2,5-Disubstituted-1,3,4-oxadiazoles: Thymidine phosphorylase inhibitors

Article abstract of DOI:10.1007/s00044-013-0588-2

A series of 2,5-disubstituted-1,3,4-oxadiazoles (1-16) were synthesized via microwave irradiation and screened against thymidine phosphorylase enzyme. Four members of the series have demonstrated thymidine phosphorylase inhibitory activities with IC₅₀ values between 37 and 320 μM. Pyridinyl-containing 1,3,4-oxadiazoles exhibited an enhanced inhibitory potential, while phenyl analogs did not show substantial inhibitory potential. Compound 9, a dipyridinyl residue having an IC₅₀ value of 37 ± 0.76 μM was found to be the most potent in the series superior to standard inhibitor 7-deazaxanthine (IC₅₀ = 39.28 ± 0.76 μM). Graphical abstract: [InlineMediaObject not available: see fulltext.]

Full text of DOI:10.1007/s00044-013-0588-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0588-2
ORIGINAL RESEARCH
2,5-Disubstituted-1,3,4-oxadiazoles: thymidine phosphorylase
inhibitors
•
Khalid Mohammed Khan Mubeen Rani Nida Ambreen
•
•
•
•
•
Muhammad Ali Sajjad Hussain Shahnaz Perveen
Muhammad Iqbal Choudhary
Received: 27 November 2012 / Accepted: 29 March 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of 2,5-disubstituted-1,3,4-oxadiazoles
(1–16) were synthesized via microwave irradiation and
screened against thymidine phosphorylase enzyme. Four
members of the series have demonstrated thymidine
phosphorylase inhibitory activities with IC50 values
between 37 and 320 lM. Pyridinyl-containing 1,3,4-oxa-
diazoles exhibited an enhanced inhibitory potential, while
phenyl analogs did not show substantial inhibitory poten-
tial. Compound 9, a dipyridinyl residue having an IC50
value of 37 0.76 lM was found to be the most potent in
the series superior to standard inhibitor 7-deazaxanthine
(IC50 = 39.28 0.76 lM).
malignant disorders (Sivridis et al., 2002). There are dif-
ferent cells that express thymidine phosphorylase, there-
fore, might have different role in angiogenic progression
(Cole et al., 1999). Thymidine phosphorylase/platelet-
derived endothelial cell growth factor (PD-ECGF) is a
stimulator of angiogenesis (Moghaddam et al., 1995). PD-
ECGF was first isolated from human platelets and then
from placenta tissues (Yu et al., 2008; Miyazono et al.,
1987). It is now well understood that angiogenic activity is
due to thymidine phosphorylase enzyme, however, previ-
ously this activity was thought to be present in platelets
(Somjen et al., 1999). It was later on found that PD-ECGF
is similar to thymidine phosphorylase which is responsible
for catalyzing phosphorylation of thymidine and associated
analogs reversibly to 2-deoxy-D-ribose-1-phosphate and
their respective bases. Upon rapid dephosphorylation,
2-deoxy-D-ribose-1-phosphate diffuses out of the cell in the
form of 2-deoxy-D-ribose. It is anticipated that 2-deoxy-D-
ribose is indispensable for the angiogenic activity (Nakaj-
ima et al., 2004).
Keywords Oxadiazoles ꢀ Thymidine phosphorylase
inhibition ꢀ Angiogenesis
Introduction
Thymidine phosphorylase (EC 2.4.2.4) has proved to be an
angiogenic factor that is mainly expressed in cancer cells
and tumor-associated macrophages in most of the human
In solid tumors such as breast cancer, ovary, colorectal
and renal carcinoma, expression of this enzyme has been
found abnormal (Ikeda et al., 2002; Brown et al., 2000; Toi
et al., 2005). The information related to thymidine phos-
phorylase pathologies suggests that compounds, which can
effectively block the activity of thymidine phosphorylase
could be helpful in controlling various cancers.
K. M. Khan (&) ꢀ M. Rani ꢀ N. Ambreen ꢀ S. Hussain ꢀ
M. I. Choudhary
H. E. J. Research Institute of Chemistry, International Center
for Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
As of date several PD-ECGF/thymidine phosphorylase
inhibitors have been designed. 6-Aminouracil and 6-am-
inothymine-based inhibitors were of the ever first inhibitors
of thymidine phosphorylase (Balzarini et al., 1998). Sev-
eral modifications of these lead molecules were made for
better inhibition efficacies. These investigations have led to
the development of 7-deazaxanthine (7-DX), a purine
derivative, with inhibitory potential against E. coli enzyme
e-mail: khalid.khan@iccs.edu; hassaan2@super.net.pk
M. Ali
Department of Chemistry, COMSATS Institute of Information
Technology, Abbottabad 22060, KPK, Pakistan
S. Perveen
PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr.
Salim-uz-zaman Siddiqui, Karachi 75280, Pakistan
123

Med Chem Res
O
O
3
which was found similar to those of reference compounds
(Langen et al., 1967). Which was based on the observation
that 2, 3, and 4 positions of the pyrimidine ring of the
7-deazaxanthine molecule interacts with amino acids in the
thymine/thymidine-binding sites. Addition of pyrimidine
ring filled the apparent space in the vicinity of residue
Phe210 which created extra stabilizing (Langen et al.,
1967).
4
N
N
(ii)
(i) or
5
2
R
NHNH2 R'
OH
R
O
R'
1
(i) POCl3 (ii) POCl3, Al2O3
Scheme 1 Route for the synthesis of 2,5-disubstituted-1,3,4-oxadi-
azoles 1–16
1,3,4-Oxadiazole has a significant importance in het-
erocycles and imparted its importance as construction
motif for the development of newer drugs. They displayed
remarkable biological activity profiles in variety of studies
(Cledualdo et al., 2012; Kagthara et al., 1999; Poonam
et al., 2012). 2,5-Disubstituted-1,3,4-oxadiazoles are
known for various biological activities such as antimicro-
bial, antiviral, antiprotozoal, genotoxic, antiproteolysis,
pain relievers, CNS suppressants, antitumors as well as
lipid peroxidation inhibitors (Somani et al., 2008; El-
Emam et al., 2004; Maslat et al., 2002; Mohan et al., 2004;
Chaudhary et al., 2006; Yale and Losee, 1966; Khan et al.,
2001; Chauhan et al., 2003; Zheng et al., 2003; Naga-
lakshmi, 2008; Rakesh et al., 2009; Maillard et al., 1962).
This class of compounds has also proven their potential as
corrosion inhibitors by acting essentially as cathodic
inhibitors (Benabdellah et al., 2012). This class of com-
pounds has also proved their potential for development of
hydroxamic acid-based histone deacetylase inhibitors
(Harish et al., 2011). In continuation of our medicinal
chemistry research, we prepared a collection of different
2,5-disubstituted-1,3,4-oxadiazole derivatives which were
screened against thymidine phosphorylase enzyme. To the
best of our knowledge, this is newer class of compounds
that has been identified as thymidine phosphorylase
inhibitors.
solid support which does not affect the yield and speed of
reaction. The rapid and high-yielding reaction proceeded
smoothly in 1–11 min. The conventional mode requires
this reaction to be completed in 4–10 h under reflux. The
products were isolated through solvent extraction after the
treatment of crude reaction mixture with cold aqueous
sodium bicarbonate. The solvent was dried over Na2SO4
and evaporated on a rotary evaporator under reduced
pressure. Crude products were purified by column chro-
matography using ethyl acetate as eluent (Khan et al.,
2004).
Results and discussion
All 2,5-disubstituted 1,3,4-oxadiazoles (1–16) were evalu-
ated against thymidine phosphorylase enzyme according to
the literature protocol Table 1 (Krenitsky and Bushby,
1979).
Literature shows that imidazole rings of biodirective
nitroimidazolyl prodrugs at normal intracellular pH of 7.2
were expected to exist largely in their cationic forms which
interact strongly with dianionic monohydrogen phosphate-
binding site of the thymidine phosphorylase enzyme
(Krenitsky and Bushby, 1979). Similarly, such intracellular
pH levels, the pyridine analogs are protonated, into py-
ridinium ions which interact with dianionic phosphate-
binding site. These binding interactions might be also
possible through potassium salt bridges, as the assay pro-
tocol involves a potassium phosphate buffer.
Experimental procedure
We have previously reported the synthesis of 1,3,4-oxadi-
azoles by using microwave radiations (Khan et al., 2004).
Later on we reported the tyrosinase inhibitory potential of
this class of compounds (Khan et al., 2005). This study was
undertaken to determine thymidine phosphorylase inhibi-
tory properties of 2,5-disubstituted-1,3,4-oxadiazoles in
order to search for new anti-cancer compounds.
Out of 16 compounds, only four (4, 9, 13, and 14)
showed varying a degree of inhibitory activity. Compound
9 was found to be the active most member of this series
with an IC50 value = 37 0.76 lM. Surprisingly, a clo-
sely related compound 1 was devoid of any activity.
Another structurally related compound 4 showed a lower
activity with an IC50 value 247.31 2.20 lM. Compound
13 was the second most active compound of this series
(IC50 = 89.23 0.24 lM). Compound 14, with similar
structure to compound 13 demonstrated a low activity
(IC50 = 320 0.84 lM) against thymidine phosphory-
lase. The less or complete devoid of activity of most of the
Chemistry
2,5-Disubstituted-1,3,4-oxadiazoles (1–16) were prepared
from respective hydrazides and carboxylic acids under
microwave conditions using phosphorous oxychloride
(POCl3) (Scheme 1). Neutral alumina oxide was used as
123

Med Chem Res
Table 1 In vitro thymidine phosphorylase inhibition of compounds 1–16
Compound no.
Structures
IC50 (lM SEM)
Compound no.
Structures
IC50 (lM SEM)
N
N
N
O
N
1
NA
9
37 0.76
O
N
N
N
N
N
N
N
2
NA
10
NA
O
CHCl
O
CHCl
2
2
N
N
N
N
N
3
4
5
NA
11
12
13
NA
O
CCl
3
O
CCl
3
N
N
N
N
N
247.31 2.20
NA
O
O
N
CH
CH
3
3
N
N
N
N
N
NA
89.23 0.24
O
O
N
N
N
N
N
N
6
7
8
NA
NA
NA
14
15
16
320 0.84
O
O
N
N
N
N
NA
O
O
N
OCH
OCH
3
3
N
N
N
NA
OCH
3
OCH
3
O
O
N
OCH
3
OCH
3
OCH
3
OCH
3
Standard
7-Deazaxanthine
39.28 0.76
SEM standard error of the mean, NA not active
compounds of this series may be due appropriate sub-
stituent on the oxadiazole ring and results proves that
pyridine substitution on 2 and 5 position is suitable for
thymidine phosphorylase inhibitory properties. In other
words, pyridine rings at 2 and 5 position of oxadiazole
residue induces thymidine phosphorylase inhibitory
potential into the molecule. However, replacement of only
one pyridine ring by other aromatic ring sharply decline or
makes the molecule completely inactive (Table 1).
Apparently pyridine substituents on both side of oxadiazole
ring (compound 9) provide a fruitful opportunity to interact
with the enzyme. In short, it has been observed that the
occupation of a bulky group at positions 2 and 5 of 1,3,4-
oxadiazole resulted in inhibition of enzyme.
monitoring the effect of increasing concentrations of
compounds in the assay described below. The IC50 values
were calculated from inhibition data by using EZ-Fit
Enzyme Kinetic Program (Perrella Scientific Inc., Amherst,
USA).
Thymidine phosphorylase assay protocol
Thymidine phosphorylase/PD-ECGF (human and E. coli)
activity was determined by measuring the absorbance at
290 nm spectrophotometrically. The original method
described by Krenitsky and Bushby (1979) was modified. In
brief, total reaction mixture of 200 lL contained 145 lL of
potassium phosphate buffer (pH 7.4), 30 lL of enzyme
(human and E. coli) at concentration 0.05 and 0.002 U,
respectively, were incubated with 5 lL 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 sub-
strate particles. Substrate (20 lL, 1.5 mM), was dissolved in
Determination of IC50 assay
The concentration of test compounds that inhibited the
enzyme activity by 50 % (IC50 values) were determined by
123

Med Chem Res
0
0
0
0
0
0
potassium phosphate buffer, was immediately added to plate
and continuously read after 10, 20, and 30 min in microplate
reader (spectra max, molecular devices, CA, USA). All
assays were performed in triplicate.
J5 (4 ,6 ) = 7.8 Hz, H-30/50), 7.54 (t, 1H, J4 (3 ,5 ) = 7.8 Hz,
H-40), 5.78 (s, 1H, H-100); 13C-NMR (100 MHz, DMSO-
d6): d 165 (C-2/5), 163.1 (C-2), 129.1 (C-300/500), 128.6 (C-
400), 127.3 (C-200/600), 122.8 (C-100), 70.2 (C-10); IR (KBr)
m
max: 1567, 3062, 1662, 628, 603 cm-1; UV (methanol):
General experiment
kmax 248 (log e = 2.46) nm; EI MS (m/z): 233 (M?4, 3),
231 (M?2, 10), 229 (M?, 28), 223 (31), 175 (48) 136 (29),
105 (100), 83 (95), 77 (8), 87 (3), 66 (69); Anal. calcd. for
C9H6Cl2N2O: C, 47.19; H, 2.64; N, 12.23. Found: C, 47.11;
H, 2.34; N, 12.03.
The reactions were monitored by TLC which was per-
formed on 2.0 9 5.0 cm aluminum sheets pre-coated 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. The hydrazides used as
intermediates in this study were either purchased or syn-
thesized. Phosphorous oxychloride and carboxylic acids
were commercially available (Fluka Aldrich). All the
synthesized compounds gave satisfactory CHN analysis.
The formations of compounds were confirmed by com-
parative TLC with authentic samples and by comparing
spectroscopic data with literature (Khan et al., 2004).
2-(Trichloromethyl)-5-(phenyl)-1,3,4-oxadiazole (3) Yield:
88 %; Rf = 0.37 (ethyl acetate:acetone, 8:2); 1H NMR
0
0
0
0
(400 MHz, DMSO-d6): d 8.13 (dd, 2H, J2 ,3 = J6 ,5 = 7.8,
J2 ,4 = J6 ,4 = 1.1, Hz, H-20/60), 7.98 (t, 2H, J3 (2 ,4 )
0
0
0
0
0
0
0
=
J5 (4 ,6 ) = 7.8 Hz, H-30/50), 7.63 (t, 1H, J4 (3 ,5 ) = 7.8 Hz,
H-40); 13C NMR (100 MHz, DMSO-d6): d 165.1 (C-5), 163.1
(C-2), 129.3 (C-300/500), 128.5 (C-400), 127.1 (C-200/600), 122.8
(C-100), 97.4 (C-10); IR (KBr) mmax: 1560, 3060, 1662, 628,
603 cm-1; UV (methanol): kmax 245 (log e = 2.46) nm; EI
MS (m/z): 269 (M?6, 0.20), 267 (M?4, 6), 265 (M?2, 15),
263 (M?, 18), 227 (68), 145 (69), 105 (100), 68 (51); Anal.
calcd. for C9H5Cl3N2O: C, 41.02; H, 1.19; N, 10.63;.
Found:C, 40.97; H, 1.14; N, 10.50.
0
0
0
0
0
0
General procedure for the synthesis of 2,5-disubstituted-
1,3,4-oxadiazoles (1–16)
Acid hydrazide (3.65 mmol), aliphatic or aromatic acid
(3.65 mmol) and alumina (14.71 mol) were finely ground
with
a
mortar and pestle. Phosphorous oxychloride
2-(400-Methylphenyl)-5-(phenyl)-1,3,4-oxadiazole (4) Yield:
79 %; Rf = 0.39 (ethyl acetate:acetone, 8:2); 1H NMR
(5.36 mmol) was added to this mixture and then subjected for
microwave irradiation with different time intervals. After
completion of the reaction, the mixture was then poured into
ice-cold water. The precipitate was filtered and washed with a
10 % solution of NaHCO3, and then purified by column
chromatography using ethyl acetate as an eluent to afford 2,5-
disubstituted-1,3,4-oxadiazoles as slightly yellowish solids.
0
0
0
(400 MHz, DMSO-d6):
0
d
8.14 (t, 2H, J3 (2 ,4 )
0
=
J5 (4 ,6 ) = 8.3 Hz, H-30/50), 7.79 (d, 2H, J2 ,3 = J6 ,5
0
=
0
0
0
0
8.3 Hz, H-20/60), 7.54 (t, 1H, J4 (3 ,5 ) = 7.8 Hz, H-40),
0
0
0
7.63 (d, 2H, J2 ,3 , = J6 ,5 = 7.8 Hz, H-200/600), 7.14 (d,
00 00
00 00
2H, J3 ,2 = J5 ,6 = 7.8 Hz, H-300/500), 3.7 (s, 3H, CH3);
13C NMR (100 MHz, DMSO-d6): d 164.4 (C-2/5), 142.5
(C-100), 133.5 (C-10), 131.7 (C-400), 129.1 (C-30/50), 128.6
(C-40), 127.3 (C-20/60), 127.1 (C-200/600), 126.2 (C-300/500),
21.8 (C-70); IR (KBr) mmax: 1563, 3059, 1660, 628,
601 cm-1; UV (methanol): kmax 267 (log e = 3.11) nm;
EI MS (m/z): 236 (M?,7), 221 (54), 164 (34), 105 (61), 91
(46), 77 (100); Anal. calcd. for C15H12N2O: C, 76.25; H,
5.12; N, 11.86. Found: C, 76.05; H, 5.02; N, 11.66.
00 00
00 00
2-(300-Pyridinyl)-5-(phenyl)-1,3,4-oxadiazole (1) Yield:
87 %; Rf = 0.42 (ethyl acetate:acetone, 8:2); 1H NMR
00 00
(400 MHz, DMSO-d6): d 8.74 (d, 1H, J2 ,4 = 1.1 Hz,
H-200), 8.49 (dd, 1H, J4 ,5 = 8.3, J4 ,6 = 1.2, Hz, H-400),
00 00
00 00
8.17 (dd, 2H, J5 ,4 = 8.3, J5 ,6 = 4.9, Hz, H-500), 8.10
00 00
00 00
(dd, 1H, J6 ,5 = 4.9, J6 ,4 = 1.2, Hz, H-600); 7.65 (m, 5H,
H-20/30/40/50/60); 13C-NMR (100 MHz, DMSO-d6): d 165
(C-2/5), 152.5 (C-200), 147.8 (C-600), 134 (C-400), 133.7 (C-
10), 129.1 (C-30/50), 128.6 (C-40), 127 (C-20/60), 124.2 (C-
300), 123.8 (C-500); IR (KBr) mmax: 1587, 3055, 1661, 626,
603; UV (methanol): kmax 262 (log e = 3.17) nm; EI MS
(m/z): 223 (M?, 4), 175 (66), 135 (35), 107 (48), 78 (100),
68 (38); Anal. calcd. for C13H9N3O: C, 69.95; H, 4.06; N,
18.82. Found: C, 66.38; H, 3.97; N, 17.55.
00 00
00 00
2-(100-Naphthyl)-5-(phenyl)-1,3,4-oxadiazole (5) Yield:
79 %; Rf = 0.39 (ethyl acetate:acetone, 9:1); 1H NMR
00 00
(500 MHz, DMSO-d6): d 8.46 (dd, 1H, J8 ,7 = 8.4,
J8 ,6 = 1.3 Hz, H-800), 8.38 (d, 1H, J4 ,3 = 8.4, Hz,
00 00
00 00
H-400), 8.19 (t, 1H, J3 (2 ,4 ) = 8.0 Hz, H-300), 7.68 (d, 1H,
00 00 00
J5 ,6 = 8.4 Hz, H-500), 7.60 (d, 1H, J2 ,3 = 7.8 Hz,
00 00
00 00
H-200), 7.51 (t, 1H, J6 (5 ,4 ) = 8.4 Hz, H-600), 7.48 (t, 2H,
00 00 00
J7 (6 ,8 ) = 7.8 Hz, H-700); 7.40 (dd, 2H, J2 ,3 = J6 ,5
=
00 00 00
0
0
0
0
2-(Dichloromethyl)-5-(phenyl)-1,3,4-oxadiazole (2) Yield:
83 %; Rf = 0.36 (ethyl acetate:acetone, 8:2); 1H NMR
8.3 Hz, 2H, J2 ,4 = J6 ,4 = 1.2, Hz, H-20/60), 7.34 (t, 2H,
0 0 0 0
J3 (2 ,4 ) = J5 (4 ,6 ) = 7.8 Hz, H-30/50), 7.30 (t, 1H, J4 (3 ,5 )
0
0
0
0
0
0
0
0
0
=
(400 MHz, DMSO-d6): d 7.93 (dd, 2H, J2 ,3 = J6 ,5 = 8.3,
7.8 Hz, H-40); 13C NMR (125 MHz, DMSO-d6): d 164.3
0
0
0
0
J2 ,4 = J6 ,4 = 1.2, Hz, H-20/60), 7.65 (t, 2H, J3 (2 ,4 )
(C-2/5), 135.8 (C-10), 134.3 (C-100), 133.5 (C-100), 132.4
0
0
0
0
0
0
0
=
123

Med Chem Res
(C-90), 129.1 (C-300/500), 128.6 (C-400), 128.2 (C-40/50/80),
127.3 (C-200/600), 126.2 (C-60/70), 125.8 (C-30), 124.1 (C-20);
IR (KBr) mmax: 1557, 3058, 1659, 628, 604 cm-1; UV
(methanol): kmax 288 (log e = 3.17) nm; EI MS (m/z): 272
(M?, 9), 214 (16), 154 (32), 126 (14), 105 (100), 76 (58), 50
(19); Anal. calcd. for C18H12N2O: C, 79.39; H, 4.44; N,
10.29. Found: C, 79.09; H, 4.14; N, 10.20.
C17H16N2O4: C, 65.38; H, 5.16; N, 8.97. Found: C, 65.12;
H, 5.06; N, 8.35.
2-(300-Pyridinyl)-5-(30-pyridinyl)-1,3,4-oxadiazole (9) Yield:
89 %; Rf = 0.3 (ethyl acetate:acetone, 8:2); 1H NMR
0
0
00 00
=
(500 MHz, DMSO-d6): d 9.22 (d, 2H, J2 ,4 = J2 ,4
1.1 Hz, H-20/200), 8.78 (dd, 2H, J4 ,5 = J4 ,5 = 8.3,
0
0
00 00
J4 ,6 = J4 ,6 = 1.2, Hz, H-40/400), 8.35 (dd, 2H, J5 ,4
=
0
2-(200-Naphthyl)-5-(phenyl)-1,3,4-oxadiazole (6) Yield:
82 %; Rf = 0.34 (ethyl acetate:acetone, 9:1); 1H-NMR
0
0
00 00
0
J5 ,4 = 8.3, J5 ,6 = J5 ,6 = 4.9, Hz, H-50/500), 7.63 (dd,
00 00
0
0
00 00
2H, J6 ,5 = J6 ,5 = 4.9, J6 ,4 = J6 ,4 = 1.2 Hz, H-60/
600); 13C NMR (125 MHz, DMSO-d6): d 164.7 (C-2/5),
152.5 (C-20/200), 147.8 (C-60/600), 134.5 (C-40/400), 124.5
(C-30/300), 123.7 (C-50/500); IR (KBr) mmax: 3061, 1659,
1563, 1286 cm-1; UV (methanol): kmax 208 (log e = 2.41)
nm; EI MS (m/z): 224 (M?, 12), 160 (9), 106 (100), 93
(80); Anal. calcd. for C12H8N4O: C, 64.28; H, 3.60; N,
24.99. Found: C, 64.19; H, 3.59; N, 24.88.
0
0
00 00
0
0
00 00
00 00
(500 MHz, DMSO-d6): d 8.46 (dd, 1H, J8 ,7 = 8.4,
J8 ,6 = 1.3 Hz, H-800), 8.38 (d, 1H, J4 ,3 = 8.4, Hz, H-400),
00 00
00 00
8.19 (s, 1H, H-100), 7.68 (t, 1H, J7 (6 ,8 ) = 8.4 Hz, H-700), 7.60
00 00 00
(d, 1H, J5 ,6 = 7.8 Hz, H-500), 7.51 (d, 1H, J3 ,4 = 8.4 Hz,
00 00
00 00
H-300), 7.48 (t, 1H, J6 (5 ,7 ) = 8.4 Hz, H-600); 7.40 (dd, 2H,
00 00 00
J2 ,3 = J6 ,5 = 8.3, J2 ,4 = J6 ,4 = 1.2, Hz, H-20/60), 7.34 (t,
0
0
0
0
0
0
0
0
2H, J3 (2 ,4 ) = J5 (4 ,6 ) = 7.8 Hz, H-30/50), 7.30 (t, 1H,
0
0
0
0
0
0
J4 (3 ,5 ) = 7.8 Hz, H-40); 13C NMR (125 MHz, DMSO-d6): d
165.1 (C-2/5), 133.7 (C-20), 133.5 (C-100), 133.3 (C-100), 133.1
(C-90), 129.1 (C-300/500), 128.6 (C-400), 128.1 (C-50/80), 127.7
(C-10), 127.4 (C-200/600), 126.2 (C-06/70) 124 (C-30); IR (KBr)
0
0
0
2-(Dichloromethyl)-5-(30-pyridinyl)-1,3,4-oxadiazole (10) Yield:
85 %; Rf = 0.34 (ethyl acetate:acetone, 9:1); 1H NMR
(500 MHz, DMSO-d6): d 9.20 (d, 1H, J2 ,6 = 1.1 Hz, H-20),
9.12 (br.d, 1H, J6 ,5 = 4.9 Hz, H-60), 8.87 (br.d, 1H,
0
0
m
max: 1560, 3060, 1659, 625, 603 cm-1;UV (methanol): kmax
288 (log e = 3.17) nm; EI MS (m/z): 272 (M?, 57), 214 (16),
154 (32), 105 (100), 76 (58); IR (KBr) mmax: 3071, 1564, 1660,
1276; Anal. calcd. for C18H12N2O: C, 79.39; H, 4.44; N, 10.29.
Found: C, 79.09; H, 4.10; N, 10.10.
0
0
J = 8.3 Hz, H-40), 8.69 (dd, 1H, J5 ,4 = 8.3, J5 ,6 = 5.0 Hz,
H-50), 3.39 (s, 1H, H-100); 13C NMR (125 MHz, DMSO-d6):
d 163.8 (C-5), 162.5 (C-2), 152.1 (C-200), 147.5 (C-600),
133.7 (C-400), 124.1 (C-300), 123 (C-500), 71 (C-10); IR (KBr)
0
0
0
0
max: 3061, 2998, 1664, 1564, 1281 cm-1, 757; UV
2-(400-Methoxyphenyl)-5-(phenyl)-1,3,4-oxadiazole (7) Yield:
79 %; Rf = 0.39 (ethyl acetate:acetone, 8:2); 1H NMR
m
(methanol): kmax 209 (log e = 2.42) nm; EI MS (m/z): 234
(M?4, 6), 232 (M?2, 19), 230 (M?, 29), 194 (66), 159 (29),
146 (63), 106 (73), 78 (100), 51 (85); Anal. calcd. for
C8H5Cl2N3O: C, 41.77; H, 2.19; N, 18.27. Found: C, 41.75;
H, 2.11; N, 18.22.
0
0
0
0
(400 MHz, DMSO-d6): d 9.25 (d, 2H, J2 ,3 = J6 ,5 = 7.6,
Hz, H-20/60), 8.12 (br.d, 2H, J2 ,3 = J6 ,5 = 7.8 Hz, H-200/
600), 7.48 (m, 3H, H-30/40/50), 7.42 (m, 2H, H-300/500), 3.93 (s,
3H, OCH3); 13C NMR (100 MHz, DMSO-d6): d 163.8
(C-2/5), 160.7 (C-40), 133.4 (C-100), 129.1 (C-300/500), 128.8
(C-10), 128.4 (C-400), 127.2 (C-200/600), 115.5 (C-20/60),
114.8 (C-3/050); IR (KBr) mmax: 1565, 3055, 1657, 628,
602 cm-1; UV (methanol): kmax 267 (log e = 3.11) nm; EI
MS (m/z): 252 (M?,7), 223 (54), 196 (34), 135 (100), 105
(46); Anal. calcd. for C15H12N2O2: C, 71.42; H, 4.79; N,
11.10. Found: C, 70.72; H, 4.59; N, 11.03.
00 00
00 00
0
2-(Trichloromethyl)-5-(3 -pyridinyl)-1,3,4-oxadiazole (11) Yield:
91 %; Rf = 0.34 (ethyl acetate:acetone, 9:1); 1H NMR
0
0
(500 MHz, DMSO-d6): d 9.23 (d, 1H, J2 ,4 = 1.1 Hz,
H-20), 9.12 (br.d, 1H, J6 ,5 = 4.9 Hz, H-60), 8.86 (br.d, 1H,
0
0
J4 ,5 = 8.3 Hz, H-40), 8.63 (dd, 1H, J5 ,4 = 8.3,
0
0
0
0
J5 ,6 = 4.8 Hz, H-50); 13C NMR (125 MHz, DMSO-d6): d
164 (C-2), 163.5 (C-5), 152.8 (C-20), 147.9 (C-60), 134.2
(C-40), 125.1 (C-30), 124 (C-50), 97.8 (C-100); IR (KBr)
0
0
2-(300,400,500-Trimethoxyphenyl)-5-(phenyl)-1,3,4-oxadiazole
(8) Yield: 79 %; Rf = 0.39 (ethyl acetate); 1H NMR
m
max: 1562, 3059, 1663, 1283, 781 cm-1; UV (methanol):
kmax 207 (log e = 2.41) nm; EI MS (m/z): 270 (M?6, 1),
268 (M?4, 6), 266 (M?2, 18), 264 (M?, 13), 158 (29),
118.5 (39), 106 (43), 78 (100), 68 (49), 51 (41); Anal.
calcd. for C8H4Cl3N3O: C, 36.33; H, 1.52; N, 15.89.
Found: C, 36.30; H, 1.43; N, 15.86.
0
0
0
0
=
(400 MHz, CD3OD-d4): d 9.20 (d, 2H, J2 ,3 = J6 ,5
4.9 Hz, H-20/60), 8.87 (m, 3H, H-30/40/50); 7.49 (s, 2H,
H-200/600), 3.93 (s, 6H, –2OCH3), 3.88 (s, 3H, OCH3); 13C
NMR (100 MHz, CD3OD-d4): d 165.7 (C-2/5), 154.5
(C-30/50), 139.8 (C-40), 133.7 (C-100), 129.1 (C-300/500),
128.6 (C-400), 127.3 (C-200/600), 120.2 (C-10), 104.8 (C-20/
60), 60.8 (C-80), 56.7 (70/90); IR (KBr) mmax: 1566, 3061,
1660, 627, 601 cm-1; UV (methanol): kmax 283 (log e =
3.17) nm; EI MS (m/z): 312 (M?, 9), 281 (11), 250 (13), 78
(100), 77 (53), 68 (38), 51 (79); Anal. calcd. for
00
2-(4 -Methylphenyl)-5-(30-pyridinyl)-1,3,4-oxadiazole (12) Yield:
81 %; Rf = 0.43 (ethyl acetate:acetone, 9:1); 1H NMR
(500 MHz, DMSO-d6): d 9.14 (d, 1H, J2 ,6 = 1.1 Hz, H-20),
0
0
9.04 (br.d, 1H, J6 ,5 = 4.9 Hz, H-60), 8.78 (br.d, 1H,
0
0
123

Med Chem Res
2-(400-Methoxyphenyl)-5-(30-pyridinyl)-1,3,4-oxadiazole
(15) Yield: 81 %; Rf = 0.43 (ethyl acetate:acetone, 9:1);
J4 ,5 = 8.3 Hz, H-40), 8.54 (dd, 1H, J5 ,4 = 8.3, J5 ,6
=
0
0
0
0
0
0
4.6 Hz, H-50), 7.49 (br.d, 2H, J2 ,6 = J6 ,5 = 7.8 Hz,
00 00
00 00
1H NMR (500 MHz, DMSO-d6): d 9.14 (d, 1H, J2 ,6
H-200/600), 7.32 (d, 2H, J3 ,4 , = J5 ,6 = 7.8 Hz, H-300/500),
6.12 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6): d 165.1
(C-2/5), 152.8 (C-20), 147.5 (C-60), 142.6 (C-100), 134.2 (C-
40), 131.5 (C-400), 127.2 (C-200/400), 126.2 (C-300/500), 124.3
(C-30), 123.9 (C-50), 23.4 (C-700); IR (KBr) mmax: 3063, 2912,
1663, 1561, 1283 cm-1; UV (methanol): kmax 206 (log e =
2.41) nm; EI MS (m/z): 237 (M?, 13), 222 (28), 146 (58),
106 (43), 78 (100), 77 (62), 68 (47); Anal. calcd. for
C14H11N3O: C, 70.87; H, 4.67; N, 17.71. Found: C, 70.69;
H, 4.60; N, 17.63.
0
0
=
00 00
00 00
1.1 Hz, H-20), 9.04 (br. d, 1H, J6 ,5 = 4.9 Hz, H-60), 8.78
0
0
(br.d, 1H, J4 ,5 = 8.3 Hz, H-40), 8.54 (dd, 1H, J5 ,4 = 8.3,
0
0
0
0
J5 ,6 = 4.9 Hz, H-50), 7.49 (br.d, 2H, J2 ,3 = J6 ,5
=
00 00
0
0
00 00
7.8 Hz, H-200/600), 7.32 (m, 2H, H-300/500); 13C NMR
(125 MHz, DMSO-d6): d 164 (C-2/5), 160.2 (C-40), 152.5
(C-200), 147.8 (C-600), 134.1 (C-400), 129.2 (C-10), 124.2 (C-
300), 123.8 (C-500), 116 (C-20/60), 115 (C-30/50), 57.1 (C-70); IR
(KBr) mmax: 3063, 2912, 1663, 1561, 1283 cm-1; UV
(methanol): kmax 206 (log e = 2.41) nm; EI MS (m/z): 253
(M?, 13), 222 (28), 146 (58), 106 (43), 78 (100), 77 (62), 68
(47); Anal. calcd. for C14H11N3O2: C, 66.40; H, 4.38; N,
16.59. Found: C, 66.29; H, 4.38; N, 16.49.
2-(100-Naphthyl)-5-(30-pyridinyl)-1,3,4-oxadiazole (13) Yield:
83 %; Rf = 0.34 (ethyl acetate:acetone, 8:2); 1H-NMR
(400 MHz, DMSO-d6): d 9.22 (d, 1H, J2 ,6 = 1.1 Hz, H-20),
0
0
2-(300,400,500-Trimethoxyphenyl)-5-(30-pyridinyl)-1,3,4-oxadi-
9.15 (br.d, 1H, J6 ,5 = 4.9 Hz, H-60), 8.89 (br.d, 1H,
0
0
azole (16) Yield: 79 %; Rf = 0.39 (ethyl acetate); 1H
J4 ,5 = 8.3 Hz, H-40), 8.60 (dd, 1H, J5 ,4 = 8.3, J5 ,6
=
0
0
0
0
0
0
0
0
=
NMR (400 MHz, CD3OD-d4): d 9.20 (d, 1H, J2 ,6
4.9 Hz, H-50), 8.46 (dd, 1H, J8 ,7 = 8.4, J8 ,6 = 1.3 Hz,
00 00
00 00
1.1 Hz, H-20), 9.13 (br.d, 1H, J6 ,5 = 4.9 Hz, H-60), 8.87
0
0
H-800), 8.38 (d, 1H J4 ,3 = 8.4 Hz, H-400),, 8.29 (t, 1H,
00 00
(br.d, 1H, J4 ,5 = 8.3 Hz, H-40), 8.65 (dd, 1H, J5 ,4 = 8.3,
0
0
0
0
J7 (6 ,8 ) = 8.0 Hz, H-700), 8.19 (t, 1H, J3 (2 ,4 ) = 8.0 Hz,
00 00 00
00 00 00
J5 ,6 = 4.9 Hz, H-50), 7.49 (s, 2H, H-200/600), 3.93 (s, 2H,
–2OCH3), 3.88 (s, 3H, OCH3); 13C NMR (100 MHz,
CD3OD-d4): d 165.1 (C-2/5), 153.5 (C-300/500), 152.5
(C-20), 147.8 (C-60), 139.3 (C-400), 134 (C-40), 123.7 (C-
300), 123.1 (C-50), 120.6 (C-100), 104.5 (C-200/600), 60.7
(C-800), 57.2 (C-700/900); IR (KBr) mmax: 3059, 1665, 1562,
1284, 1198 cm-1; UV (methanol): kmax 283 (log e = 3.17)
nm; EI MS (m/z): 313 (M?, 9), 282 (11), 251 (13), 220
(18), 146 (37), 106 (73), 78 (100), 77 (53), 68 (38), 51 (79);
Anal. calcd. for C16H15N3O4: C, 61.34; H, 4.83; N, 13.41.
Found: C, 61.30; H, 4.80; N, 13.34.
0
0
H-300), 7.68 (d, 1H, J2 ,3 = 8.0 Hz, H-200), 7.60 (dd,
00 00
1H, J5 ,6 = 8.4, J5 ,7 = 1.4 Hz, H-500), 7.51 (t, 1H,
00 00
00 00
J6 (5 ,7 ) = 8.4 Hz, H-600); 13C-NMR (100 MHz, DMSO-
00 00 00
d6): d 166 (C-2/5), 153.7 (C-200), 147.8 (C-600), 137 (C-10),
134.4 (C-100), 134.1 (C-400), 133.1 (C-90), 129.1 (C-40/50/80),
126.6 (C-60/70), 125 (C-30), 124.2 (C-20), 123.8 (C-300), 122.5
(C-500); IR (KBr) mmax: 3063, 1561, 1661, 1281, 637,
609 cm-1; UV (methanol): kmax 289 (log e = 3.17) nm; EI
MS (m/z): 273 (M?, 57), 146 (47), 106 (77), 78 (100), 77
(63), 68 (49), 51 (67); Anal. calcd. for C17H11N3O: C, 74.71;
H, 4.06; N, 15.38. Found: C, 74.65; H, 4.02; N, 15.31.
2-(200-Naphthyl)-5-(30-pyridinyl)-1,3,4-oxadiazole (14) Yield
1
0.81 g (81 %); Rf = 0.34 (ethyl acetate:acetone, 9:1); H
Conclusion
In this study, compound 9 has been identified as a potent
thymidine phosphorylase inhibitor with an IC50 value of
37 0.76 lM. This compound may serve as lead for
further development as this compound is an important
addition to new inhibitors of thymidine phosphorylase in
the presence of existing plethora of inhibitors.
NMR (400 MHz, DMSO-d6): d 9.25 (d, 1H, J = 1.1 Hz,
H-20), 9.16 (br.d, 1H, J6 ,2 = 4.9 Hz, H-60), 8.71 (br.d, 1H,
0
0
J4 ,5 = 8.3 Hz, H-40), 8.49 (dd, 1H, J5 ,4 = 8.3,
0
0
0
0
J5 ,6 = 4.9 Hz, H-50), 8.32 (br.s, 1H, H-100), 8.13 (d, 1H,
0
0
J4 ,3 = 8.1 Hz, H-400), 8.03 (br.d, 1H, J3 ,4 = 8.1 Hz,
00 00
00 00
H-300), 7.82 (dd, 1H, J5 ,6 = 7.8, J5 ,7 = 1.6 Hz, H-500),
00 00
00 00
7.76 (dd, 1H, J8 ,7 = 7.9, J = 1.6 Hz, H-800), 7.54 (t, 1H,
00 00
Acknowledgments The authors are thankful to OPCW, Netherlands
(Project No. L/ICA/ICB/173681/12) for financial support.
J7 (6 ,8 ) = 7.9 Hz, H-700), 7.45 (t, 1H, J6 (5 ,7 ) = 7.9 Hz,
H-600); 13C-NMR (100 MHz, DMSO-d6): d 166 (C-2/5),
153.7 (C-200), 147.8 (C-600), 134.4 (C-200/400), 133.7 (C-100),
133.1 (C-90), 132.1 (C-40), 127.6 (C-5 /80), 126 (C-60/70),
00 00 00
00 00 00
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