Synthesis, thymidine phosphorylase inhibition and molecular modeling studies of 1,3,4-oxadiazole-2-thione derivatives

Article abstract of DOI:10.1016/j.bioorg.2015.04.003

Thymidine phosphorylase (TP) inhibitors have attracted great attention due to their ability to suppress the tumors formation. In our ongoing research, a series of 1,3,4-oxadiazole-2-thione (1-12) has been synthesized under simple reaction conditions in good to excellent yields (86-98%) and their TP inhibition potential has also been evaluated. The majority of synthesized compounds showed moderate thymidine phosphorylase inhibitory activity with IC50 values ranging from 38.24 ± 1.28 to 258.43 ± 0.43 μM, and 7-deazaxanthine (7DX) was used as a reference compound (IC50 38.68 ± 4.42). The TP activity was very much dependent on the C-5 substituents; among this series the compound 6 bearing 4-hydroxyphenyl group was found to be the most active with IC50 38.24 ± 1.28 μM. Molecular docking studies revealed their binding mode.

Full text of DOI:10.1016/j.bioorg.2015.04.003

Accepted Manuscript
Synthesis, thymidine phosphorylase inhibition and molecular modeling studies
of 1,3,4-oxadiazole-2-thione derivatives
Sohail Anjum Shahzad, Muhammad Yar, Marek Bajda, Lubna Shahzadi,
Zulfiqar Ali Khan, Syed Ali Raza Naqvi, Sadaf Mutahir, Nasir Mahmood,
Khalid Mohammed Khan
PII:
S0045-2068(15)00030-9
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.04.003
YBIOO 1802
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
24 December 2014
Please cite this article as: S.A. Shahzad, M. Yar, M. Bajda, L. Shahzadi, Z.A. Khan, S.A.R. Naqvi, S. Mutahir, N.
Mahmood, K.M. Khan, Synthesis, thymidine phosphorylase inhibition and molecular modeling studies of 1,3,4-
oxadiazole-2-thione derivatives, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.04.003
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Synthesis, thymidine phosphorylase inhibition and molecular modeling
studies of 1,3,4-oxadiazole-2-thione derivatives
a,*
b,*
c
b
Sohail Anjum Shahzad , Muhammad Yar , Marek Bajda , Lubna Shahzadi , Zulfiqar Ali
d
d
e
f
g
Khan , Syed Ali Raza Naqvi , Sadaf Mutahir , Nasir Mahmood , Khalid Mohammed Khan
a
b
Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
Interdisciplinary Research Centre in Biomedical Materials, COMSATS Institute of Information Technology,
Lahore, 54000, Pakistan
c
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical College,
Medyczna 9, 30-688 Cracow, Poland
d
Department of Chemistry, Government College University, Faisalabad 38000, Pakistan
e
Department of Chemistry, GC University, Lahore 54000, Pakistan
f
Department of Allied Health Sciences and Chemical Pathology; Department of Human Genetics and Molecular
Biology, University of Health Sciences, Lahore, Pakistan
gHussain Ibrahim Jamal Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi,
Karachi, 75270, Pakistan
*Corresponding authors: E-mail addresses: sashahzad@ciit.net.pk (S. A. Shahzad); drmyar@ciitlahore.edu.pk (M. Yar).
Abstract: Thymidine phosphorylase (TP) inhibitors have attracted great attention due to their
ability to suppress the tumors formation. In our ongoing research, a series of 1,3,4-oxadiazole-2-
thione (1-12) has been synthesized under simple reaction conditions in good to excellent yields
(86-98%) and their TP inhibition potential has also been evaluated. The majority of synthesized
compounds showed moderate thymidine phosphorylase inhibitory activity with IC50 values
ranging from 38.24 + 1.28 to 258.43 ± 0.43 μM , and 7-deazaxanthine (7DX) was used as a
reference compound (IC50 38.68 ± 4.42). The TP activity was very much dependent on the C-5
substituents; among this series the compound 6 bearing 4-hydroxyphenyl group was found to be
the most active with IC50 38.24 + 1.28 μM . Molecular docking studies revealed their binding
mode.
Keywords: Thymidine phosphorylase, oxadiazole-2-thione, molecular modeling, anti-cancer

Introduction
Although angiogenesis is a very critical process in a repairing of tissues and organs it is a highly
undesirable phenomenon during the tumor formation. It is believed that, tumor growth could be
1,2
blocked by stopping angiogenesis. Regarding the mechanism of the action of thymidine
phosphorylase (TP), it triggers the reversible phosphorolysis of thymidine to produce thymine
3,4
and 2� -deoxy-D-ribose 1-phosphate. Subsequently, 2� -deoxy-D-ribose 1-phosphate shows
dephosphorylation reaction and as a result 2� -deoxy-D-ribose is produced. It is possible that the
2
� -deoxy-D-ribose stimulates the production of vascular endothelial growth factor (VEGF),
which initiates a number of processes, for example, invites endothelial cells for secretion of
matrix metalloproteinases, proliferation, and also migration to tumor tissue. All these actions
5
help in the formation of new blood vessels, which could cause cancer metastasis. This is the
reason why anti-angiogenic substances are highly desirable.
The 2� -deoxy-D-ribose is considered a valuable target to suppress the tumor growth, and TP
5,6
inhibitors are able to reduce the production of 2� -deoxy-D-ribose. In such scenario, one can
easily understand the advantages of TP inhibitors in the control of cancer and that is the reason
5,7,8
recently a number of efforts have been reported on the development of TP inhibitors.
One of
the leading candidate in this field is the 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil
hydrochloride (TPI). It is a pyrimidine based compound and the most active human TP inhibitor,
9
-12
whereas, 7-deazaxanthine (7DX) is the first known TP inhibitor as shown in Figure 1.
O N
2
O
OMe
O
N
4
N
3
N
N
NH
Cl
NH
H
NH
2
5
S
O
N
S
N
H
O
N
O
1
R
N
H
O
H
Me
H2N
This work
Our previous work
TP inhibition: IC50 14.40 ± 2.45 μM
Cl
TPI
7DX
Figure 1. Chemical structure of known TP inhibitors; TPI, 7DX, our previously reported potent
TP inhibitor and newly proposed TP inhibitors
1
3-16
Oxadiazole motif is well known due to its huge importance in medicinal chemistry.
We
have recently identified and reported the TP inhibitory potential of 1,3,4-oxadiazole-2-thiones

17
Mannich base derivatives, derived from the compounds listed in the current paper. There it was
observed that the oxadiazole motif played a crucial role in the inhibitory process along with the
contributions from the various substituents around the molecules. In that effort we synthesized
the Mannich base derivatives of 1,3,4-oxadiazole-2-thiones, which indeed required an extra
1
7-
synthetic step and additional chemicals. As part of our ongoing medicinal chemistry interests,
2
3
24,25
in current research, we have synthesized the simple 1,3,4-oxadiazole-2-thione derivatives
and analyzed their TP inhibition potential. We have also performed molecular modeling studies
for all the synthesized compounds to rationalize their binding modes with the TP.
1
. Results and Discussion
1
.1. Chemistry
1
7
In our current research, a series of 1,3,4-oxadiazoline-2-thione derivatives 1-12 bearing
different level of C-5 substituents was prepared by condensing respective hydrazides with carbon
disulfide in the presence of potassium hydroxide and ethanol on alumina as a solid support as
24
shown in Scheme 1. Neutral alumina oxide was used as solid support which does not affect the
25
yield and speed of reaction The reaction proceeded and completed efficiently under microwave
irradiation within 7 minutes. The pure solid products were isolated as precipitates, which were
washed with 50% aqueous ethanol and needed no further chromatographic techniques for
purification. Also, it can be seen from percentage yield that different substituents influence the
conversion rate and shorten the reaction time (please see Table 1). All final compounds were
structurally characterized by IR, NMR, EIMS and elemental analysis.
CS2
O
N
NH
KOH / EtOH
R
NHNH2
MW
R
O
S
1-12
86-98%
Scheme 1: Synthetic protocol of 1,3,4-oxadiazole-2-thione derivatives 1-12.

1
.2. Thymidine phosphorylase inhibition activities
In this study, to develop and understand structure–activity relationship (SAR) twelve derivatives
of oxadiazole-2-thione were synthesized bearing different degree of aryl substituents at C-5
position. Among the tested compounds, compound 6 having 4-hydroxyphenyl at C-5 position
was found to be the most active with an IC50 value 38.24 ± 1.28 μM (entry 6, Table 1), which was
decreased to IC50 68.37±1.23 μM in the case of its analogue 2 having 2-hydroxyl group at C-5
(
entry 2 and 6, Table 1). Similarly, compound 7, having methoxy group at para position showed
IC50 72.43 ± 0.48 μM while its trimethoxy analogue 8 exhibited slightly enhanced enzyme
inhibition activity (IC50 63.43 ± 0.92), this is may be due to combined greater inductive effect by
three methoxy substituents.
Again very interesting results have been seen on the basis of position of substituents. For
example, compound 10 and 11, they both contain chloro substituents in phenyl ring but IC50
value of compound 11 having chloro group at para position was 63.97 ± 0.73 μM , and the IC50
value dramatically increased to 258.43 ± 0.43 μM in case of compound 10 having chloro group at
meta position. The results also revealed the electron-withdrawing polar group like nitro
completely hinders the enzyme inhibition activity and resulted as precipitates.
Table 1: Showing percentage yield and thymidine phosphorylase inhibition activity of
synthesized oxadiazole derivatives
N
NH
5
S
O
R
a
Comp.
R
Yield %
IC ( μM )
5
0
1
90
50.00 + 0.78

HO
2
3
4
89
86
96
68.37 ± 1.23
74.34 ± 0.73
91.78 ± 0.97
H N
2
CH3
5
6
7
CH3
98
91
94
-
OH
38.24 + 1.28
72.43 + 0.48
OCH3
OCH3
OCH
OCH3
NO2
8
9
3
91
63.43 ± 0.92
93
95
PPT
Cl
1
0
258.43 ± 0.43
63.97 ± 0.73
1
1
2
Cl
Br
92
93
1
63.97 ± 0.73
7
-Deazaxanthine
Standard
38.68± 4.42
a
Enzyme inhibition IC50 values are means of three independent experiments (mean ± SEM, n = 3)
1
.3. Molecular modeling studies
Analysis of the binding mode for the novel derivatives 1-12 was performed according to the
1
7
previously described method. Thymidine phophorylase (TP) from Escherichia coli of high
resolution (1.50 A; PDB code: 4EAD) was used for docking studies upon initial preparation.

This enzyme structure represents the most closed form, characteristic for the most complexes
with inhibitors, bound with 3� -azido-2� -fluoro-dideoxyuridine (ONP). Docking validation was
based on two reference compounds from crystal structures – ONP and TPI (5-chloro-6-[1-(2-
iminopyrrolidinyl)methyl]-uracil) and it was shown that docking runs were able to reproduce
original arrangement of the ligand with low rmsd (root mean square deviation) value below 1.
Further, 7-deazaxanthine (7DX) - assay reference compound – was docked to TP. It was
observed that NH and CO groups formed hydrogen bonds with Lys190, Ser186 and Arg171
while the whole molecule created π- πstacking with Tyr168 residue.
N NH
O
N N
O
R
S
R
SH
tautomer II
tautomer I
N N
O
N N
+ H
O+
3
+
H O
2
R
SH
R
S
O
unionized II
ionized II
Figure 2. The novel compounds can occur in tautomeric forms (top). The ionization of lactim
form (bottom).
Initial calculations revealed that all compounds 1-12 occurred in physiological conditions in
ionized form II (Figure 2) and therefore such form was docked into thymidine phosphorylase. It
was noted that the binding mode of novel compounds was highly dependent on the substituents
in the phenyl ring (Figure 3). In case of the most active compound 6, phenyl ring created CH-π
interactions with Phe210. The hydroxyl group in position 4 formed hydrogen bond with Arg171.
The oxadiazole moiety was engaged in two hydrogen bonds. The oxygen atom interacted with
water molecule while nitrogen atom in position 4 created H-bond with hydroxyl group of
Tyr168. The orientation of the inactive compound 5 was reversed. The phenyl ring created π- π
stacking with Tyr168 while the oxygen atom from oxadiazole ring formed hydrogen bond with
Ser186. Comparing both compounds 5 and 6, it’s worth to note that inhibitor 6 provided more
interactions of greater importance within active center than inactive derivative 5. This could

explain the observed differences in the activity. Moreover, other derivatives 1-4 and 7-12
presented variable binding mode. It seems that even the most potent compound 6 is not specific
inhibitor of thymidine phosphorylase but it remains a good starting point for further optimization
to obtain the highly active derivatives.
Figure 3. The binding mode of the compounds 6 (left) and 5 (right)
2
. Conclusion
We synthesized 5-substituted-1,3,4-oxadiazole-2-thione derivatives via a procedure which is
much more efficient and has the ability to tolerate a variety of substituents. Ease of separation of
pure product and high yields are unique features of this used method. All the synthesized
compounds were tested for their TP inhibition, most of the compounds exhibited moderate to
good inhibition activity. Compound 6 has shown best inhibition results (IC50 38.24 + 1.28 μM )
and molecular docking studies explained its binding pattern with the TP. We believe these TP
inhibitors will find wide attraction in the field of anti-cancer drug development research.
3
. Experimental
3
.1. General Methods
The ultraviolet spectra were measured in chloroform on a Lambda 5 UV/Vis.
spectrophotometer (Perkin-Elmer). IR spectra (KBr discs) were recorded on a Bruker FT-IR
IFS48 spectrophotometer. EI mass spectra data were recorded with various MAT 711 (70eV)
1
spectrophotometers and data are tabulated as m/z. H-NMR spectra were recorded in
CD
COCD , CD OD and DMSO-d using Bruker AC (300 MHz and 400 MHz, 500 MHz)
3 3 3 6
spectrometers, respectively. Splitting patterns were as follows s (singlet), d (doublet), dt (doublet

of triplet), dd (double doublets), t (triplet), and m (multiplet). Chemical shifts are reported in δ
ppm) and coupling constants are given in Hz. The progress of all reactions was monitored by
(
TLC, which was performed on 2.0 X 5.0 cm aluminum sheets precoated with silica gel 60F254 to
a thickness of 0.25 mm (Merck). The chromatograms were visualized under ultraviolet light
(
254-366 nm) or iodine vapors. All the reagents were commercially available (Flulka, Aldrich,
and Wako).
3
.2. General procedure for the synthesis of 5-substituted -1,3,4-oxadiazole-2-thione (1-12)
A mixture of respective hydrazide (10 mmol), potassium hydroxide (0.56 g, 10 mmol) and
alumina were finely ground in a glove box with a mortar and pestle. Then carbon disulfide (1.2
mL, 20 mmol) was added to this mixture in a pyrex glass vial, which was placed in a
screw‐capped thick‐walled Teflon® vessel. Microwave‐irradiation (MW domestic type oven 900
W with a frequency 2450 MHz, Dawlance, Pakistan) was applied for 7 minutes. After the
completion of reaction (TLC analysis), ethanol was added into reaction mixture and filtered.
Filtrate was evaporated; distilled water was added to semi‐solid material and acidified with
hydrochloric acid to pH = 4. Precipitates so obtained were filtered and dried to afford off white
solid compound 2a‐r and then recrystallized from ethanol:water (50:50) mixture (Table 1).
5
-phenyl-1,3,4-oxadiazole-2(3H)-thione (1)
Yield: 1.43 g, (80%). R
f
= 0.61 (Ethyl acetate/hexane = 1:1).FTIR (KBr) νmax: 3431 (NH), 1641
+
(
C=N), 1328 (C=S), 1020 (C-O). EI-MS, (m/z %): 178 (M , 25), 118 (100), 105 (25), 103 (41),
1
7
7 (81), 51 (82). H NMR (400 MHz, CDCl
3
, drops of CD
3
OD): 14.70 (bs, 1H, NH), 7.90 (d,
2
H, δ, J = 7.3 Hz), 7.47-7.59 (m, 3H). Anal. calcd. for C
8
H N OS: C, 53.92; H, 3.39; N, 15.72;
6 2
found: C, 53.86; H, 3.41; N, 15.65.
5-(2′‐Hydroxyphenyl‐1,3,4‐oxadiazole‐2(3H)‐thione (2)
°
Yield: 1.33 g (89%). M.p.: 200‐201 C. R
f
= 0.67 (Ethyl acetate:hexane = 1:1). FT‐IR (KBr,
+
-
1
ν
max, cm ): 3364 (NH), 1618 (C=N), 1309 (C=S), 1051 (C‐O‐C). MS (m/z, %): 194 (M , 100),
1
1
1
34 (1), 121 (99), 119 (16), 93 (7), 65 (8). H NMR (400 MHz, Acetone‐d
6
, δ, ppm): 14.33 (bs,
H, NH), 8.84 (br s, 1H, OH), 7.61 (m, 1H, H‐4´), 7.08 (d, 1H, H‐3´), 7.07 (dd, 1H, J = 8.1 Hz,
J = 6.4 Hz, H‐5´), 7.01 (1H, J = 7.7 Hz, J = 5.0, J = 1.5 Hz, H‐6´). Anal. calcd. for
S: C, 49.47; H, 3.11; N, 14.42. Found: C, 49.49; H, 3.15; N, 14.46%.
8 6 2 2
C H N O

5-(2′‐Amino phenyl)‐1,3,4‐oxadiazole‐2(3H)‐thione (3):
°
Yield: 1.31 g (86 %). M.p.: 156‐157 C. R
f
= 0.68 (Ethyl acetate:hexane = 1:1). FT‐IR (KBr,
1
+
-
max, cm ): 1054 (C‐O‐C), 1616 (C=N), 3585 (NH). MS (m/z, %): 193 (M , 17), 177 (100), 133
ν
1
(
47), 120 (15), 118 (33), 92 (15), 76 (10). H NMR (500 MHz, DMSO‐d
6
, δ, ppm): 14.63 (bs,
), 7.61 (dd, 1H, J = 7.2 Hz,
.7 Hz, H‐3´), 7.55 (dd , 1H, J = 7.0 Hz, 1.9 Hz, H‐4´), 7.48 (t, 1H, J = 7.0 Hz, H‐5´). Anal.
OS: C, 49.73; H, 3.65; N, 21.75. Found: C, 49.71; H, 3.63; N, 21.74%.
1
1
H, NH), 7.89 (dd, 1H, J = 7.0 Hz, 1.5 Hz, H‐6´), 7.88 (bs, 2H, NH
2
8 7 3
calcd. for C H N
5-(3′‐Methylphenyl‐1,3,4‐oxadiazole‐2(3H)‐thione (4):
Yield: 1.44 g (96%). M.p.: 148‐149 °C. R
f
= 0.69 (Ethyl acetate:hexane = 1:2). FT‐IR (KBr, νmax,
1
+
-
cm ): 3400 (NH), 1635 (C=N), 1319 (C=S), 1022 (C‐O). MS (m/z, %): 192 (M , 49), 132 (100),
1
1
16 (10), 104 (17), 91 (59), 77 (17), 65 (34), 63 (20), 51 (24). H NMR (500 MHz, CDCl
3
, drops
of CD
bs, 2H, H‐ 3´/4′), 2.4 (s, CH
C, 56.28; H, 4.17; N, 14.62%.
3
OD, δ, ppm): 14.45 (bs, 1H, NH), 7.71 (s, 1H, H‐2′), 7.67 (d, 1H, J = 6.1 Hz, H‐2′), 7.39
(
3
). Anal. calcd. for C OS: C, 56.23; H, 4.19; N, 14.57. Found:
9 8 2
H N
5-(4′‐Methylphenyl‐1,3,4‐oxadiazole‐2(3H)‐thione (5):
Yield: 1.47 g (98%). M.p.: 159‐160 °C. R
f
= 0.69 (Ethyl acetate:hexane = 1:2). FT‐IR (KBr, νmax,
1
+
-
cm ): 3409 (NH), 1636 (C=N), 1333 (C=S), 1016 (C‐O). MS (m/z, %): 192 (M , 52), 132 (100),
1
1
19 (20), 117 (10), 104 (12), 102 (2), 91 (45), 65 (16). H NMR (400 MHz, CDCl
3
, drops of
CD
3
OD, δ, ppm): 14.53 (bs, 1H, NH), 7.74 (d, 2H, J =8.1 Hz H‐2´/6´), 7.30 (d, 2H, J = 8.1 Hz,
). Anal. calcd. for C OS: C, 56.23; H, 4.19; N, 14.57. Found: C,
6.28; H, 4.14; N, 14.52%.
H‐3´/5´), 2.38 (s, 3H, CH
3
9 8 2
H N
5
5(4′‐Hydroxyphenyl)‐1,3,4‐oxadiazole‐2(3H)‐thione (6):
1
Yield: 1.36 g (91%). M.p.: 233‐235 °C. H NMR (400 MHz, CDCl , drops of CD OD, δ, ppm):
3
3
1
1.4 (s, 1H, SH), 7.18 (d, 2H, J =8.46 Hz, Ar-H), 7.08 (d, 2H, J = 8.46 Hz, Ar-H), 5.12 (s, 1H,
OH). Anal. calcd. for C S: C, 49.47; H, 3.11; N, 14.42. Found: C, 49.52; H, 3.18; N,
8
6 2 2
H N O
14.47%.
5-(4′‐Methoxyphenyl)‐1,3,4‐oxadiazole‐2(3H)‐thione (7):

Yield: 1.41 g (94%). M.p.: 190‐191 °C. R
f
= 0.65 (Ethyl acetate:hexane = 1:1). FT‐IR (KBr, νmax,
1
+
-
cm ): 3399 (NH), 1659 (C=N), 1333 (C=S), 1019 (C‐O). MS (m/z, %): 208 (M , 100), 148 (54),
1
1
35 (12), 133 (88), 107 (3), 105 (20), 92 (10), 77 (13), 64 (14), 51 (18). H NMR (500 MHz,
CDCl
3
, drops of CD
H, J =8.8 Hz, H‐3´/5´), 3.61 (s, 3H, OCH
3.45. Found: C, 51.96; H, 3.83; N, 13.49%.
3
OD, δ, ppm):14.23 (br s, 1H, NH), 7.84(d, 2H, J = 8.8 Hz, H‐2´/6´), 7.05 (d,
2
1
3
). Anal. calcd. for C S: C, 51.91; H, 3.87; N,
9 8 2 2
H N O
5-(3′,4′,5′‐Trimethoxyphenyl)‐1,3,4‐oxadiazole‐2(3H)‐thione (8):
Yield: 1.36 g (91%). M.p.: 175‐176 °C. R = 0.59 (Ethyl acetate:hexane = 1:1). FT‐IR (KBr, νmax,
f
1
-
cm ): 3171 (NH), 1579 (C=N), 1331 (C=S), 1041 (C‐O‐C). MS (m/z, %): 268 (100), 208 (32),
1
1
93 (70), 178 (12), 167 (6), 152 (7), 135 (13). H NMR (300 MHz, DMSO‐d
6
, δ, ppm): 14.81 (br
‐3´/5´), 3.72 (s, 3H, OCH ‐4´). Anal. calcd.
S: C, 49.24; H, 4.51; N, 10.44. Found: C, 49.28; H, 4.56; N, 10.47%.
s, 1H, NH), 7.09 (s, 2H, H‐2´,6´), 3.84 (s, 6H, OCH
3
3
for C11
H
12
N
2
O
4
5-(4′‐Nitrophenyl)‐1,3,4‐oxadiazole‐2(3H)‐thione (9):
Yield: 1.39 g (93%). M.p.: >250 °C (Decompose). R
f
= 0.64 (Ethyl acetate:hexane = 1:1). FT‐IR
1
+
-
KBr, νmax, cm ): 3366 (NH), 1632 (C=N), 1328 (C=S), 1086 (C‐O‐C). MS (m/z, %): 223 (M ,
(
1
7
9), 163 (100), 150 (4), 133 (21), 117 (28), 105 (4), 104 (12), 102 (9), 76 (34). H NMR (400
, drops of CD OD, δ, ppm): 14.73 (bs, 1H, NH), 8.40 (d, 2H, J = 8.7 Hz, H‐3′,5′),
.17 (d, 2H, J = 8.7 Hz, H‐ 2′,6′). Anal. calcd. for C S: C, 43.05; H, 2.26; N,
MHz, CDCl
3
3
8
1
8 5 3 3
H N O
8.83.Found: C, 43.01; H, 2.22; N, 18.86%.
5-(3′‐Chlorophenyl‐1,3,4‐oxadiazole‐2(3H)‐thione (10):
Yield: 1.42 g (95%). M.p.: 178‐179 °C. R = 0.69 (Ethyl acetate:hexane = 1:1). FT‐IR (KBr, νmax,
f
1
+
+
-
cm ): 1063 (C‐O‐C), 1608 (C=N), 3594 (NH). MS (m/z, %): 214 (M , 48), 212 (M , 100), 179
1
(
3), 155 (6), 154 (37), 153 (11), 152 (100), 141 (5), 139 (17), 137 (12), 117 (7), 102 (8), 76 (7).
H‐NMR (500 MHz, DMSO‐d
.8 Hz, H‐ 2´), 7.70 (dd, 1H, J =7.9 Hz, J = 1.2 Hz, H‐4´), 7.60 (t, 1H, J = 7.9 Hz, J = 7.9 Hz,
H‐3´). Anal. calcd. for C ClN OS: C, 45.18; H, 2.37; N, 13.17. Found: C, 45.23; H, 2.42; N,
6
): 14.49 (bs, 1H, NH), 7.85 (d, 1H, J = 1.6 Hz, H‐6´), 7.83 (d, J =
7
8
H
5
2
13.15%.
5-(4′‐Chlorophenyl‐1,3,4‐oxadiazole‐2(3H)‐thione (11):

Yield: 1.38 g (92%). M.p.: 173‐174 °C. R = 0.69 (Ethyl acetate:hexane = 1:1). FT‐IR (KBr, νmax,
f
1
+
+
-
cm ): 1023 (C‐O‐C), 1669 (C=N), 3348 (NH). MS (m/z, %): 214 (M ,35), 212 (M , 100), 179
1
(
3),154 (30), 152 (82), 141 (4), 139 (14), 137 (14), 117 (5), 102 (8), 76 (7). H NMR (500 MHz,
DMSO‐d
6
, δ, ppm): 14.52 (bs, 1H, NH), 7.88 (d, 2H, J = 8.6 Hz, H‐2´/6´), 7.65 (d, 2H, J = 8.6
ClN OS: C, 45.18; H, 2.37; N, 13.17. Found: C, 45.15; H,
Hz, H‐3´/5´). Anal. calcd. for C
8
H
5
2
2
.39; N, 13.14%.
5-(4′‐Bromophenyl‐1,3,4‐oxadiazole‐2(3H)‐thione (12):
Yield: 1.39 g (93%). M.p.: 230‐231 °C. R = 0.66 (Ethyl acetate:hexane = 1:2). FT‐IR (KBr,
f
1
+
+
-
max, cm ): 1073 (C‐O‐C), 1633 (C=N), 3280 (NH). MS (m/z, %): 258 (M , 60), 256 (M , 59),
ν
198 (47), 196 (46), 185 (9), 184 (69), 183 (15), 181 (6), 157 (7), 155 (8), 117 (6), 76 (7), 50 (8).
1
H NMR (500 MHz, CDCl
3
, drops of CD
3
OD, δ, ppm): 14.59 (bs, 1H, NH), 7.8 (d, 2H, J = 8.4
BrN OS: C, 34.37; H, 1.96; N,
Hz, H‐2′/6′), 7.7 (d, 2H, J = 8.4 Hz, H‐3′/5′). Anal. calcd. for C
8
H
5
2
1
0.90. Found: C, 34.32; H, 1.98; N, 10.96%.
3
.3. Docking studies
2
9
Corina online tool was applied to creating three-dimensional structure of compounds.
3
0
Protonation states were predicted by Marvin online tool Gasteiger-Marsili charges were
3
1
assigned by Sybyl-X 1.1 following check of atom types and protonation states of the ligands.
Finally, analyzed structures were saved in the mol2 format.
Escherichia coli TP from 4EAD crystal structure was prepared in two steps. Initially, sulfate
ion was replaced by phosphate in its dihydrogen form, the N- and C-terminal amino acids were
set as charged and hydrogen atoms were added to the protein, water and ligands using Sybyl-X
1
.1. Then, all histidine residues were protonated at Ne, ligand molecules except phosphate
removed, and binding site defined as all amino acid residues within 10 Å from ONP using
3
2
Hermes 1.5 The presence of water molecules within 5 Å from ONP was also taken into
account. They were set as toggle.
3
3
Docking was performed using Gold 5.1 program. A standard set of genetic algorithm with
population size 100 and number of operations 100 000 was applied. As a result, 20 poses for
each ligand were obtained and sorted according to GoldScore values. Results were visualized by
3
4
PyMOL.

3
.4. Procedure for Thymidine Phosphorylase Inhibition
TP/PD-ECGF (E. coli TP (Sigma T6632) activity was determined by measuring the absorbance
35
at 290 nm spectrophotometrically. The original method reported by Krenitsky was modified.
Briefly, a total reaction mixture of 200 µl contained 145 µl of potassium phosphate buffer (pH
7
.4), 30 µl of enzyme (E. coli TP (Sigma T6632) at concentration 0.05 and 0.002 U, respectively,
were incubated with 5 µl of test materials for 10 min at 25 °C in microplate reader. After
incubation, pre read at 290 nm was taken to deduce the absorbance of substrate molecules.
Substrate (20 µl, 1.5 mM) was dissolved in potassium phosphate buffer and was immediately
added to plate and continuously read after 10, 20, and 30 min in micro-plate reader. All assays
were performed in triplicate. The plate reader used was SpectraMax Plus 384 while 96-wells
plate was used and the value of blank was subtracted from experimental wells to eliminate the
back ground absorbance.
Acknowledgments
The authors are grateful to the Higher Education Commission (HEC) Pakistan for providing
financial support under TTS program. Dr Ather Farooq Khan for his assistance in this research
work.
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Graphical ab
str
act
CS2
O
N
NH
KOH / EtOH
NHNH2
MW
O
S
HO
6
HO
The binding mode of the compounds 6

Highlights
•
•
•
•
Efficient synthesis of 1,3,4-oxadiazole-2-thione derivatives
Thymidine phosphorylase inhibition studies
Molecular docking studies for binding mode investigations
Compound 6 bearing 4-hydroxyphenyl group was found to be the most active