their acetylcholinesterase and alkaline phosphatase inhibition studies. Most of the tested compounds showed promising activities, amongst them (6k) and (6q) showed excellent acetylcholinesterase inhibitory activity with IC50 0.2410.012 and 0.2600.013M, respectively, as compared with those of standard drug whereas the compound (6p), with IC50 0.0440.001M, was found to be the most potent inhibitor of alkaline phosphatase.

Full text of DOI:10.1071/CH12134

CSIRO PUBLISHING
Aust. J. Chem. 2012, 65, 1413–1419
Full Paper
http://dx.doi.org/10.1071/CH12134
Synthesis, Acetylcholinesterase and Alkaline
Phosphatase Inhibition of Some New 1,2,4-Triazole
and 1,3,4-Thiadiazole Derivatives
A
A
B
Imtiaz Khan, Muhammad Hanif, Muhammad Tahir Hussain,
C
C
Aftab Ahmed Khan, Muhammad Adil S. Aslam,
Nasim Hasan Rama,
A D
,
C D
,
and Jamshed Iqbal
A
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan.
Department of Applied Sciences, National Textile University, Faisalabad 37610,
Pakistan.
B
C
Department of Pharmaceutical Sciences, COMSATS Institute of Information
Technology, Abbottabad 22060, Pakistan.
D
Corresponding authors. Email: nhrama@qau.edu.pk; drjamshed@ciit.net.pk
A new series of 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones (6a–s) and 2,5-disubstituted-1,3,4-thiadiazoles
(7a–h) was synthesized by intramolecular dehydrocyclization of various 1,4-disubstituted thiosemicarbazide derivatives
(5a–s) by refluxing in 4 N aqueous sodium hydroxide and by overnight stirring with polyphosphoric acid, respectively.
The structures of these compounds were characterized by IR, 1H and 13C NMR, elemental analysis and mass spectroscopic
studies. All the synthesized compounds were screened for their acetylcholinesterase and alkaline phosphatase inhibition
studies. Most of the tested compounds showed promising activities, amongst them (6k) and (6q) showed excellent
acetylcholinesterase inhibitory activity with IC50 0.241 ꢀ 0.012 and 0.260 ꢀ 0.013 mM, respectively, as compared with
those of standard drug whereas the compound (6p), with IC50 0.044 ꢀ 0.001 mM, was found to be the most potent inhibitor
of alkaline phosphatase.
Manuscript received: 4 March 2012.
Manuscript accepted: 18 May 2012.
Published online: 27 August 2012.
Introduction
tartrate are the current approved drugs for the treatment of
Alzheimer’s patients.[9] Apart from marketed drugs, several
acetylcholinesterase inhibitors such as cambogin (A) and gutti-
ferone F (B) have been isolated from Symphonia globulifera
leaves possessing acetylcholinesterase inhibitory activity with
IC50 values of 1.13 ꢀ 0.06 and 0.95 ꢀ 0.01 mM, respectively.[10]
However, acetylcholinesterase inhibitors present some limita-
tions, such as their short half-lives and excessive side effects
caused by activation of peripheral cholinergic systems, as well
as hepatotoxicity, which is the most frequent and important side
effects of these drug therapies.[11–13] For this reason, alternative
and complementary therapies need to be developed.
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 monopho-
sphates and regulates the functions of many biological sys-
tems.[14–16] The widespread occurrence of ALP in nature
suggests its involvement in fundamental biochemical processes,
however, there is no positive evidence regarding its physiologi-
cal function, or the nature of the natural substrates. Hydrolysis of
phosphoesters, phosphate transferase activity, protein phospha-
tase activity, phosphate transport, modulation of organic cation
transport, and involvement in cell proliferation have been
Acetylcholinesterase is a serine hydrolase (AChE, acetylcholine
hydrolase, EC 3.1.1.7) that plays an essential role in the cho-
linergic synapses. Hydrolysis of the neurotransmitter acetyl-
choline (ACh) in the nervous system by acetylcholinesterase is
known to be one of the most efficient enzyme catalytic reac-
tions. The basis of this high efficiency has been sought by means
of ligand-binding studies using various substrates and has led to
the suggestion that the active centre is composed of a cationic
esteratic subsite containing the active serine, an anionic site
which accommodates the choline moiety of ACh and a periph-
eral anionic site (PAS).[1,2] The primary physiologic role of the
AChE peripheral site is to accelerate the hydrolysis of acetyl-
choline at low substrate concentrations.[3,4]
The role of the cholinergic system has been an intensive issue
of interest in Alzheimer’s disease, which is a neurodegenerative
disorder causing deterioration of memory and other cognitive
functions.[5,6] In Alzheimer’s disease, a cholinergic deficiency
in the brain has been reported.[7,8] Therefore, the synthesis and
study of inhibitors of acetylcholinesterase may aid to the
development of therapeutically useful compounds to treat such
neurological disorders. Acetylcholinesterase inhibitors donepe-
zil hydrochloride, galantamine hydrobromide and rivastigmine
Journal compilation Ó CSIRO 2012
www.publish.csiro.au/journals/ajc

1414
I. Khan et al.
H
N
OCH3
OH
NH2
NH2NH2·H2O
n
O
n
O
n
CH3OH
H2SO4
R
R
R
O
CH3OH/Reflux
(3a–e)
(1a–e)
(2a–e)
SCN
CH3OH
R
Ј
Reflux
(4a–c)
R
S
Ј
S
H
N
RЈ
4 N NaOH
N
N
H
N
H
n
O
R
n
R
Reflux
N
NH
(5a–s)
(6a–s)
PPA
stirring
R
Ј
S
n
NH
R
N
N
(7a–h)
RЈ
R
RЈ
n
R
RЈ
n
R
n
6a 3-OCH3
4-Cl
4-OCH3
1
1
2
2
0
0
1
2
2
6j 4-CH3
0
0
0
0
0
0
0
0
0
0
7a 4-CH3
2,6-diCH3
0
0
0
0
0
0
0
2,6-diCH3
2,6-diCH3
2,3-diCH3
2,4-diCH3
2,3-diCH3
2,6-diCH3
2,4-diCH3
2,3-diCH3
2,6-diCH3
2,4-diCH3
2,6-diCH3
2,3-diCH3
2,4-diCH3
2,3-diCH3
2,6-diCH3
2,4-diCH3
2,4-diCH3
b
c
d
e
f
g
h
i
3-OCH3
3-OCH3
3-OCH3
3,4-diOCH3
3,4-diOCH3
3-OCH3
k
l
2-Cl
b
2-Cl
4-Cl
4-Cl
4-OCH3
4-Cl
4-OCH3
2-OCH3
2-OCH3
c
4-Cl
3-NO2
H
4-Cl
3-Cl
2-CH3
3-Cl
4-Cl
m
n
o
p
q
r
d
e
f
g
h
3-NO2
H
4-Cl
3-Cl
4-NO2
3-OCH3
3,4,5-triOCH3 3-OCH3
s
Scheme 1.
suggested as possible functions of ALP.[17,18] The biological
action of the alkaline phosphatase in serum is associated with
metabolic bone (hypophosphatasia) and liver diseases and also
is used as a marker of osteoblastic differentiation.[19] Alkaline
phosphatases may potentially be employed as therapeutic agents
and therapeutic targets and show several uses in clinical medi-
cine and in biotechnology. Literature investigation has revealed
that the theophylline (C) and pyrazole (D) are the widely used
alkaline phosphatase inhibitors.
Five-membered heterocyclic compounds, particularly
azoles, occupy a unique place in the realm of natural and
synthetic organic chemistry.[20] 1,2,4-Triazoles represent an
overwhelming and rapid developing field in modern hetero-
cyclic chemistry. From literature, it is predictable that 1,2,4-
triazoles represent important pharmacophores, and have a wide
range of therapeutic properties. They play a vital role as
medicinal agents owing to different biological activities and
an extensive exploration on this moiety can be helpful for
obtaining better therapeutic molecules.[21]
In view of above facts and in continuation of our previous
studies,[22,23] it was contemplated to evaluate some new triazole
derivatives with different substitution patterns for acetyl cho-
linesterase and alkaline phosphatase inhibitory activities. This
paper describes the synthesis of nine triazole derivatives (6a–i)
and enzyme inhibitory activities of nineteen triazoles (6a–s) and
eight thiadiazoles (7a–h). The synthesis of ten triazoles (6j–s)
and eight thiadiazoles (7a–h) is described elsewhere.[22]
Results and Discussion
Chemistry
The synthetic route for the target compounds 4,5-disubstituted-
2,4-dihydro-3H-1,2,4-triazole-3-thiones (6a–i) is illustrated in
Scheme 1. Substituted aromatic esters (2a–e) were synthesized
by the reaction of the corresponding substituted aromatic acids
(1a–e) with methanol in the presence of catalytic amount of
sulfuric acid. Esters (2a–e) were converted to the corresponding
acid hydrazides (3a–e) by refluxing with hydrazine hydrate
(80 %) in methanol.[24–27] Different substituted anilines were
converted into corresponding dithiocarbamate salts by stirring
with concentrated ammonia solution and pure carbon disulfide
in methanol. The intermediate dithiocarbamate salts were not
isolated but converted directly to substituted phenylisothio-
cyanates (4a–e) by overnight stirring with lead nitrate solu-
tion.[28] The compounds (4a–e) were isolated by steam
distillation. Thiosemicarbazide derivatives (5a–i) were synthe-
sized by the condensation of the corresponding acid hydrazides
(3a–e) and phenylisothiocyanates (4a–c). These compounds
were used as key intermediate for the synthesis of 1,2,4-triazole
derivatives. The 4,5-disubstituted-2,4-dihydro-3H-1,2,4-
triazole-3-thiones (6a–i) were synthesized by intramolecular
dehydrative cyclization of thiosemicarbazides (5a–i) when
refluxed in 4 N sodium hydroxide solution, followed by neu-
tralization with concentrated HCl.[22] The structure elucidations
of the newly synthesized compounds were carried out by FTIR,
1H NMR and 13C NMR spectroscopy. Further confirmations of

Evaluation of Some Triazoles and Thiadiazoles
1415
Table 1. Acetylcholinesterase inhibition activities of the synthesized
compounds (6a–i) and literature compounds 6j–s and 7a–h
Table 2. Alkaline phosphatase inhibition activities of the synthesized
compounds (6a–i) and literature compounds 6j–s and 7a–h
Compound
ICA50 [mM] ꢀ s.e.m.B or
Ki [mM]
Compounds
ICA50 [mM] ꢀ s.e.mB or
Ki [mM]
(% Inhibition)C
(% Inhibition)C
6a
2.89 ꢀ 0.41
(19 ꢀ 3)c
1.78 ꢀ 0.16
4.68 ꢀ 0.54
(22 ꢀ 4)c
–
2.63 ꢀ 0.31
6a
(19)c
(15)c
–
6b
–
6b
–
6c
1.62 ꢀ 0.14
6c
(11)c
–
6d
4.27 ꢀ 0.44
6d
(18)c
–
6e
–
6e
(11)c
–
6f
–
6f
(8)c
–
6g
4.76 ꢀ 0.14
–
4.34 ꢀ 0.13
6g
(11)c
–
6h
–
6h
(26)c
–
6i
4.34 ꢀ 0.42
3.96 ꢀ 0.32
6i
(5)c
–
6j
(0)c
–
6j
(8)c
–
6k
0.241 ꢀ 0.012
0.221 ꢀ 0.011
6k
(13)c
–
c
6l
(6.2 ꢀ 2)
–
6l
(19)A
(9)c
–
6m
(14 ꢀ 5)c
(0.5 ꢀ 0.1)c
(10 ꢀ 2)c
–
6m
–
6n
–
6n
(6)c
–
6o
–
6o
6.7 ꢀ 5.7
0.061 ꢀ 0.001
10 ꢀ 2
(16)c
4.92 ꢀ 4.61
6p
(11 ꢀ 3)c
–
6p
0.044 ꢀ 0.001
6q
0.260 ꢀ 0.013
0.314 ꢀ 0.015
21.9 ꢀ 5.2
(2 ꢀ 0.1)c
(8.4 ꢀ 2)c
(4 ꢀ 1)c
0.242 ꢀ 0.012
6q
7.3 ꢀ 2
6r
0.286 ꢀ 0.013
6r
–
6s
20.1 ꢀ 4.2
6s
(16)c
–
7a
–
7a
(15)c
–
7b
–
–
7b
(17)c
–
7c
7c
–
–
–
7d
–
–
7d
–
7e
2.22 ꢀ 0.75
27.1 ꢀ 4.3
0.351 ꢀ 0.013
(14 ꢀ 4)c
2.03 ꢀ 0.55
24.75 ꢀ 3.3
0.320 ꢀ 0.012
–
7e
71 ꢀ 8
0.15 ꢀ 0.02
20 ꢀ 2
(19)c
52 ꢀ 7
0.11 ꢀ 0.02
14 ꢀ 2
–
7f
7f
7g
7g
7h
7h
Neostigmine methylsulfate
Donepezil
69.1 ꢀ 8.2
0.021 ꢀ 0.004
63.1 ꢀ 7.1
0.019 ꢀ 0.004
KH2PO4
Theophylline
3.11 ꢀ 0.03
47 ꢀ 0.51
1.5
91 ꢀ 0.51
–, not determined
AThe IC50 presented here is the concentration that inhibited 50 % of the
–, not determined
AThe IC50 presented here is the concentration that inhibited 50 % of the
enzyme activity.
enzyme activity.
Bs.e.m. ¼ Standard mean error of 3 experiments.
CThe % inhibition of the enzyme activity caused by 1 mM of the tested
compounds, given in parentheses.
Bs.e.m. ¼ standard mean error of 3 experiments.
CThe % inhibition of the enzyme activity caused by 0.1 mM of the tested
compounds, given in parentheses.
found to be more active than the standard neostigmine methyl-
sulfate. The replacement of this group by meta-methoxy group
at aryl ring A and para-chloro group at aryl ring B resulted in the
slight decrease in the acetylcholinesterase inhibition (IC50
1.78 ꢀ 0.16 mM) and Ki value 1.62 ꢀ 0.14 mM as shown by
compound (6c). By increasing the number of substituents in
the aryl ring A, for instance 3,4-dimethoxy groups, resulted in
the reduction of acetylcholinesterase inhibitory activity and by
increasing the length of the carbon chain between the positions 5
of triazole ring and methoxyphenyl group from 0 to 2, would
result in the increase in acetylcholinesterase inhibition potential
of the compounds. The triazole class of compounds was found to
be significantly active against acetylcholinesterase. In the thia-
diazole class, the presence of the meta-chloro group at aryl ring
A resulted in the maximum inhibitory activity as exhibited by
compound (7g).
the compounds were carried out by mass spectrometry and
microanalysis.
Pharmacology
Acetylcholinesterase Inhibition
The acetylcholinesterase (E.C. 3.1.1.7 from Rabbit brain)
inhibition activity of the synthesized compounds (6a–s) was
evaluated quantitatively by Ellman’s method.[29] The tested
compounds showed acetylcholinesterase inhibition activity
and Ki value ranging from IC50 0.241 ꢀ 0.012 to 27.1 ꢀ 4.3,
and 0.22 ꢀ 0.011 to 24.75 ꢀ 3.3 mM respectively, whereas stan-
dard drug neostigmine methylsulfate and Donepezil showed
IC50 value 69.1 ꢀ 8.2 mM and Ki value 63.1 ꢀ 7.1 mM and
0.021 ꢀ 0.004 mM and Ki value 0.019 ꢀ 0.004 mM respectively.
Results of acetylcholinesterase inhibitory activity of the synthe-
sized compounds (6a–s) and (7a–h) are given in Table 1. It is
clear from the results that the activity of compounds is associated
with the position of the substituents on both the rings A and B.
The compound (6k) having ortho-chloro group at aryl ring A
with an IC50 value 0.241 ꢀ 0.012 mM showed maximum inhibi-
tory activity. Similarly, the compounds (6q–r) possessing methyl
and chloro groups at the ortho and para position at ring A were
In general, the triazole class of compounds showed maxi-
mum acetylcholinesterase inhibitory activity as compared with
thiadiazoles.
Alkaline Phosphatase Inhibition
A spectrophotometric method by Iqbal[30] was used to carry
out the inhibitory studies. Some of the triazole derivatives

1416
I. Khan et al.
showed excellent inhibitory activity in the micromolar range
towards ALP, with (6p) being the most potent among them
showing maximum inhibitory activity as compared with the
standard drug. The results of alkaline phosphatase inhibition
studies of the synthesized compounds are given in Table 2.
Among the synthesized compounds (6a–s) and (7a–h), only
four were potent inhibitors of alkaline phosphatase. Others
either precipitated out or had low inhibitory activity. In the
triazole series, compound (6p) bearing a chloro group at the
meta position showed maximum potency having IC50 ¼ 0.061 ꢀ
0.001 mM and Ki value 0.044 ꢀ 0.001 mM, whereas for (6o)
the inhibition decreases (IC50 ¼ 6.7 ꢀ 5.7 mM) upon shifting the
chloro substituent to the para position.
In the thiadiazole series, the potent compound (7f) has IC50
0.15 ꢀ 0.02 mM as the para position bears a chloro group in
comparison to (7g) (IC50 ¼ 20 ꢀ 2 mM), which exhibits chloro
functionality at the meta position. So, the pattern suggests that
inhibition increases when the chloro is substituted at the para
position. Other compounds such as (6q) and (7e) have rather less
inhibitory activity, which suggests that other substituents are
rather unfavourable for inhibition.
hydrazides (3a–e) by refluxing with hydrazine hydrate (80 %)
in methanol as described in literature.[24–27]
General Procedure for the Synthesis of Substituted
Phenylisothiocyanates (4a–e)
Substituted anilines were converted to their dithiocarbamate
salts, which were oxidized to corresponding phenylisothiocya-
nates (4a–e) by the standard procedure.[28]
General Procedure for the Synthesis of 1,4-Disubstituted
Thiosemicarbazides (5a–i)
The corresponding acid hydrazide (3) (0.0068 mol) was
dissolved in methanol (30 mL) and added slowly to the solution
of substituted isothiocyanate (0.0066 mol) in methanol (10 mL).
The reaction mixture was refluxed for 10–12 h and monitored by
TLC. After consumption of the starting materials, the mixture
was cooled to room temperature. Evaporation of solvent under
reduced pressure left crude 1,4-disubstituted thiosemicarbazide
(5) as an oil, which solidified on cooling.[22] It was purified by
recrystallization from a mixture of ethyl acetate and petroleum
ether to yield thiosemicarbazide (5).
4-(4-Chlorophenyl)-1-(2-(3-methoxyphenyl)acetyl)
thiosemicarbazide 5a
Conclusions
In the present study, we reported a new series of triazole deri-
vatives. The acetylcholinesterase inhibitory activity of the
synthesized compounds was evaluated. Some of the synthesized
compounds were found to be significant inhibitors of acetyl-
cholinesterase. The compounds (6k), (6q,r) and (7g) showed
excellent acetylcholinesterase inhibitory activity as compared
with those of standard drug.
The synthesized compounds were also evaluated for
alkaline phosphatase inhibition. The compounds (6p) and (7f)
were found to be the most promising inhibitors of alkaline
phosphatase.
White solid (1.59 g, 67 %), mp 156–1578C. Rf 0.25 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3321, 3208,
1661, 1619, 1595, 1557, 1258. dH (300 MHz, DMSO-d6)10.10
(s, 1H, NH-C¼O), 9.86 (s, 1H, NH-C¼S), 9.74 (s, 1H,
NH-C¼S), 7.50 (d, J 8.7, 2H, Ar-H), 7.41 (d, J 8.7, 2H,
Ar-H), 7.20 (t, J 7.8, 1H, Ar-H), 6.91–6.79 (m, 3H, Ar-H),
3.54 (s, 3H, OCH3), 3.40 (s, 2H, CH2). dC 172.11, 169.91,
159.61, 138.60, 137.97, 137.33, 129.65, 128.48, 122.07, 121.67,
115.40, 115.17, 55.44, 41.48. Anal. Calc. for C16H16ClN3O2S:
C 54.93, H 4.61, N 12.01, S 9.17. Found: C 55.21, H 4.52,
N 12.12, S 9.15 %.
Experimental
4-(4-Methoxyphenyl)-1-(2-(3-methoxyphenyl)acetyl)
General
thiosemicarbazide 5b
The purity of the synthesized compounds were ascertained by
thin-layer chromatography and the Rf values were determined
by employing pre-coated silica gel aluminium plates, Kieslgel
60 F254 from Merck (Germany), using petroleum ether : ethyl
acetate (8 : 2 as an eluent. Melting points were measured on a
Gallenkamp melting point apparatus (MP-D) and are uncor-
rected. The IR spectra were recorded on FTS 3000 MX, Bio-Rad
Merlin (Excalibur model) spectrophotometer.
NMR spectra were recorded on a Bruker Avance 300 MHz
spectrometer using DMSO-d6 solution with TMS as an internal
standard. The multiplicities were expressed as s ¼ singlet, d ¼
doublet, t ¼ triplet, q ¼ quartet, dt ¼ doublet of triplet and cou-
pling constant (J) in Hertz (Hz). Mass spectra were recorded on
Agilent Technologies 6890N gas chromatograph and an inert
mass selective detector 5973 mass spectrometer. The elemental
analyses were performed on Leco CHNS-932 Elemental Ana-
lyzer, Leco Corporation (USA).
White solid (1.53 g, 65 %), mp 149–1508C. Rf 0.34 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3359, 3206,
1682, 1593, 1542, 1262. dH (300 MHz, DMSO-d6) 10.11 (s, 1H,
NH-C¼O), 9.76 (s, 1H, NH-C¼S), 9.50 (s, 1H, NH-C¼S), 7.30–
7.11 (m, 3H, Ar-H), 6.99–6.72 (m, 5H, Ar-H), 3.74 (s, 3H,
OCH3), 3.44 (s, 2H, CH2), 3.31 (s, 3H, OCH3). dC 174.31,
159.16, 138.60, 137.37, 136.11, 129.65, 129.01, 128.48, 125.37,
122.07, 115.39, 112.47, 55.17, 55.21, 42.75. Anal. Calc. for
C17H19N3O3S: C 59.11, H 5.54, N 12.17, S 9.28. Found: C
59.01, H 5.65, N 12.21, S 9.12 %.
4-(4-Chlorophenyl)-1-(3-(3-methoxyphenyl)propanoyl)
thiosemicarbazide 5c
White solid (1.53 g, 62 %), mp 173–1758C. Rf 0.29 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3300, 3179,
1671, 1593, 1542, 1252. dH (300 MHz, DMSO-d6) 9.94 (s, 1H,
NH-C¼O), 9.69 (s, 1H, NH-C¼S), 9.54 (s, 1H, NH-C¼S), 7.47
(d, J 8.7, 1H, Ar-H), 7.38 (d, J 8.7, 1H, Ar-H), 7.20 (t, J 8.1, 1H,
Ar-H), 7.01 (t, J 2.1, 1H, Ar-H), 6.81–6.70 (m, 4H, Ar-H), 3.41
(s, 3H, OCH3), 2.82 (t, J 6.9, 2H, CH2), 2.54 (t, J 6.9, 2H, CH2).
dC 172.82, 159.47, 139.89, 138.17, 137.03, 129.65, 128.52,
123.17, 121.54, 115.30, 115.11, 111.41, 55.36, 37.43, 33.24.
Anal. Calc. for C17H18ClN3O2S: C 56.12, H 4.99, N 11.55, S
8.81. Found: C 55.98, H 4.85, N 11.32, S 8.73 %.
General Procedure for the Synthesis of Aromatic Esters
(2a–e) and Acid Hydrazides (3a–e)
Substituted aromatic esters (2a–e) were synthesized by the
reaction of corresponding substituted aromatic acids (1a–e) in
the presence of catalytic amount of sulfuric acid, the esters
(2a–e) were converted into corresponding aromatic acid

Evaluation of Some Triazoles and Thiadiazoles
1417
4-(4-Methoxyphenyl)-1-(3-(3-methoxyphenyl)
propanoyl)thiosemicarbazide 5d
Ar-H), 6.74 (dd, J 8.4, 2.4, 1H, Ar-H), 6.67 (d, J 7.8, 1H, Ar-H),
3.68 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 3.71 (t, J 6.9, 2H, CH2),
3.40 (t, J 6.9, 2H, CH2). dC 172.03, 165.17, 152.56, 149.31,
137.43, 132.55, 129.34, 125.77, 121.86, 118.73, 115.24, 113.65,
111.14, 110.21, 55.72, 53.44, 42.12, 38.60. Anal. Calc. for
C18H21N3O3S: C 60.15, H 5.89, N 11.69, S 8.92. Found:
C 59.86, H 5.69, N 11.51, S 9.14 %.
White solid (1.61 g, 66 %), mp 180–1828C. Rf 0.28 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3327, 3269,
1680, 1593, 1542, 1257. dH (300 MHz, DMSO-d6) 10.18 (s, 1H,
NH-C¼O), 9.73(s, 1H, NH-C¼S), 9.71 (s, 1H, NH-C¼S), 7.48 (d,
J 8.4, 1H, Ar-H), 7.40 (d, J 8.4, 1H, Ar-H), 7.23 (t, J 7.8, 1H, Ar-
H), 7.03 (t, J 2.1, 1H, Ar-H), 6.89–6.71 (m, 4H, Ar-H), 3.72 (s, 3H,
OCH3), 3.69 (s, 3H, OCH3), 2.78 (t, J 6.8, 2H, CH2), 2.54 (t, J 6.8,
2H, CH2). dC 171.56, 159.61, 138.60, 137.34, 133.46, 130.71,
129.65, 128.48, 126.54, 122.07, 115.39, 112.47, 55.24, 53.37,
36.14, 34.75. Anal. Calc. for C18H21N3O3S: C 60.15, H 5.89,
N 11.69, S 8.92. Found: C 59.85, H 6.05, N 11.38, S 8.72 %.
4-(3-Methoxyphenyl)-1-(3-(3,4,5-trimethoxyphenyl)
propanoyl)thiosemicarbazide 5i
White solid (2.02 g, 71 %), mp 155–1568C. Rf 0.40 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3385, 3216,
1675, 1625, 1605, 1496, 1255. dH (300 MHz, DMSO-d6) 11.02
(s, 1H, NH-C¼O), 9.88 (s, 1H, NH-C¼S), 9.75 (s, 1H, NH-C¼S),
7.44 (s, 2H, Ar-H), 7.26 (t, J 8.1, 1H, Ar-H), 7.13 (s, 1H, Ar-H),
7.01 (d, J 7.2, 1H, Ar-H), 6.80 (d, J 7.1, 1H, Ar-H), 3.81 (s, 9H,
OCH3), 3.72 (s, 3H, OCH3), 3.41 (t, J 6.9, 2H, CH2), 3.30 (t, J 6.9,
2H, CH2). dC 187.01, 167.12, 156.33, 151.23, 146.33, 138.21,
134.22, 128.23, 125.67, 118.17, 114.87, 111.28, 110.31, 63.78,
55.47, 43.31, 35.29. Anal. Calc. for C20H25N3O5S: C 57.26,
H 6.01, N 10.02, S 7.64. Found: C 57.21, H 5.89, N 10.21, S 7.41 %.
4-(4-Chlorophenyl)-1-(3,4-dimethoxybenzoyl)
thiosemicarbazide 5e
White solid (1.84 g, 74 %), mp 165–1678C. Rf 0.24 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3400, 3180,
1668, 1596, 1510, 1262. dH (300 MHz, DMSO-d6) 10.30 (s, 1H,
NH-C¼O), 9.61 (s, 1H, NH-C¼S), 9.54 (s, 1H, NH-C¼S), 7.59
(dd, J 8.4, 1.8, 1H, Ar-H), 7.54 (d, J 1.8, 1H, Ar-H), 7.32
(d, J 9.0, 2H, Ar-H) 7.05 (d, J 8.4, 1H, Ar-H), 6.93 (d, J 9.0, 2H,
Ar-H), 3.81 (s, 3H, OCH3), 3.71 (s, 3H, OCH3). dC 173.71,
166.10, 152.19, 148.56, 137.44, 132.57, 127.99, 125.16, 121.85,
113.64, 111.62, 111.26, 53.60, 52.81. Anal. Calc. for
C16H16ClN3O3S: C 52.53, H 4.41, N 11.49, S 8.76. Found:
C 52.75, H 4.69, N 11.77, S 8.50 %.
General Procedure for the Synthesis of 1,2,4-Triazol-
3-thiones 6a–i
The corresponding thiosemicarbazide (5) (0.0014 mol) was
refluxed in aqueous sodium hydroxide solution (4 N, 25 mL).
The progress of the reaction was monitored by TLC. After
completion of reaction (12–14 h), the reaction mixture was
cooled to room temperature and filtered. The filtrate was
neutralized with hydrochloric acid (4 N) to precipitate the
1,2,4-triazole-3-thione (6), which was filtered and recrystallized
from aqueous ethanol.[22]
1-(3,4-Dimethoxybenzoyl)-4-(4-methoxyphenyl)
thiosemicarbazide 5f
White solid (1.84 g, 75 %), mp 174–1758C. Rf 0.36 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3364, 3185,
1664, 1599, 1511, 1270. dH (300 MHz, DMSO-d6) 10.42 (s, 1H,
NH-C¼O), 9.83 (s, 1H, NH-C¼S), 9.78 (s, 1H, NH-C¼S),
7.60–7.33 (m, 6H, Ar-H), 7.06 (d, J 8.4, 1H, Ar-H), 3.82
(s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.65 (s, 3H, OCH3). dC
172.42, 166.03, 152.20, 148.56, 136.89, 132.57, 128.04, 125.16,
121.86, 113.66, 111.62, 111.26, 54.28, 53.32, 52.72. Anal. Calc.
for C17H19N3O4S: C 56.50, H 5.30, N 11.63, S 8.87. Found:
C 56.22, H 5.07, N 11.48, S 8.69 %.
5-(3-Methoxybenzyl)-4-(4-chlorophenyl)-2,4-dihydro-
1,2,4-triazole-3-thione 6a[31]
White solid (0.36 g, 78 %), mp 169–1708C. Rf 0.31 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3294, 1561,
1543, 1483, 1272. dH (300 MHz, DMSO-d6) 13.74 (s, 1H, NH),
7.14 (t, J 9.0, 1H, Ar-H), 7.13 (dt, J 9.0, 2.1, 2H, Ar-H), 7.01 (dt,
J 9.0, 2.1, 2H, Ar-H), 6.75 (dd, J 9.0, 2.1, 1H, Ar-H), 6.53 (d, J
7.5, 1H, Ar-H), 6.46 (t, J 2.1, 1H, Ar-H), 3.67 (s, 3H, OCH3),
3.43 (s, 2H, CH2). dC 168.40, 159.62, 151.55, 136.28, 134.56,
132.93, 130.71, 129.88, 129.76, 121.25, 114.71, 113.05, 55.34,
31.88. m/z (GCMS, %) 331 (100), 316 (9), 298 (14), 283 (5), 121
(38), 111 (22), 91 (31), 77 (27), 51 (18). Anal. Calc. for
C16H14ClN3OS: C 57.91, H 4.25, N 12.66, S 9.66. Found:
C 57.65, H 4.56, N 12.87, S 9.54 %.
4-(2-Methoxyphenyl)-1-(2-(3-methoxyphenyl)acetyl)
thiosemicarbazide 5g
White solid (1.60 g, 68 %), mp 189–1918C. Rf 0.23 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3412, 3146,
1691, 1614, 1546, 1258. dH (300 MHz, DMSO-d6) 10.37 (s, 1H,
NH-C¼O), 9.68 (s, 1H, NH-C¼S), 9.57 (s, 1H, NH-C¼S), 7.10–
6.99 (m, 4H, Ar-H), 7.01 (t, J 7.2, 1H, Ar-H), 6.47 (dd, J 8.4, 2.1,
1H, Ar-H), 6.41 (dd, J 8.4, 2.1, 1H, Ar-H), 6.39 (s, 1H, Ar-H),
3.71 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.37 (s, 2H, CH2); dC
173.21, 165.17, 154.35, 147.51, 137.21, 133.67, 128.14, 126.34,
125.47, 122.16, 120.39, 114.61, 111.63, 111.03, 55.47, 53.31,
43.52. Anal. Calc. for C17H19N3O3S: C 59.11, H 5.54, N 12.17,
S 9.28. Found: C 59.22, H 5.66, N 12.30, S 9.45 %.
5-(3-Methoxybenzyl)-4-(4-methoxyphenyl)-
2,4-dihydro-1,2,4-triazole-3-thione 6b
White solid (0.33 g, 73 %), mp 171–1738C. Rf 0.30 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3289, 1605,
1574, 1517, 1267. dH (300 MHz, DMSO-d6) 13.68 (s, 1H, NH),
7.12 (dt, J 8.4, 2.1, 2H, Ar-H), 7.11 (d, J 1.8, 1H, Ar-H), 7.08
(dt, J 9.0, 2.1, 2H, Ar-H), 6.72 (dd, J 8.4, 2.1, 1H, Ar-H), 6.65
(d, J 7.2, 1H, Ar-H), 6.46 (t, J 2.1, 1H, Ar-H), 3.80 (s, 3H,
OCH3), 3.64 (s, 3H, OCH3), 3.33 (s, 2H, CH2). dC 168.64,
160.15, 154.87, 150.28, 138.91, 129.88, 129.11, 126.43, 122.20,
119.34, 114.21, 111.98, 55.78, 55.31, 31.92. m/z (GCMS, %)
327 (100), 312 (8), 294 (8), 279 (4), 146 (10), 121 (29), 91 (21),
77 (21), 51 (8). Anal. Calc. for C17H17N3O2S: C 62.36, H 5.23,
N 12.83, S 9.79. Found: C 62.06, H 5.45, N 12.64, S 9.70 %.
4-(2-Methoxyphenyl)-1-(3-(3-methoxyphenyl)
propanoyl)thiosemicarbazide 5h
White solid (1.78 g, 73 %), mp 186–1878C. Rf 0.26 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3425, 3212,
1682, 1612, 1585, 1251. dH (300 MHz, DMSO-d6) 10.47 (s, 1H,
NH-C¼O), 9.83 (s, 1H, NH-C¼S), 9.78 (s, 1H, NH-C¼S), 7.27
(t, J 7.6, 1H, Ar-H), 7.12 (d, J 2.1, 1H, Ar-H), 7.20–6.82 (m, 4H,

1418
I. Khan et al.
5-{2-(3-Methoxyphenyl)ethyl}-4-(4-chlorophenyl)-
2,4-dihydro-1,2,4-triazole-3-thione 6c
1523, 1498, 1289. dH (300 MHz, DMSO-d6) 13.74 (s, 1H, NH),
7.03 (t, J 8.7, 1H, Ar-H), 7.21–6.90 (m, 4H, Ar-H), 6.74 (dd, J
8.4, 3.0, 1H, Ar-H), 6.46 (d, J 8.3, 1H, Ar-H), 6.39 (t, J 1.9, 1H,
Ar-H), 3.59 (s, 3H, OCH3), 3.54 (s, 3H, OCH3), 2.41 (s, 2H,
CH2). dC 168.63, 159.50, 154.98, 152.05, 136.39, 131.82,
130.53, 129.72, 122.10, 121.22, 120.98, 114.98, 112.95,
112.83, 55.48, 53.38, 35.91. m/z (GCMS, %) 327 (50), 312
(2), 294 (100), 161 (4), 146 (10), 121 (30), 91 (21), 77 (29), 51
(18). Anal. Calc. for C17H17N3O2S: C 62.36, H,5.23, N 12.83, S
9.79. Found: C 62.41, H 5.29, N 12.61, S 9.56 %.
White solid (0.36 g, 75 %), mp 163–1648C. Rf 0.25 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3263, 1594,
1534, 1523, 1284. dH (300 MHz, DMSO-d6) 13.70 (s, 1H, NH),
7.62 (dt, J 8.7, 2.1, 2H, Ar-H), 7.40 (dt, J 6.6, 3.0, 2H, Ar-H),
7.15 (t, J 8.1, 1H, Ar-H), 6.74 (dd, J 9.3, 1.8, 1H, Ar-H), 6.63 (d,
J 8.4, 1H, Ar-H), 6.61 (s, 1H, Ar-H), 3.65 (s, 3H, OCH3), 3.30–
2.75 (m, 4H, 2 ꢂ CH2). dC 168.00, 159.73, 151.93, 141.96,
134.62, 132.02, 130.66, 129.95, 129.86, 120.87, 114.34,
112.18, 55.32, 37.48, 31.79. m/z (GCMS, %) 345 (92), 330
(4), 312 (8), 297 (2), 175 (10), 150 (6), 121 (100), 91 (65), 75
(25). Anal. Calc. for C17H16ClN3OS: C 59.04, H 4.66, N 12.15, S
9.27. Found: C 59.33, H 4.36, N 12.39, S 9.46 %.
5-{2-(3-Methoxyphenyl)ethyl}-4-(2-methoxyphenyl)-
2,4-dihydro-1,2,4-triazole-3-thione 6h
White solid (0.35 g, 73 %), mp 120–1228C. Rf 0.36 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3286, 1586,
1509, 1501, 1230. dH (300 MHz, DMSO-d6) 13.69 (s, 1H, NH),
7.53 (t, J 8.7, 1H, Ar-H), 7.61–7.10 (m, 4H, Ar-H), 6.71 (dd, J
8.4, 2.9, 2H, Ar-H), 6.35 (t, J 1.9, 1H, Ar-H), 3.59 (s, 3H, OCH3),
3.56 (s, 3H, OCH3), 3.34 (t, J 7.4, 2H, CH2), 3.31 (t, J 7.4, 2H,
CH2). dC 168.31, 152.49, 142.83, 131.94, 130.72, 129.88,
122.21, 121.32, 120.88, 116.64, 114.39, 113.29, 112.09,
111.00, 55.37, 53.44, 37.25, 34.63. m/z (GCMS, %) 341 (100),
326 (5), 308 (5), 293 (4), 175 (4), 121 (35), 91 (25), 77 (15).
Anal. Calc. for C18H19N3O2S: C 63.32, H 5.61, N 12.31, S 9.39.
Found: C 63.54, H 5.40, N 12.49, S 9.19 %.
5-{2-(3-Methoxyphenyl)ethyl}-4-(4-methoxyphenyl)-
2,4-dihydro-1,2,4-triazole-3-thione 6d
White solid (0.34 g, 71 %), mp 178–1808C. Rf 0.25 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3286, 1596,
1544, 1515, 1293. dH (300 MHz, DMSO-d6) 13.67 (s, 1H, NH),
7.27 (dt, J 8.7, 1.8, 2H, Ar-H), 7.14 (t, J 7.8, 1H, Ar-H), 7.07 (dt,
J 9.0, 3.3, 2H, Ar-H), 6.73 (dd, J 8.7, 2.1, 1H, Ar-H), 6.63 (d, J 7.8,
1H, Ar-H), 6.60 (s, 1H, Ar-H), 3.55 (s, 3H, OCH3), 3.52 (s, 3H,
OCH3), 2.70 (t, J6.0, 2H, CH2), 2.62 (t, J 6.0, 2H, CH2). dC 168.24,
160.11, 159.72, 152.25, 142.07, 129.89, 126.58, 123.47, 120.86,
115.00, 114.33, 112.16, 55.92, 55.33, 37.41, 34.70. m/z (GCMS,
%) 341 (100), 326 (5), 308 (6), 293 (3), 179 (12), 121 (46), 91 (26),
77 (16). Anal. Calc. for C18H19N3O2S: C 63.32, H 5.61, N 12.31,
S 9.39. Found: C 63.09, H 5.66, N 12.51, S 9.30 %.
5-{2-(3,4,5-Trimethoxyphenyl)ethyl}-4-(3-
methoxyphenyl)-2,4-dihydro-1,2,4-triazole-3-thione 6i
White solid (0.41 g, 74 %), mp 208–2108C. Rf 0.23 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3200, 1595,
1534, 1528, 1289. dH (300 MHz, DMSO-d6) 13.73 (s, 1H, NH),
7.46 (t, J 8.1, 1H, Ar-H), 7.11 (dd, J 8.4, 2.4, 1H, Ar-H), 6.94 (t,
J 1.8, 1H, Ar-H), 6.90 (dt, J 8.1, 1.6, 1H, Ar-H), 6.50–6.44 (m,
2H, Ar-H), 3.69–3.65 (m, 12H, OCH3), 3.37 (t, J 7.2, 2H, CH2),
3.31 (t, J 7.2, 2H, CH2). dC 167.98, 151.43, 153.17, 152.06,
136.32, 136.18, 135.13, 130.65, 120.75, 115.39, 114.60, 105.89,
55.62, 54.25, 51.29, 36.82, 35.65. m/z (GCMS, %) 401 (38), 386
(8), 287 (13), 181 (100), 148 (12), 92 (6), 77 (6). Anal. Calc. for
C20H23N3O4S: C 59.83, H 5.77, N 10.47, S 7.99. Found:
C 59.54, H 5.51, N 10.40, S 8.09 %.
4-(4-Chlorophenyl)- 5-(3,4-dimethoxyphenyl)-
2,4-dihydro-1,2,4-triazole-3-thione 6e
White solid (0.37 g, 76 %), mp 234–2368C. Rf 0.29 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3273, 1587,
1534, 1512, 1257. dH (300 MHz, DMSO-d6) 14.08 (s, 1H, NH),
7.60 (dt, J 8.7, 2.1, 2H, Ar-H), 7.42 (dt, J 8.7, 3.0, 2H, Ar-H),
6.95 (d, J 8.1, 1H, Ar-H), 6.87 (dd, J 6.1, 1.8, 1H, Ar-H), 6.83
(d, J 2.1, 1H, Ar-H), 3.65 (s, 3H, OCH3), 3.54 (s, 3H, OCH3).
dC 168.79, 150.83, 148.70, 137.51, 134.48, 131.29, 129.86, 126.84,
118.12, 113.71, 111.99, 111.92, 55.70, 54.23. m/z (GCMS, %) 347
(100), 332 (12), 304 (10), 183 (8), 163 (14), 120 (12), 111 (12), 75
(18). Anal. Calc. for C16H14ClN3O2S: C 55.25, H 4.06, N 12.08,
S 9.22. Found: C 55.44, H 4.36, N 12.00, S 9.35 %.
Pharmacological Procedures
Acetylcholinesterase Inhibition Assay
The inhibitory activities of newly synthesized novel com-
pounds were determined spectrophotometrically using acet-
ylthiocholine as substrate by modifying the method of
Ellman.[29] The assay solution consisted of 20 mL of 50 mM
Tris–hydrochloride buffer, containing 0.1 M sodium chloride,
0.02 M magnesium chloride (pH 8.0) and 50 mL of 3 mM 5,50-
dithio-bis(2-nitrobenzoic acid). Increasing concentration of test
compounds (10 mL) were added to the assay solution and pre-
incubated for 15 min at 258C with the enzyme. The enzymatic
reaction was started by adding 10 mL of acetylethiocholinchlor-
ide as a substrate and again incubated for 5 min. The hydrolysis
of acetylthiocholine was determined by monitoring the forma-
tion of the yellow 5-thio-2-nitrobenzoate anion as a result of the
reaction with 5,50-dithio-bis(2-nitrobenzoic acid) with thiocho-
lines, catalyzed by enzymes at a wavelength of 412 nm. For the
non-enzymatic reaction, the assays were carried out with a blank
containing all components except acetylcholinesterase. The
reaction rates were compared and the percentage inhibition
owing to the presence of tested inhibitors was calculated.
5-(3,4-Dimethoxyphenyl)-4-(4-methoxyphenyl)-
2,4-dihydro-1,2,4-triazole-3-thione 6f
White solid (0.35 g, 73 %), mp 221–2238C. Rf 0.28 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3297, 1576,
1517, 1489, 1256. dH (300 MHz, DMSO-d6) 13.90 (s, 1H, NH),
7.27 (d, J 8.7, 2H, Ar-H), 7.15 (d, J 9.0, 2H, Ar-H), 7.11–6.70
(m, 3H, Ar-H), 3.65 (s, 3H, OCH3), 3.63 (s, 3H, OCH3), 3.54 (s,
3H, OCH3). dC 169.12, 151.00, 151.00, 150.72, 148.63, 136.52,
130.49, 127.84, 121.60, 118.42, 111.88, 111.82, 55.96, 55.93,
53.67. m/z (GCMS, %) 343 (100), 328 (10), 179 (5), 163 (18),
147 (15), 120 (10), 92 (10), 77 (11), 51 (4). Anal. Calc. for
C17H17N3O3S: C 59.46, H 4.99, N 12.24, S 9.34. Found:
C 59.41, H 4.67, N 12.20, S 9.55 %.
5-(3-Methoxybenzyl)-4-(2-methoxyphenyl)-
2,4-dihydro-1,2,4-triazole-3-thione 6g
White solid (0.32 g, 71 %), mp 156–1588C. Rf 0.24 (petro-
leum ether/ethyl acetate, 8 : 2). nmax (KBr)/cmꢁ1 3225, 1591,

Evaluation of Some Triazoles and Thiadiazoles
1419
Neostigmine methylsulfate and Donepezil were used as stan-
dard inhibitors. Each concentration was analyzed in three
independent experiments run in triplicate, The Cheng–Prusoff
equation was used to calculate the Ki values from the IC50 values
determined by the nonlinear curve-fitting program Prism 5.0
(GraphPad, San Diego, CA, USA).
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N. Sewald, Molecules 2007, 12, 1548. doi:10.3390/12081548
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J. Dolan-Ureno, JAMA 1992, 268, 2523. doi:10.1001/JAMA.1992.
03490180055026
Alkaline Phosphatase Inhibition Assay
Initial screening of the newly synthesized compounds was
performed at a concentration of 0.1 mM of test compounds. For
potentially active compounds, full concentration-inhibition
curves were determined. To screen putative inhibitors, activity
of calf intestinal alkaline phosphatase (CIALP) was measured
by a spectrophotometric assay as previously described by
J. Iqbal.[30] The reaction mixture comprised of 50 mM TRIS-
HCl, 5 mM MgCl2, 0.1 mM ZnCl2 (pH 9.5), the inhibitors
(0.1 mM with final DMSO 1 % (v/v) and the mixture was pre-
incubated for 10 min by adding 5 mL of CIALP (0.025 U mLꢁ1).
Then, 10 mL of substrate (0.5 mM p-NPP) was added to initiate
the reaction and the assay mixture was incubated again for
30 min at 378C. 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 inhibitor activity of each sample containing the
inhibitor was compared with the control sample (without inhibi-
tor). The compounds that exhibited more than 50 % inhibitory
activities at calf intestinal alkaline phosphatase were further
evaluated for the determination of inhibition constants. Inhibi-
tory activities of the potent compounds were determined by a
range of concentrations of inhibitors spanning three orders of
magnitude. All of the experiments were repeated three times in a
triplicate manner. KH2PO4 and Theophylline were used as the
standard inhibitors of calf ALP. The Ki values from the IC50
values determined by the nonlinear curve-fitting program Prism
5.0 (GraphPad, San Diego, CA, USA).
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Neurology 1998, 50, 136. doi:10.1212/WNL.50.1.136
[14] M. H. Le Du, J. L. Millan, J. Biol. Chem. 2002, 277, 49808.
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G. Azzar, R. Buchet, J. Biol. Chem. 2005, 280, 37289. doi:10.1074/
JBC.M504260200
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Acknowledgements
The authors are grateful to Dr Ghayoor Abbas Chotana for his valuable
suggestions. This work was financially supported by the Higher Education
Commission (HEC) of Pakistan under the National Research Support
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