1,3,4-Oxadiazole-2(3H)-thione and its analogues: A new class of non-competitive nucleotide pyrophosphatases/phosphodiesterases 1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.09.011

A series of 1,3,4-oxadiazole-2 (3H)-thiones and 1,3,4-thiadiazole-2 (3H)-thiones were synthesized and evaluated for their inhibitory activities against the two nucleotide pyrophosphatase phosphodiesterase 1 enzymes. Dixon, as well as Lineweaver-Burk plots

Full text of DOI:10.1016/j.bmc.2009.09.011

Bioorganic & Medicinal Chemistry 17 (2009) 7816–7822
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,3,4-Oxadiazole-2(3H)-thione and its analogues: A new class of
non-competitive nucleotide pyrophosphatases/phosphodiesterases 1 inhibitors
a
a
a
a
Khalid M. Khan a, , Naheed Fatima , Maimona Rasheed , Saima Jalil , Nida Ambreen ,
*
Shahnaz Perveen b, M. Iqbal Choudhary a
a H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
b PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2009
Revised 5 September 2009
Accepted 10 September 2009
Available online 19 September 2009
A series of 1,3,4-oxadiazole-2 (3H)-thiones and 1,3,4-thiadiazole-2 (3H)-thiones were synthesized and
evaluated for their inhibitory activities against the two nucleotide pyrophosphatase phosphodiesterase
1 enzymes. Dixon, as well as Lineweaver–Burk plots, and their secondary replots have indicated that
the inhibition was of pure non-competitive type, against both snake venom and pure human
recombinant enzymes as the Vmax values decreases without affecting the Km values. 5-[4-(t-Butyldimeth-
ylsilyloxy)-phenyl]-1,3,4-thiadiazole-2 (3H)-thione (17) and [4-(t-butyldimethylsilyloxy)-phenyl]-1,3,4-
oxadiazole-2 (3H)-thione (1) were found to be the most active compounds with IC50 values 66.47 and
368 lM, respectively. The Ki values were 100 lM and 360 lM against the snake venom and human
recombinant NPP1 enzyme, respectively. Most active compounds were found to be non-toxic in neutro-
phil viability assay.
Keywords:
Oxadiazole
Thiadiazole
Phosphodiesterase 1
Snake venom
Ó 2009 Elsevier Ltd. All rights reserved.
Nucleotide pyrophosphatase/
phosphodiesterase-1 (NPP1)
1. Introduction
activities.18 Some of them are also known to be strong inhibitors of
monoamine oxidase,19 cyclooxygenase,20 lipoxygenase,20 and
Nucleotide pyrophosphatases/phosphodiesterases (EC 3.1.4.1,
NPP1) catalyze the release of nucleoside-50-monophosphates by
the hydrolysis of a variety of nucleotides or nucleotide derivatives.1
They exist both as membrane proteins with an extra-cellular active
site or as soluble proteins in body fluids. They are widely distrib-
uted in mammalian intestinal mucosa, liver cells, serum, and snake
venom, as well as in various plants.1–3 NPP1 or PC-1 (plasma cell
membrane glycoprotein) is a key regulator of calcification of bone
and other tissues. Over-expression of NPP1 has been associated
with chondrocalcirosis,4 while under-expression causes severe
periarticular calcification in mice5,6 and the syndrome of idiopathic
infantile arterial calcification in humans.
succinate dehydrogenase19 enzymes and also active as carbonic
anhydrase inhibitors.21
In this manuscript, we report the detail snake venom and
human NPP1 (PC-1) inhibitory activities, inhibition kinetics, and
cytotoxicity, of twenty-six 1,3,4-oxadiazole-2(3H)-thione and
1,3,4-thiadiazole-2(3H)-thione derivatives.
A total of 26 1,3,4-oxadiazole-2(3H)-thione and 1,3,4-thiadia-
zole-2(3H)-thiones were synthesized.22 To best of our knowledge,
it is the first report of the inhibitory potential of this class of com-
pounds against NPP1. The results of preliminary screening, IC50
values, kinetic parameters, and cytotoxicity clearly indicate that
these compounds have significant potential for inhibition of NPP1.
Only a few studies have been conducted on the inhibition of
nucleotide pyrophosphatases/phosphodiesterases by natural or
synthetic compounds.7–16 During this study, we randomly evalu-
ated 1,3,4-oxadiazole-2(3H)-thione and 1,3,4-thiadiazole-2(3H)-
thione derivatives for the inhibition of the nucleotide pyrophos-
phatases/phosphodiesterases.
2. Experimental
All reagents were obtained from Aldrich and Sigma Chemicals
and used without further purification. The redistilled and de-ion-
ized water was used in all experiments.
A variety of biological activities are conducted for 1,3,4-oxadiaz-
ole-2(3H)-thione and their sulfur analogs 1,3,4-thiadiazole-2
(3H)-thione. This includes bactericidal17 fungicidal,18 and herbicidal
2.1. Enzyme inhibitory assays
2.1.1. Snake venom phosphodiesterase
Activity against snake venom phosphodiesterase 1 (Sigma P
4631) (EC 3.1.4.1) was assayed by using the reported method23,24
with the following modifications. Tris–HCl buffer 33 mM pH 8.8,
* Corresponding author. Tel.: +92 21 4824910; fax: +92 21 4819018.
E-mail address: hassaan2@super.net.pk (K.M. Khan).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.011

K. M. Khan et al. / Bioorg. Med. Chem. 17 (2009) 7816–7822
7817
30 mM Mg-acetate with 0.000742 U/well final concentrations using
microtiter plate assay and 0.33 mM bis-(p-nitro phenyl) phosphate
(Sigma N-3002) as a substrate. Cysteine and EDTA25–27 (E. Merck)
2.1.2. Human recombinant NPP1 (PC-1)
Water soluble human recombinant NPP1 (catalytic extracellular
domain) was obtained as gift from Professor J. W. Goding, Monash
University, Melbourne, Australia. NPP1 was assayed by the hydro-
lysis of p-nitrophenyl phenyl phosphonate (Sigma N-2881) as de-
scribed by Kelly et al.28 The assay was performed in a buffer
consisting of 20 mM Tris–HCl, pH 8.0, 0.5 M NaCl and 1 mM CaCl2.
Enzyme activity was monitored spectrophotometrically at 37 °C on
were used as positive controls, having IC50 = 748
l
M
0.15 and
274 0.07, respectively. After 30 min of incubation, the enzyme
l
M
activity was monitored at 37 °C on a microtitre plate spectropho-
tometer (Molecular Devices, USA) by following the release of p-
nitrophenol from p-nitrophenyl phosphate at 410 nm. All the
reactions were performed in triplicate, and the initial rates were
measured as the rates of changes in the OD/min (optical density/
min) and used in subsequent calculations.
a
microtitre plate spectrophotometer (Molecular Devices) at
405 nm, by following the release of p-nitrophenol and assays were
conducted in triplicate.
R2
4
N
3
N
2
1
X
5
R1
S
Compounds
Compd.
X
O
O
R1
R2
H
CH3
5-[4-t-Butyldimethylsilyloxy phenyl]-1,3,4-
oxadiazole-2(3H)-thione
O
Si C(CH3)3
CH3
1
2
H
N
N
Bis(1,3,4-oxadiazol-2-propyl)-5-thione
Bis(1,3,4-oxadiazol-2-methyl)-5-thione
H
(H2C)3
S
O
H
N
N
3
4
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H2C
S
O
5-[4-Benzyloxyphenyl]-1,3,4-oxadiazole-
O
2(3H)-thione
5-[4-Hydroxyphenyl]-1,3,4-oxadiazole-
OH
5
2(3H)-thione
N
6
5-[4-Pyridyl]-1,3,4-oxadiazole-2(3H)-thione
5-[3-Benzyloxyphenyl]-1,3,4-oxadiazole-
O
7
H
H
2(3H)-thione
OH
5-[3-Hydroxyphenyl]-1,3,4-oxadiazole-
8
2(3H)-thione
5-[4-Benzyloxyphenyl]-3-octyl-1,3,4-
O
-(CH2)7-CH3
9
oxadiazole-2(3H)-thione
5-[4-Benzyloxyphenyl]-3-benzyl-1,3,4-
O
O
-CH2-Ph
10
11
12
13
14
15
oxadiazole-2(3H)-thione
5-[4-Benzyloxyphenyl]-3-pentyl-1,3,4-
-(CH2)4-CH3
oxadiazole-2(3H)-thione
5-Cyclohexyl-1,3,4-oxadiazole-2(3H)-thione
H
H
Ph
Ph
5-Diphenylmethyl-1,3,4-oxadiazole-2(3H)-
CH
O
thione
5-[4-Benzyloxyphenyl]-3-benzoyl-1,3,4-
O
oxadiazole-2(3H)-thione
5-[4-Benzyloxyphenyl]-3-(2-hydroxyethyl)-
O
-CH2-CH2OH
1,3,4-oxadiazole-2(3H)-thione
Scheme 1.

7818
K. M. Khan et al. / Bioorg. Med. Chem. 17 (2009) 7816–7822
5-[1-Naphthyl]-1,3,4-oxadiazole-2(3H)-
16
17
18
19
20
21
22
23
24
25
26
O
S
S
S
S
S
S
S
S
S
S
H
H
thione
CH3
5-[4-t-Butyldimethylsilyloxy phenyl]-1,3,4-
thiadiazole-2(3H)-thione
O
Si C(CH )3
3
CH3
5-[4-Hydroxyphenyl]-1,3,4-thiadiazole-
OH
H
2(3H)-thione
N
5-[4-Pyridyl]-1,3,4-thiadiazole-2(3H)-thione
Bis(1,3,4-thiadiazole-2-methyl)-5-thione
5-Phenyl-1,3,4-thiadiazole-2(3H)-thione
H
H
H
N
N
H2C
S
S
H
5-[4-Hydroxyphenyl]-3-benzyl-1,3,4-
OH
-CH2-Ph
-CH2-CH3
thiadiazole-2(3H)-thione
5-[4-Propoxyphenyl]-3-propyl-1,3,4-
O
CH3
thiadiazole-2(3H)-thione
Ph
Ph
5-Diphenylmethyl-1,3,4-thiadiazole-2(3H)-
CH
H
thione
5-[4-Hydroxyphenyl]-1,3,4-thiadiazole-
OH
-(CH2)2-CH3
-CH2-Ph
2(3H)-thione
5-[4-Benzyloxyphenyl]-3-benzyl-1,3,4-
O
thiaadiazole-2(3H)-thione
Scheme 1 (continued)
2.1.3. Enzyme kinetic studies
2.1.5. Statistical analysis
Kinetic parameters were calculated using EZ-Fit Enzyme Kinet-
ics Program (Perrella Scientific Inc., Amherst, USA). The assay con-
ditions for the measurements of the residual activities of all
inhibitors were identical, except that fixed concentrations of inhib-
iting compounds were used in the assay medium. Dissociation con-
stants (Ki values) were determined by the interpretation of Dixon
plots. Lineweaver–Burk plots and their secondary plots using ini-
tial velocities were drawn over substrate concentrations between
0.166 and 0.666 mM for snake venom and 1.0 and 2.5 mM for hu-
man recommended PC-1.
Assays were conducted in triplicate at each concentration of the
inhibitors. Graphs were plotted using GRAFIT program (Leather Bar-
row, R.J. GraFit; 4.09 ed; Erithacus Software Ltd.; Tains, U.K.). Val-
ues of the correlation coefficient, slope, intercept and their
standard errors were obtained by the linear regression analysis
using the same software. The correlation coefficient for all the lines
of all graphs was >0.99 at each point in the constructed graphs,
which represents the means of three experiments.
2.2. Cell viability assay
2.1.4. Determination of type of inhibition
2.2.1. Isolation of human neutrophils
Two different methods were applied to monitor the effect of
the inhibitor on both Km and Vmax values. This was done firstly
by plotting the reciprocal of the rate of the reactions against
the reciprocal of the substrate concentration as Lineweaver–Burk
plot29 and secondly by the Dixon plot in which the reciprocal of
the rate of the reactions was plotted against the inhibitor concen-
trations.30 The secondary re-plot of the Lineweaver–Burk was also
constructed as 1/Vmax or slope against different concentrations of
the respective inhibitor. The secondary replot of Dixon plot was
constructed as the slope of each line of substrate concentration
in original Dixon plot against the reciprocals of the substrate
concentrations.
The types of inhibition were determined by the graphical anal-
ysis of Dixon plots, Lineweaver-Burk plots and their secondary
plots. Ki Values (the constant of dissociation of the enzyme-inhib-
itor complex into free enzyme and inhibitor) were determined by
the interpretation of Dixon plot, Lineweaver–Burk plot, and its sec-
ondary replots by using initial velocities.
Heparinized fresh venous blood was drawn from healthy volun-
teers in a local blood bank and neutrophils were isolated by the
method of Siddiqui et al. 31 Briefly whole blood was mixed with Fi-
coll paque and left for the sedimentation of unwanted red blood
cells. After 30 min, buffy coat was layered on the Ficoll (3 mL) in
the centrifuge tube and centrifuged for 30 min at 1500 rpm. After
discarding the supernatant, unwanted RBCs was further lysed by
mixing it with hypotonic ammonium chloride solution (0.83%),
centrifuged and the neutrophils with MHS and resuspended at
1 ꢀ 107 cells/mL.
2.2.2. Assay protocol
We used the modified method of Berridge et al.32 Briefly, the
isolated human neutrophils (1 ꢀ 107 cells/mL) were incubated
with the various conc of 200–12.5 lg/mL test compounds for
30 min. WST-1 (0.25 mM) was added and incubated in shaking
water bath at 37 °C. After 3 h incubation, absorbance at 450 nm
was measured with spectraMAX 340 (Molecular Devices, USA)

K. M. Khan et al. / Bioorg. Med. Chem. 17 (2009) 7816–7822
7819
Table 2
micro plate reader. The OD is the mean of the five experimental
In vitro human recombinant phosphodiesterase
derivatives 1–9, and 17–19
1
activity of oxadiazole thiones
replicates. % Cell viability was calculated by using the following
formula:
a
Compound
IC50
(l
M) SEMb
Ki*
a
(l
M) SEMb
% Viability of cells
1
2
3
368 2.2
467 1.1
>1000
360 0.14
150 0.025
—
¼ fðOD test compound ꢀ 100=OD controlÞ ꢁ 100g ꢁ 100:
4
5
6
7
8
9
17
18
19
628 3.7
Inactive
Inactive
494 3.4
Inactive
Inactive
>1000
850 0.02
—
—
360 0.3
—
—
—
3. Results and discussion
A series of 26 1,3,4-oxadiazole-2(3H)-thiones and their sulfur
analogs 1,3,4-thiadiazole-2(3H)-thione were synthesized accord-
ing to the Scheme 1. The structures of all the compounds were de-
duced on the basis of spectroscopic techniques, such as UV, IR,
NMR, and mass spectrometry.
All 1,3,4-oxadiazole-2(3H)-thione, and their sulfur analogs
1,3,4-thiadiazole-2(3H)-thione (Scheme 1), were evaluated for
in vitro snake venom NPP1 inhibitory activity, whereas cysteine
and EDTA were used as positive controls or standards. 5-[4-
(t-Butyldimethylsilyloxy)-phenyl]-1,3,4-thiadiazole-2(3H)-thione
(17) was found to be the most active compound in the series
900 3.5
998 1.2
850 0.8
—
Ki*a = is the mean of five values calculated from Lineweaver–Burk plot, its secondary
replots and Dixon plot.
— = Not tested due to weak enzyme inhibitory activity.
a
IC50 is the 50% inhibitory concentration.
SEM = Standard mean error of 3–5 experiments.
b
(IC50 = 66.47 3.12 lM) against the snake venom phosphodiester-
ase, more potent than the standards. The other compounds 1–5
and 18–20 were also found to be active with IC50 values in the
range of 383–960 lM. Compounds 6–9 exhibited a weak inhibitory
activity whereas no inhibitory potential was displayed by remain-
ing compounds. Results of snake venom NPP1 inhibitory assay are
presented in Table 1.
tween 467–998 lM. However, other compounds showed either
only weak or no activity (Table 2). Only those compounds which
showed good inhibitory potential against NPP1 were subjected to
kinetic analysis. Graphical representations compound 1, as an
example of non-competitive inhibition against the snake venom
Compounds which showed good to moderate inhibitory activity
against the snake venom NPP1 were selected to study against hu-
man NPP1. 5-[4-(t-Butyldimethylsilyloxy)-phenyl]-1,3,4-oxadiaz-
ole-2(3H)-thione (1) demonstrated an excellent activity against
the human NPP1 enzyme (IC50 = 368 2.2 lM). Compounds 2, 4,
7, 18, and 19 were also found to be active with the IC50 values be-
0.09
0.06
0.03
0
Table 1
In vitro snake venom phosphodiesterase 1 activity of oxadiazole thione derivatives 1–
16
a
Compound
IC50
(
l
M) SEMb
Ki*a M) SEMb
(l
1
2
3
4
5
383 0.09
429 0.12
850 0.2
900 0.6
960 0.32
>1000
375 1.2
450 0.8
500 2.5
1400 3.1
1150 3.4
—
-2
-1
0
1
2
3
4
5
6
7
1/S (mM)
6
7
>1000
—
8
9
1000 0.52
>1000
1050 2.8
—
—
—
—
—
—
—
0.016
0.012
0.008
0.004
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
66.47 3.12
390 0.24
660 0.7
839 0.31
>1000
Inactive
Inactive
>1000
Inactive
Inactive
-○- Slope
-•- 1/Vmaxapp
—
100 0.5
350 1.0
525 0.8
1050 0.38
—
—
—
—
—
—
-600 -400 -200
0
200
400
600
Compound 1 (µM)
Ki* = is the mean of five values calculated from Lineweaver–Burk plot, its secondary
replots and Dixon plot.
Figure 1. Steady state inhibition of snake venom NPP1 by compound 1. (A)
Lineweaver–Burk plot in the absence (s) and presence of 150 M; (d) 300 M (h)
and 600 M(j) of compound 1, (B) respective secondary replots of the Lineweaver–
Burk plot, that is, 1/Vmaxapp and slope versus various concentrations of compound 1.
Each point in the graph represents the mean of three experiments.
l
l
— = Not tested due to weak inhibitory activity
l
a
IC50 is the 50% inhibitory concentration.
SEM = Standard mean error of 3–5 experiments.
b

7820
K. M. Khan et al. / Bioorg. Med. Chem. 17 (2009) 7816–7822
0.09
0.09
0.06
0.03
0
0.06
0.03
0
-600 -400 -200
0
200
400
600
-0.2
0
0.2
0.4
1/S (mM)
0.6
0.8
1
Compound 1 (µM)
0.09
0.06
0.03
0
0.09
0.06
0.03
0
-○- Slope
-•- 1/Vmaxapp
-2
-1
0
1
2
3
4
5
6
7
-600
-300
0
300
600
1/S (mM)
Compound 1 (µM)
Figure 2. Steady state inhibition of snake venom phosphodiesterase by compound
1. (A) Dixon plot at four fixed bis-(p-nitrophenyl) phosphate concentrations: (s)
1.5 mM; (d)3.0 mM; (h) 4.5 mM and (j) 6.0 mM, (B) secondary replots of the
Dixon plot: slope versus reciprocal of substrate concentrations. Each point in the
graph represents the mean of three experiments.
Figure 3. Steady state inhibition of human PC-1 by compound 1. (A) Lineweaver–
Burk plot in the absence (s) and presence of 200 M (d), 500 M (h), and 700 M;
(j) of compound 1, (B) respective secondary replots of the Lineweaver–Burk plot,
that is, 1/Vmaxapp and slope versus various concentrations of compound 1. Each
point in the graph represents the mean of three experiments.
l
l
l
and pure human recombinant NPPI enzyme, are shown in Figures
1–5.
383 0.09 lM) may be due to replacement of sulfur by oxygen in
the heterocyclic ring. In other words, hydrogen at C-3 and sulfur
in the ring and t-butyldimethylsilyloxyphenyl at C-5 of ring are
mainly responsible for the activity. Comparison of activities of clo-
3.1. Structure–activity relationship (SAR) studies against snake
venom nucleotide pyrophosphatases/phosphodiesterases
(NPP1)
sely related compounds 5 (IC50 = 960 0.32
lM), 8 (IC50 = 1000),
and 18 (IC50 = 390 0.24 M), clearly indicated the importance of
l
sulfur in the ring, as well as hydrogen at C-3. The complete loss
of inhibitory potential in compounds 22 and 25 may be due to
aforementioned. The weak or lack of inhibitory activity in com-
pounds 4, 9–11, 14, 15, 23, and 26 may be due to absence of sulfur
and unsubstituted position 3, or both. The difference in the activi-
ties of compounds 2, 3, and 20 with IC50 values 429, 850, and
Twenty-six 1,3,4-oxadiazole-2(3H)-thione and 1,3,4-thiadia-
zole-2(3H)-5-thione analogs were screened for their snake venom
NPP1 inhibitory potential. Snake venom is a complex mixture of
enzymes and protein. Many of these proteins are harmless to hu-
mans, but some are toxins. The composition of snake venom differs
from species to species. It was found that the presence of an oxy-
gen or sulfur in five-membered heterocyclic ring, as well as the
substitution of various groups at C-5 play an important role in
the activity against the snake venom NPP1. It was discovered that
the presence of hydrogen at C-3 of the heterocyclic ring contributes
in the inhibitory activity, in addition to the above-mentioned
factor. Similarly the presence of a sulfur atom in the ring also influ-
ences the inhibitory potential. 5-[4-(t-Butyldimethylsilyloxy)-phe-
317 lM, respectively, also indicated that sulfur in ring is responsi-
ble of activity, along with a few other structural features. It infer-
ence was supported by difference in the activities of compounds
6 and 19. Compounds 12, 13, 16, 21, and 24 were found to be com-
pletely inactive due to reasons mentioned above.
3.2. Structure–activity relationship (SAR) studies against
human nucleotide pyrophosphatases/phosphodiesterases
(NPP1)
nyl]-1,3,4-thiadiazole-2(3H)-thione (17), which have
hydrogen at C-3 and sulfur in the ring found to be the most active
member of the series (IC50 = 66.47 3.12 M) against the snake ve-
a free
l
Phosphodiesterases are used to interfere with the prey’s cardiac
system, mainly to lower the blood pressure. Based on the prelimin-
ary activities results against the snake venom nucleotide pyrophos-
phatases/phosphodiesterases (NPP1), we selected compounds 1–9
nom phosphodiesterase, better than the two standards used in the
study. The low activity of the structurally related 5-[4-(t-butyldi-
methylsilyloxy)-phenyl]-1,3,4-oxadiazole-2(3H)-thione (1) (IC50
=

K. M. Khan et al. / Bioorg. Med. Chem. 17 (2009) 7816–7822
7821
in ring is also participating in the activity. The strong activities of
compounds 2, 4, and 7 against human NPP1 as compared to their
activities against the snake venom NPP1, may be due to presence
of oxygen in ring. The low activities of compounds 18, and 19 may
be due to absence of oxygen in the ring.
0.09
0.06
0.03
0
3.3. Evaluation of cytotoxicity
The cytotoxicities of the active compound were evaluated on
human neutrophils by measuring reduction of WST-1 salt. WST-1
tetrazolium salt readily reduced to Formazan dye in the presence
of cellular enzymes of metabolically active neutrophils. Five con-
centrations ranges 200–12.5 lg/mL of compounds 1, 17, and 20
were used to determine the viability of the cells. These compounds
-600 -400 -200
0
200 400 600 800
were found to be largely non-toxic as neutrophils showed over 90%
Compound 1 (µM)
viability up to 200
lg/mL concentration.
3.4. Kinetic studies
0.09
0.06
0.03
0
On the basis of IC50 values, compounds 1–5, 8, 17–20, which
showed inhibitory potential against the snake venom NPP1 and
human NPP1 (compound 1, 2, 4, 7, 18 and 19), were selected for ki-
netic studies.31,32 The Ki values and mode of inhibition are pre-
sented in Tables 1 and 2. The Ki values (inhibitory constants)
were determined directly from the Dixon plots. These values were
also confirmed by plotting the 1/Vmaxapp against different concen-
trations of respective inhibitor. 1/Vmaxapp values were calculated
at each intersection point of lines of every inhibitory concentration
on the Y-axis of the Lineweaver–Burk plot. Secondly the slope of
each line of inhibitory concentration on the Lineweaver–Burk plot
was plotted against inhibitor concentrations. All the compounds
were found to be pure non-competitive type of inhibitors as the
Vmax values decreased without affecting the Km values. Graphical
representation of 5-[4-t-butyldimethylsilyloxy phenyl]-1,3,4-oxa-
diazole-2(3H)-thione (1), as an example of non-competitive
inhibition against snake venom and pure human recombinant phos-
phodiesterase 1 enzyme are presented in Figures 1–5. Compound 1
binds to the enzyme at a site other than the enzymes active site. This
affects the rate of the reaction catalyzed by the enzyme because the
presence of the inhibitor causes a change in the structure and shape
of the enzyme. This change in shape means the enzyme is no longer
able to bind with the structure correctly.
-0.2
0
0.2
0.4
1/S (mM)
0.6
0.8
1
Figure 4. Steady state inhibition of human PC-1 by compound 1. (A) Dixon plot at
four fixed p-nitrophenyl phosphonate concentrations: (s) 0.4 mM; (d) 0.6 mM; (h)
0.8 mM and (j) 1.0 mM, (B) secondary replots of the Dixon plot: slope versus
reciprocal of substrate concentrations. Each point in the graph represents the mean
of three experiments.
120
100
80
Acknowledgements
60
We are grateful to Professor J. W. Goding, Monash University,
Melbourne, Australia, for a generous gift of water-soluble human
recombinant NPP1 (catalytic extra cellular domain) and also for
reviewing this manuscript. We also acknowledge the help of the
Fatmid Foundation for the blood samples.
40
20
17
1
20
0
0
50
100
150
200
250
conc (ug/mL)
References and notes
Figure 5. Graph shows
% cell viability of freshly isolated human neutrophils
1. Razzell, W. E. Methods Enzymol. 1963, 6, 236.
2. Bollen, M.; Gijsbers, R.; Ceulemans, H.; Stalmans, W.; Stefan, C. Crit. Rev.
Biochem. Mol. Biol. 2000, 35, 393.
(1 ꢀ 107 cells/mL) in the presence of various concentrations (200–12.5
compound 1, 17, and 20.
lg/mL) of
3. Goding, J. W.; Grobben, B.; Slegers, H. Biochim. Biophys. Acta 2003, 1638, 1.
4. Johnson, K.; Hashimoto, S.; Lotz, M.; Pritzker, K.; Goding, J.; Terkeltaub, R.
Arthritis Rheum. 2001, 44, 1071.
5. Okawa, A.; Nakamura, I.; Goto, S.; Moriya, H.; Nakamura, Y.; Ikegawa, S. Nat.
Genet. 1998, 19, 271.
6. Sali, A.; Favaloro, J. M.; Terkeltaub, R.; Goding, J. W. In Proceedings of the Second
International Workshop on Ecto-ATPases and Related Ectonucleotidases;
Diepenbeek, Belgium; Shaker Publishing BV: Maastricht, The Netherlands,
1999.
7. Ahmed, V. U.; Abbasi, M. A.; Hussain, H.; Akhtar, M.; Farooq, U.; Fatima, N.;
Choudhary, M. I. Phytochemistry 2003, 63, 217.
and 17–19 for screening against the human NPP1. Compounds
(inhibitors) are potential candidates for the therapy of snake bites.
Results of inhibitory assay are presented in Table 2. The two factors
emerge out of the experimental results, that is, suitable substitution
at C-5, and hydrogen at C-3 are responsible for human NPP1 inhibi-
tion. 5-[4-(t-Butyldimethylsilyloxy)-phenyl]-1,3,4-oxadiazole-2(3H)-
thione (1) (IC50 = 368 2.2
lM) was found to be most active
8. Iqbal, K.; Anis, I.; Muhktar, N.; Malik, A.; Fatima, N.; Choudhary, M. I.
Heterocycles 2003, 60, 151.
compound against the human NPP1, clearly indicating that oxygen

7822
K. M. Khan et al. / Bioorg. Med. Chem. 17 (2009) 7816–7822
9. Ahmed, V. U.; Abbasi, M. A.; Zubair, M.; Fatima, N.; Farooq, U.; Choudhary, M. I.
Helv. Chim. Acta 2004, 87, 67.
10. Choudhary, M. I.; Fatima, N.; Abbasi, A.; Jalil, S.; Ahmad, V. U.; Rahman, A. U.
Bioorg. Med. Chem. 2004, 12, 5793.
11. Tahir, M. M.; Brezina, D.; Fatima, N.; Choudhary, M. I.; Rahman, A. U. J. Inorg.
Biochem. 2004, 2, 589.
12. Abbasi, M. A.; Ahmed, V. U.; Zubair, M.; Fatima, N.; Farooq, U.; Hussain, S.;
Lodhi, M. A.; Choudhary, M. I. Planta Med. 2004, 70, 1189.
13. Rahman, A. U.; Naz, H.; Fadimatou; Makhmoor, T.; Yasin, A.; Fatima, N.; Ngounou,
F. N.; Kimbu, S. F.; Sondengam, B. L.; Choudhary, M. I. J. Nat. Prod. 2005, 68, 189.
14. Ahmed, V. U.; Zubair, M.; Abbasi, M. A.; Kousar, F.; Fatima, N.; Choudhary, M. I.
Z. Naturforsch. 2005, 60b, 1101.
15. Choudhary, M. I.; Fatima, N.; Khan, K. M.; Jalil, S.; Iqbal, S.; Rahman, A. U. Bioorg.
Med. Chem. 2006, 14, 8066.
16. Mostafa, M.; Nahar, N.; Mosihuzzaman, M.; Sokeng, S.; Fatima, N.; Rahman, A.
U.; Choudhary, M. I. Nat. Prod. Res. 2006, 20, 686.
21. (a) Almajan, G. L.; Barbuceanu, S. F. A.; Scozzafava, A.; Supuran, C. T. J. Enz.
Inhib. Med. Chem. 2008, 23, 101; (b) Mohammed, K.; Hamid, A.; Atef, A.; Hafez,
A.; Nawal, A.; El-Koussi; Nadia, M.; Mahfouz; Innocenti, A.; Supuran, C. T. Bioor.
Med. Chem. 2007, 15, 6975; (c) Supuran, C. T.; Scozzafava, A.; Saramet, I.;
Banciu, M. D. J. Enz. Inhib. 1998, 13, 177.
22. Amtul, Z.; Rasheed, M.; Choudhary, M. I.; Rosanna, S.; Khan, K. M.; Atta-ur-
Rahman Biochem. Biophys. Res. Commun. 2004, 319, 1053.
23. Francoeur, A. M.; Assalian, A. Biochemica 1996, 3, 19.
24. Mamillapalli, R.; Haimovitz, R.; Ohad, M.; Shinitzky, M. FEBS Lett. 1998, 436,
256.
25. Razzell, W. E.; Khorana, H. G. J. Biol. Chem. 1959, 234, 2105.
26. Nakabayashi, T.; Matsuoka, Y.; Ikezawa, H.; Kimura, Y. Int. J. Biochem. 1994, 26,
171.
27. López-Gómez, J.; Costas, M. J.; Ribeiro, J. M.; Fernández, A.; Romero, A.; Ávalos,
M.; Cameselle, J. C. FEBS Lett. 1998, 421, 77.
28. Kelly, S. J.; Dardinger, D. E.; Butler, L. G. Biochemistry 1975, 14, 4983.
29. Segal, I. Enzyme Kinetics, 2nd ed.; John Wiley and Sons: New York, USA, 1993.
pp 125–143.
30. Dixon, M. Biochem. J. 1953, 55, 170.
31. Siddiqui, R. A.; English, D.; Cui, Y.; Martin, M. I.; Wentlands, J.; Akard, L. J.
Leukocytes Biol. 1995, 58, 189.
17. Abdel-ghani, E. Indian J. Heterocycl. Chem. 1997, 7, 123.
18. Edwards, L. H.; U.S. 4, 259,104; Chem. Abst. 1981, 95, 25078.
19. Soni, N.; Barthwal, J. P.; Saxena, A. K.; Bhargava, K. P. J. Heterocycl. Chem. 1982,
19, 29.
20. Boschelli, D. H.; Connor, D. T.; Bornemeier, D. A.; Dyer, R. D.; Kennedy, J. A.; Kuipers, J.
P.; Okonkwo, G. C.; Schrier, D. J.; Wright, C. D. J. Med. Chem. 1993, 36, 1802.
32. Berridge, M. V.; Tan, A. S.; McCOY, K. D.; Wang, R. Biochemica 1996, 4, 14.