Synthesis and biological evaluation of novel oxadiazole derivatives: A new class of thymidine phosphorylase inhibitors as potential anti-tumor agents

Article abstract of DOI:10.1016/j.bmc.2013.12.043

Based on the fact that the thymidine phosphorylase inhibitors are considered potential anti-tumor agents, a range of novel oxadiazole derivatives 3a-3u was designed and synthesized by a simple and facile synthetic route The biological assay revealed that majority of compounds displayed modest inhibitory activity against thymidine phosphorylase at low micromolar concentrations (IC₅₀ 173.23 ± 3.04 to 14.40 ± 2.45 μM) In the current study the most active compounds were 3h and 3q with IC₅₀ values 14.40 ± 2.45 and 17.60 ± 1.07 μM, respectively Molecular docking studies were performed on the most active compounds (3h, 3k, 3o-3q) to show their binding mode

Full text of DOI:10.1016/j.bmc.2013.12.043

Bioorganic & Medicinal Chemistry 22 (2014) 1008–1015
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel oxadiazole derivatives:
A new class of thymidine phosphorylase inhibitors as potential
anti-tumor agents
b,
Sohail Anjum Shahzad a, , Muhammad Yar , Marek Bajda c,d, Bushra Jadoon a, Zulfiqar Ali Khan e,
Syed Ali Raza Naqvi e, Ahson Jabbar Shaikh a, Khizar Hayat a, Adeem Mahmmod a, Nasir Mahmood f,
Sławomir Filipek c
⇑
⇑
a Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
b Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000, Pakistan
c Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
d Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Cracow, Poland
e Department of Chemistry, Government College University, Faisalabad 38000, Pakistan
f Department of Allied Sciences and Chemical Pathology, University of Health Sciences, Lahore 54600, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the fact that the thymidine phosphorylase inhibitors are considered potential anti-tumor
agents, a range of novel oxadiazole derivatives 3a–3u was designed and synthesized by a simple and fac-
ile synthetic route. The biological assay revealed that majority of compounds displayed modest inhibitory
activity against thymidine phosphorylase at low micromolar concentrations (IC50 173.23 3.04 to
Received 16 November 2013
Revised 18 December 2013
Accepted 18 December 2013
Available online 30 December 2013
14.40 2.45
14.40 2.45 and 17.60 1.07
active compounds (3h, 3k, 3o–3q) to show their binding mode.
lM). In the current study the most active compounds were 3h and 3q with IC50 values
lM, respectively. Molecular docking studies were performed on the most
Keywords:
1,3,4-Oxadiazole-2-thione
Mannich bases
Ó 2013 Elsevier Ltd. All rights reserved.
Thymidine phosphorylase inhibitors
Anti-cancer activity
Angiogenesis
1. Introduction
tissue. These actions result in fast generation of new blood vessels
and cancer metastasis.5
Angiogenesis is the formation of new blood vessels from pre-
existing vessels and it is essential for organ growth and repair.
However, it is well known that this is a vital step in the process
of cancer growth.1,2 Thus, angiogenesis inhibitors are believed to
be potential candidates for blocking cancer growth. In particular,
thymidine phosphorylase (TP) is a pro-angiogenic factor which
catalyzes the reversible phosphorolysis of thymidine into thymine
TP inhibitors affect the production of 20-deoxy-
D-ribose and in
turn suppress tumor growth.5,6 Therefore, there is an urgent need
to develop new and potent thymidine phosphorylase inhibitors
which have the ability to suppress the formation of new blood ves-
sels and stop tumor growth. A number of efforts have been re-
ported on the development of TP inhibitors.5,7–14 The pyrimidine
derivative
5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]
uracil
and 20-deoxy-
1-phosphate undergoes further dephosphorylation to produce
20-deoxy-
-ribose which stimulates the secretion of vascular endo-
D D-ribose
-ribose 1-phosphate.3,4 The 20-deoxy-
D
R
O
O
thelial growth factor (VEGF). VEGF activates a number of processes
including endothelial cells for secretion of matrix metalloprotein-
ases, proliferation, and migration of endothelial cells to tumor
N
4
3
Cl
N
N
NH
H
NH
2
5
N
S
N
H
N
H
O
N
H
O
O
1
H2N
R
Proposed 1,3,4-
Cl
TPI
7DX
oxadiazoline-2-thione
derivatives
⇑
Corresponding authors. Tel.: +92 992 383591 6; fax: +92 992 383441 (S.A.S.);
tel.: +92 42 111001007; fax: +92 42 35321090 (M.Y.).
E-mail addresses: sohail_chem@yahoo.com (S.A. Shahzad), drmyar@ciitlahore.
edu.pk (M. Yar).
Figure 1. Chemical structure of known TP inhibitors—TPI and 7DX, and proposed
1,3,4-oxadiazole-2-thione derivatives as new class of TP inhibitors.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.043

S. A. Shahzad et al. / Bioorg. Med. Chem. 22 (2014) 1008–1015
1009
hydrochloride (TPI) is the most potent inhibitor of human TP
known till date and 7-deazaxanthine (7DX) is the first purine
derivative identified as TP inhibitor (Fig. 1).38–40 Moreover, TPI is
currently being evaluated in phase II clinical trials for solid tumors.
1,3,4-Oxadiazole ring is well known due to its number of biolog-
ical properties such as anti-inflammatory, antitumor, anticancer,
antimicrobial, antibacterial, anti-trypanosomacruzi, antifungal and
anti-HIV activities.15–23 1,3,4-Oxadiazole derivatives are also identi-
fied as potent inhibitors of dengue and West Nile virus NS2B/NS3
proteases, immunosuppressive agents, anti-hepatitis B virus com-
pounds and also urease inhibitors.24–28
In addition, certain Mannich bases derived from 1,3,4-oxadiaz-
oles are reported to possess anti-inflammatory, analgesic activity,
antifungal activity, antibacterial activity, anti-HIV activity,
anti-tuberculosis and anti-cancer activities.29–34 Such broad
spectrum of therapeutic applications of oxadiazoles and structural
resemblance with potent known inhibitors have prompted us to
synthesize and evaluate the new derivatives of 1,3,4-oxadiazole-
2-thiones as potent TP inhibitors (Fig. 1). The proposed new
oxadiazoles contain N–H, C@N, O and CS groups and R substituents
which may interact to the enzyme active sites (His85, Ser186,
Lys190, Arg171, Tyr168, Phe210) in a similar pattern like TPI.
7DX was used and considered as a reference compound in the
current in vitro TP inhibition studies. In this paper we report the
synthesis, TP inhibition, structure activity relationship (SAR) and
docking studies of new 1,3,4-oxadiazole derivatives.
instance, alkyl groups and halogens such groups as Cl and Br. All
these groups in conjunction can show important contributions to-
wards TP inhibition activity.
Compound 3h showed the lowest IC50 value of 14.40 2.45 lM
compared
to
the
standard
inhibitor
7-deazaxanthine
(38.68 4.42
lM). With the lowest IC50 value, it was the most ac-
tive compound against the enzyme and proved better than the
standard inhibitor. From TP inhibitory model studies,37 we may
conclude that either core moiety or side chain group –CH2NH–
may interact and inhibit the activity. Compounds 3a–3u were pre-
pared in order to determine the effect of varying R2 substituents at
5
(R1)-substituted-1,3,4-oxadiazole for biological activity (see
chemical structures in Table 1). A selection of R2 was designed to
explore the effect of the substituents on the phenyl ring in the
compounds 3a–3u. Therefore selected primary amines (Table 1)
were used to synthesize the corresponding Mannich bases 3a–
3u. The compound 3a (R1 = phenyl; R2 = 200-methoxyphenyl) with
a
methoxy group at ortho position exhibited IC50 value
87.24 2.08 M, which was improved to IC50 40.64 0.23 in the
l
case of its analogue having methoxy group at meta position (entry
2, Table 1). Similarly in compound 3d with R2 = 300,400-dimethyl-
phenyl showed good activity with IC50 value 40.61 3.17 lM.
The enhancement in enzyme inhibitory activity which was ob-
served may be due to positive inductive effect of two methyl
groups. A significant decrease in enzyme inhibition was observed
in the case of compound 3c (R1 = phenyl; R2 = 200-chlorophenyl)
with IC50 value 117.70 3.70
ilar to compound 3o (R1 = 20-bromophenyl; R2 = 200-chlorophenyl)
which has IC50 value 21.83 3.95 M. This lower activity is proba-
bly due to lack of bromine group at ortho position of benzene moi-
ety in compound 3c. Compound 3e with IC50 value
lM and however is structurally sim-
2. Results and discussion
2.1. Chemistry
l
117.19 2.38
69.32 0.43
l
M was found to be less potent than 3f (IC50
In our current research, a series of new 1,3,4-oxadiazoline-2-thi-
one derivatives 3a–3u bearing different level of substituents were
prepared from compounds 2a–2f according to the procedure given
in Scheme 1.18 The precursors 5-substitituted 1,3,4-oxadiazole-2-
thiones 2a–2f were prepared according to a previously reported
method from the reaction of acid hydrazide with carbon disulfide
in the presence of KOH under microwave irradiation.28,35 A variety
of N-20 substituted derivatives 3a–3u were synthesized in good
to excellent yields (43–95%) from formalin and a selected range of
primary amines (Scheme 1). All final compounds were structurally
characterized by IR, NMR, EIMS and elemental analysis.
l
M) possibly it is due to unfavorable interactions with
TP. Possibly increased inhibitory activity of 3f (R1 = 30,40,50-trime-
thoxyphenyl; CH2CH2–Ph) was observed as a result of the presence
of flexible CH2CH2–Ph chain in 3f (Table 1, entries 5 & 6). It seems
enzyme inhibition is also dependent on flexibility of side chain. In
compound 3g, the presence of R1 = 20-hydroxyphenyl and R2 = 200-
chlorophenyl resulted in substantial decrease in inhibitory activity
(IC50 = 130.50 0.87) (Table 1, entry 7). In compound 3h the pres-
ence of strongly electron donating methoxy group at postion-200
and the electron withdrawing nature of polar nitro group at posi-
tion-500 of phenyl ring has improved potency of the compound
which is in fact greater than the standard used in this study. Typ-
ically, a nitro substituent at position-500 in compound 3h appeared
to be important for activity (Table 1, entry 8). Based on this activ-
ity, and selecting 3h as a lead from our first library, we set about
probing the chemical space around the NH–Ar by including a vari-
ety of substituents of varying size at aromatic ring, whilst retaining
the 4’-methylphenyl substituent at C-5 in compounds 3h–3n (Ta-
ble 1, entries 8–14).). In comparison with compound 3m (IC50
83.19 0.50), compound 3l (133.77 0.54) has been observed to
exhibit lower activity which clearly states that dimethyls– are bet-
ter substituents as compare to dichloro– (Table 1, entries 12 and
13). Compound 3h was found to be most active in the tested series
2.2. Biological activities
Twenty one compounds (3a–3u) were evaluated for TP inhibi-
tory activity. Interestingly, all compounds were active and showed
TP inhibition with IC50 values ranged between 14.40 2.45 and
173.23 3.04 lM. Enzyme inhibition data is presented in Table 1.
7-Deazaxanthine was used as a standard inhibitor.
In order to address the structure–activity relationship and to
achieve potent TP inhibitors various degrees of substituents on
phenyl have been introduced. These substituents include electron
donating group such as methoxy and electron withdrawing group
like nitro. Hydrophobic moieties are also of our interest, for
with IC50 14.40 2.45 l
M, in this case especially 500-nitrophenyl is
considered to be an important group which has maximally in-
creased the activity (see Section 2.3 for details). In fact substituents
in the R2 has greater impact on activity, since weak enzyme inhib-
R2
Microwave
O
N
NH
NH
HCHO, EtOH
4
3
irradiation
itory activity (IC50 = 104.12 0.75 lM) was unveiled by compound
N
N
R1
S
R1
NHNH2
2
R2 NH2
43-95%
O
5
CS2, KOH,
Al2O3
3j with un-substituted phenyl group (Table 1, entry 10) as compare
to other compounds bearing substituted phenyl group (Table 1
entries 8, 9 and 11–14).
R1
S
O
1
1a-1f
2a-2f
3a-3u
The nature and position of halogen groups in the R1 and R2 have
been found to be very important in enzyme inhibition activity. For
example, compound 3q with 20-bromophenyl and 200-methoxyphenyl
21 Compounds
Scheme 1. Synthetic protocol of 1,3,4-oxadiazoline-2-thione derivatives 3a–3u.

1010
S. A. Shahzad et al. / Bioorg. Med. Chem. 22 (2014) 1008–1015
Table 1
TP inhibitory activities of Mannich bases of 5-substituted-1,3,4-oxadiazole-2-thiones 3a–3u in term of IC50 values (
l
M)
R2
NH
HCHO, EtOH, R2-NH2
N
NH
4
N
3
N
R1
S
2
5
O
43−95%
R1
S
O
1
2a-2f
3a-3u
R1
R2
Yield (%)
Temperature (°C)
Time (min)
IC50 (lM)
a
Entry
Compounds
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
Phenyl
Phenyl
Phenyl
Phenyl
200-Methoxyphenyl
300-Methoxyphenyl
200-Chlorophenyl
300,400-Dimethylphenyl
200-Methylphenyl
200-phenylethyl
95
88
90
83
80
78
77
43
83
81
62
71
74
76
93
68
94
73
51
82
87
—
RT
RT
RT
RT
RT
RT
RT
30
RT
RT
RT
30
RT
RT
RT
RT
RT
RT
RT
30
RT
—
5
4
10
8
2
5
4
30
9
8
7
12
6
2
3
7
87.24 2.08
40.64 0.23
117.70 3.70
40.61 3.17
117.19 2.38
69.32 0.43
130.50 0.87
14.40 2.45
54.72 0.47
104.12 0.75
23.70 0.97
133.77 0.54
83.19 0.50
84.40 1.38
21.83 3.95
30,40,50-Trimethoxyphenyl
30,40,50-Trimethoxyphenyl
20-Hydroxyphenyl
40-Methylphenyl
40-Methylphenyl
40-Methylphenyl
40-Methylphenyl
40-Methylphenyl
40-Methylphenyl
40-Methylphenyl
20-Bromophenyl
20-Bromophenyl
20-Bromophenyl
40-Chlorophenyl
40-Chlorophenyl
40-Chlorophenyl
40-Chlorophenyl
200-Chlorophenyl
200-Methoxy-500-nitrophenyl
400-Methylphenyl
Phenyl
200,300-Dimethyl
300,400-Dichlorophenyl
300,400-Dimethylphenyl
300-Methoxyphenyl
200-Chlorophenyl
400-Bromophenyl
200-Methoxyphenyl
200-Methylphenyl
400-Bromophenyl
200-Chlorophenyl
200-Methoxyphenyl
Standard inhibitor
7-Deazaxanthine
26.33 3.28
4
17.60 1.07
58.21 5.01
173.23 3.04
46.38 3.42
88.73 2.81
38.68 4.42
10
13
9
7
—
3s
3t
3u
a
Enzyme inhibition IC50 values are means of three independent experiments (mean SEM, n = 3).
showed quite high activity (IC50 17.60 1.07
pound 3o having 20-bromophenyl and 200-chlorophenyl also exhibited
good activity with IC50 21.83 3.95 M. However, Mannich bases 3r–
3u where R1 is 4-chlorophenyl displayed reduced activity with IC50
values (46.38 3.42 M to 173.23 3.04 M).
l
M) and similarly, com-
uracil (TPI). Both of them were crystalized with TP (PDB codes:
4EAD and 1UOU, respectively). Docking procedure reproduced origi-
nal arrangement of native ligand ONP in E. coli TP (4EAD) very well
with low rmsd value (rmsd = 0.8). In case of docking TPI to the same
enzyme, all interactions from its native complex (with human TP,
1UOU) occurred in complex with E. coli TP. To calculate rmsd value
both enzymes (E. coli and human TP) were superimposed in respect
of the most important amino acids from the active site (His85,
Ser186, Lys190, Arg171, Tyr168, Phe210) and then docked pose
was fully compared with original state. Comparison showed good
fit with relatively low rmsd. Validation confirmed that docking
parameters were set properly and enabled to start docking of
1,3,4-oxadiazole-2-thione derivatives and 7DX.
l
l
l
2.3. Molecular modelling
PDB database contains a few three-dimensional structures of en-
zyme from two different sources: human and Escherichia coli.41
Among them 4EAD complex was selected for the analysis of the
binding mode of the most active derivative 3h and the assay refer-
ence compound—7-deazaxanthine (7DX). This one, recently pub-
lished in PDB, was E. coli TP with high resolution of 1.50 Å—
enzyme of the same source as used in biological experiments. TP
Structure of 7-deazaxanthine can be treated as two condensed
parts—uracil (pyrimidine-dione) and pyrrol. Due to high similarity
to substrate, 7DX interacted in a similar manner. The NH and CO
groups created hydrogen bonds with Arg171, Ser186 and Lys190,
contains important
a and a/b domains which are essential for the
enzymatic activity as they are necessary for closing the active site
cleft for action to take place.42 This kind of change facilitates the
nucleophilic attack of phosphate onto the sugar ring.43 There are
three forms of enzyme: the most open, transition and the most
closed.42 Complexes with inhibitors—substrate analogues usually
occur in the most closed form such as 4EAD which contains 30-azi-
do-20-fluoro-dideoxyuridine (ONP). The inhibitor binds in very char-
acteristic way. The pyrimidine base part creates some hydrogen
bonds with Arg171, Ser186, Lys190 and a few water molecules, it
also stacks to Tyr168, meanwhile the sugar part interacts with
Phe210 and water molecules. The crystal structure also contains a
sulfate ion in the phosphate binding site because of crystallization
conditions. Before any calculations, the sulfate ion was replaced by
phosphate in its dihydrogen form as suggested literature.44,45 Dock-
ing of novel compounds was preceded by validation process which
was based on two reference inhibitors: 30-azido-20-fluoro-dideoxy-
uridine (ONP) and 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]
and the ring of molecule formed
strength of binding was assessed by GoldScore function and for
7-deazaxanthine obtained value 63.78 (IC50 38.68 M).
p-p stacking with Tyr168. The
l
The most active compounds from the whole series (3h, 3k, 3o–
3q) represented similar general binding mode. R2 substituent was
located in pyrimidine base region, oxadiazoline-thione in the sugar
region (middle part) and R1 substituent projected towards phos-
phate binding site. The docking runs for them were better con-
verged in comparison with other derivatives. The differences in
the binding mode concerned the substituents present in both phe-
nyl rings from R1 and R2. The most active compound 3h was ana-
lyzed in detail. It was assessed by GoldScore better than 7DX.
The score value obtained 82.60 (IC50 14.40 lM) which stayed in
accordance with experimental results. The schematic and detailed
binding mode of this derivative is presented in the Figure 2. The
highest activity of inhibitor 3h was provided by the presence of

S. A. Shahzad et al. / Bioorg. Med. Chem. 22 (2014) 1008–1015
1011
nitro group which could form H-bonds with Arg171 and Ser186.
4. Experimental
The phenyl ring from R2 substituent interacted with protonated
amine group of Lys190 and with side chain of Tyr168. The oxygen
atom from oxadiazoline ring formed hydrogen bond with
HO(Thr120). Thione and methoxy groups were directed towards
Leu117 and created hydrophobic interactions. R1 substituent could
provide some less important ‘aromatic’ hydrogen bonds (CHA
O@C(Gly89), CH—phosphate).
Comparing 7DX and 3h, the reference compound due to the
smaller molecule occupied only area where methoxy-nitrophenyl
moiety of derivative 3h was located.
4.1. 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 spectrophotom-
eter. EI mass spectra data were recorded with various MAT 711
(70 eV) spectrophotometers and data are tabulated as m/z. 1H
NMR spectra were recorded in CD3COCD3, CDCl3 and DMSO-d6
using Bruker AC (300 and 400, 500 MHz) spectrometers, respec-
tively. Splitting patterns were as follows s (singlet), d (doublet),
dt (doublet of triplet), dd (double doublets), t (triplet), and m (mul-
tiplet). Chemical shifts are reported in d (ppm) and coupling
constants are given in Hz. The progress of all reactions was moni-
tored by TLC, which was performed on 2.0 ꢀ 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. Conclusion
Twenty one new Mannich bases possessing the 1,3,4-oxadiazole
core have been effectively synthesized and most of them have been
identified as modest TP inhibitors. A number of derivatives have
been found to be more active as compared to reference compound
7DX. SAR demonstrated that the increase or decrease in TP inhib-
itory activities is very much dependent on the nature and position
of substituents in the R1 and R2. Compound 3h bearing the nitro
moiety was the most potent among the tested oxadiazoles having
Compounds 2a–2f were prepared according to our previously
reported procedures (29)
an IC50 value 14.40 2.45 lM which is about 2.5 fold more active
4.2. General procedure for the synthesis of 5-substituted-1,3,4-
oxadiazole-2-thione (2a–2f)
than 7DX. Molecular modelling not only explained the binding
mode of more active compounds (3h, 3k, 3o–3q) among the series
but it has also exhibited the importance of nitro group in the most
active compound 3h, which can possibly generate H-bonds with
Arg171 and Ser186. Thus, the current research opens a new direc-
tion for design and development of new and more potent TP inhib-
itors with promising anti-tumor activity.
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. 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 TeflonÒ vessel. Micro-
wave-irradiation (MW domestic type oven 900 W with a frequency
2450 MHz, Dawlance, Pakistan) was applied for 3–7 min. After the
completion of reaction (TLC analysis), ethanol was added into reac-
tion 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 and then recrystallized from ethanol/water
(50:50) mixture to afford compounds 2a–2f. The compounds 2a–
2f were completely characterized and data was in good match with
previous reports.28,35
4.3. General procedure for the preparation of 3-(substituted
amino methyl)-5-(substituted)-1,3,4-oxadiazoline-2-thione
(3a–3u)
Formalin 40% (0.75 ml, 0.01 mol) was added to a stirred solution
of 5-substituted 1,3,4-oxadiazole-2-thione 2a–2f (0.01 mol) in
absolute ethanol (25 ml). An ethanolic solution (10 ml) of appro-
priate amine (0.01 mol) was added portion wise to the reaction
mixture, stirred for 3–6 h at room temperature, and left overnight
in a refrigerator. The precipitate formed was filtered, washed with
cold ethanol, dried, and recrystallized from the suitable solvent.18
4.3.1. 3-(2-Methoxyphenylaminomethyl)-5-(phenyl)-1,3,4-
oxadiazoline-2-thione (3a)
Yield: 2.97 g (95%); mp: 127 °C; Rf = 0.60 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1616 (C@N), 1595
(C@C), 1438 (C@S), 1249 and 1186 (C–O–C); 1H NMR (300 MHz,
CD3COCD3): d 7.70–7.51 (m, 5H, H–Ar), 7.28 (dd, 1H, J = 6.0,
J = 1.5 Hz, H-600), 7.05 (m, 1H, H-500), 6.94 (d, 1H, J = 5.8, H-300),
6.89 (dd, 1H, J = 6.0, 5.8 Hz, H-400), 5.79 (bt, 1H, J = 7.4 Hz, NH),
5.62 (d, 2H, J = 7.4 Hz, CH2), 3.91 (s, 3H, OCH3); EI MS: m/z (rel
abund.%): 177 (25), 136 (15), 122 (7), 121 (6), 117 (100), 107 (3),
105 (25), 103 (41), 92 (24), 91 (38), 77 (81), 51 (82); Anal. Calcd
Figure 2. Schematic and detailed representation of binding mode for compound 3h.
Upper part shows the most important interactions (2 H-bonds and cation-
interaction), meanwhile lower one presents all H-bonds.
p

1012
S. A. Shahzad et al. / Bioorg. Med. Chem. 22 (2014) 1008–1015
for C16H15O2N3S (313.37): C, 61.32; H, 4.82, N, 13.41. Found: C,
61.34; H, 4.79, N, 13.44.
(t, 2H, J = 6.7 Hz, CH2); EI MS: m/z (rel abund.%): 281 (7), 267
(26), 193 (100), 150 (11), 134 (10), 133 (6), 120 (2), 105 (17),
104 (10), 77 (10); Anal. Calcd for: C20H23N3 O4S (401.48): C,
59.83; H, 5.77; N, 10.47. Found: C, 59.85; H, 5.71; N, 10.46.
4.3.2. 3-(300-Methoxyphenylaminomethyl)-5-(phenyl)-1,3,4-
oxadiazoline-2-thione (3b)
Yield: 2.75 g (88%); mp: 156–158 °C; Rf = 0.64 (ethyl acetate/
4.3.7. 3-[200-Chlorophenylaminomethyl]-5-(20-hydroxyphenyl)-
1,3,4-oxadiazoline-2-thione (3g)
hexane/chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1606 (C@N),
1590 (C@C), 1445 (C@S), 1250 and 1176 (C–O–C); 1H NMR
(300 MHz, CD3COCD3): d 7.71–7.62 (m, 5H, Ar–H0), 7.29 (1H, d,
J = 7.3 Hz, H-600), 7.37 (t, 1H, J = 7.3 Hz, H-500), 6.97 (d, 1H, J = 7.3
Hz, H-400), 6.89 (s, 1H, H-200), 6.1 (t, 1H, J = 7.2 Hz, NH), 5.57 (d,
J = 7.2 Hz, 2H, CH2), 3.91 (s, 3H, OCH3); EI MS: m/z (rel abund.%):
177 (11), 136 (8), 122 (34), 108 (12), 105 (33), 103 (100), 91
(10), 77 (18); Anal. Calcd C16H15O2N3S (313.37): C, 61.32; H, 4.82,
N, 13.41. Found: C, 61.36; H, 4.78, N, 13.45.
Yield: 2.56 g (77%); mp: 141 °C; Rf = 0.57 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1621 (C@N), 1572
(C@C), 1431 (C@S), 1243 and 1146 (C–O–C), 755; 1H NMR
(400 MHz, CDCl3): d 8.84 (br s, 1H, OH), 7.6 (m, 2H, Ar–H), 7.90
(ddd, 1H, J = 8.1, J = 5.0, J = 1.5 Hz, H-60), 7.56 (d, 1H, J = 8.9 Hz,
H-300), 7.07 (dd, 1H, J = 8.1, J = 6.4 Hz, H-50), 7.08 (d, 1H, J = 8.2 Hz,
H-30), 6.66 (ddd, 1H, J = 7.2, 2.13, 1.8 Hz, H-500), 6.61 (dd, 1H,
J = 7.2, J = 1.7 Hz, H-600), 5.84 (bt, 1H, J = 6.4 Hz, NH), 5.66 (d, 2H,
J = 6.4 Hz, CH2); EI MS: m/z (rel abund.%): 193 (3), 176 (55), 142
(3), 140 (10), 133 (29), 128, 126, 113, 111, 121 (5), 119 (16), 105
(3), 91 (47), 76 (15); Anal. Calcd C15H12ClN3O4S (333.79): C,
53.97; H, 3.62; N, 12.59. Found: C, 53.96; H, 3.60; N, 12.52.
4.3.3. 3-[200-Chlorophenylaminomethyl]-5-{phenyl}-1,3,4-
oxadiazoline-2-thione (3c)
Yield: 2.86 g (90%); mp: 122 °C; Rf = 0.58 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1626 (C@N), 1585
(C@C), 1448 (C@S), 1249 and 1196 (C–O–C); 1H NMR (300 MHz,
CD3COCD3): d 7.69–7.61 (m, 5H, Ar–H0), 7.39 (d, 1H, J = 6.4 Hz,
H-300), 7.33 (dd, 1H, J = 6.5, J = 6.1 Hz, H-500), 6.86 (m, 1H, H-400),
6.83 (dd, 1H, J = 6.6, 1.2 Hz, H-300), 6.08 (bt, 1H, J = 6.8 Hz, NH),
5.60 (d, J = 6.8 Hz, 2H, CH2); EI MS: m/z (rel abund.%): 177 (5),
142 (18), 140 (40), 128 (7), 126 (19), 113 (8), 111 (22), 105 (14),
4.3.8. 3-[200-Methoxy-500-nitrophenylaminomethyl]-5-{40-
methylphenyl}-1,3,4-oxadiazoline-2-thione (3h)
Yield: 1.59 g (43%); mp: 167 °C; Rf = 0.56 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1610 (C@N), 1590
(C@C), 1422 (C@S), 1243 and 1178 (C–O–C), 767; 1H NMR
(300 MHz, CD3COCD3): d 7.89 (s, 1H, H-600), 7.84 (d, 1H, J = 7.9
Hz, H-400), 7.51 (d, 1H, J = 7.9 Hz, H-300), 7.34 (d, 2H, J = 7.7 Hz,
H-20/60), 7.19 (d, 2H, J = 7.7 Hz, H-30/50), 5.55 (s, 2H, CH2), 3.91 (s,
3H, OCH3), 2.33 (s, 3H, CH3); EI MS: m/z (rel abund.%): 191 (52),
150 (27), 136 (18), 132 (100), 121 (34), 119 (30), 117 (10), 108
(11), 104 (15), 102 (1), 94 (13), 91 (47), 90 (38), 65 (17), 62 (6);
Anal. Calcd for: C17H16N4O4S (372.40): C, 54.83; H, 4.33; N, 15.04.
Found: C, 54.86; H, 4.37; N, 15.00.
103 (10), 77 (20), 51 (100) Anal. Calcd for
C15H12N3OSCl
(317.79): C, 56.69; H, 3.81; N, 13.22. Found; C15H12N3OSCl: C,
56.65; H, 3.84; N, 13.26.
4.3.4. 3-(300,400-Dimethylphenylaminomethyl)-5-(phenyl)-1,3,4-
oxadiazoline-2-thione (3d)
Yield: 2.58 g (83%); mp: 175 °C; Rf = 0.68 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1650 (C@N), 1599
(C@C), 1448 (C@S), 1269 and 1198 (C–O–C);
4.3.9. 3-[400-Methylphenylaminomethyl]-5-{4-methylphenyl}-
1,3,4-oxadiazoline-2-thione (3i)
1H NMR (300 MHz, CD3COCD3): d 7.78–7.64 (m, 5H, Ar–H0), 7.33
(d, 1H, J = 7.4 Hz, H-500), 7.18 (d, 1H, J = 7.4 Hz, H-600), 6.91 (s, 1H, H-
200), 5.90 (t, 1H, J = 7.5 Hz, -NH), 5.56 (d, 2H, J = 7.5 Hz, CH2), 2.76 (s,
6H, 2CH3 EI MS: m/z (rel abund.%): 177 (23), 134 (36), 120 (62), 106
(6), 105 (41), 103 (100), 90 (22), 77 (24), 75 (53); Anal. Calcd for
Yield: 2.58 g (83%); mp: 110 °C; Rf = 0.63 (ethyl acetate/hexane/
chloroform = 1:1:0.5); FTIR (KBr)
mmax: 3409 (NH), 1627 (C@N),
1342 (C@S), 1018 (C–O); 1H NMR (400 MHz, CDCl3): d 7.61 (d,
2H, J = 7.6 Hz, H-300/500), 7.74 (d, 2H, J = 8.1 Hz, H-20/60), 7.49 (d,
2H, J = 8.1 Hz, H-30/50), 7.30 (d, 2H, J = 7.6 Hz, H-200/600), 5.56 (br d,
2H, J = 6.0 Hz, CH2), 5.54 (bt, 1H, J = 6.0 Hz, NH), 2.38 (s, 3H, CH3),
2.29 (s, 3H, CH3); EI MS: m/z (rel abund.%): 191 (51), 132 (100),
120 (7), 119 (20), 117 (9), 105 (6), 104 (12), 102 (1), 91 (45), 65
(15), 91 (50); Anal. Calcd for: C17H17N3OS (311.40); C, 65.57; H,
5.50, N,13.49. Found: C, 65.59; H, 5.54; N,13.51.
C17H17ON3S (311.40): C, 65.57; H, 5.50; N, 13.49. Found: C,
65.59; H, 5.45; N, 13.54.
4.3.5. 3-(2-Methylphenylaminomethyl)-5-(3,4,5-trimethoxy
phenyl)-1,3,4-oxadiazoline2-thione (3e)
Yield: 3.09 g (80%); mp: 129 °C; Rf = 0.62 (ethyl acetate/hexane/
chloroform = 1:1: 0.5); IR (m
max, KBr, cmꢁ1): 1636 (C@N), 1575
(C@C), 1438 (C@S), 1239 and 1166 (C–O–C); 1H NMR (400 MHz,
CD3COCD3): d 7.06-7.04 (m, 4H, Ar–H), 6.66 (ddd, overlapped,
1H, H-400), 6.65 (ddd, 1H, J = 6.1, 5.9, 3.7 Hz, H-600), 5.83 (t, 1H,
J = 7.1 Hz, NH), 5.58 (d, 2H, J = 7.1 Hz, CH2), 3.83 (s, 9H, OCH3),
2.78 (s, 3H, CH3); EI MS: m/z (rel abund.%): 281 (6), 267 (100),
208 (32), 195 (45), 193 (70), 151, 150 (18), 136, 135 (13),
121, 120 (16), 107 (3), 92 (4), 91 (10), 76 (16). Anal. Calcd for:
4.3.10. 3-[Phenylaminomethyl]-5-{4-methylphenyl}-1,3,4-
oxadiazoline-2-thione (3j)
Yield: 2.40 g (81%); mp: 119 °C; Rf = 0.66 (ethyl acetate/hexane/
chloroform = 1:1:0.5); FTIR (KBr)
mmax: 3409 (NH), 1636 (C@N),
1333 (C@S), 1016 (C–O); 1H NMR (400 MHz, CDCl3): d 7.74 (d,
2H, J = 8.1 Hz H-20/60), 7.66–7.50 (m, 5H, Ar–H00), 7.30 (d, 2H,
J = 8.1 Hz, H-30/50), 5.71 (t, 1H, J = 6.7 Hz, NH), 5.53 (d, 2H, J = 6.7
Hz, CH2), 2.38 (s, 3H, CH3); EI MS: m/z (rel abund.%): 191 (51),
132 (100), 119 (20.25), 117 (9), 106 (13), 104 (12), 102 (2), 92
(3), 91 (45), 77 (21), 65 (15), 51 (10); Anal. Calcd for C16H15N3OS
(297.37): C, 64.62; H, 5.08; N, 14.13. Found: C, 64.55; H, 5.1, N,
14.16.
C19H21N3OS (387.45): C, 58.90; H, 5.46; N, 10.85. Found:
C, 58.88; H, 5.45; N, 10.86.
4.3.6. 3-[200-Phenylethylaminomethyl]-5-{30,40,50-trimethoxy
phenyl}-1,3,4-oxadiazoline-2-thione (3f)
Yield: 3.12 g (78%); mp: 132 °C; Rf = 0.64 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (
m
max, KBr, cmꢁ1): 1612 (C@N), 1592
4.3.11. 3-[200,300-Dimethylphenylaminomethyl]-5-{40-
methylphenyl}-1,3,4-oxadiazoline-2-thione (3k)
(C@C), 1418 (C@S), 1241 and 1179 (C–O–C), 755; 1H NMR
(400 MHz, CD3OCD3): d 7.28 (dd, 1H, J = 7.2, J = 3.1 Hz, H-300), 7.28
(dd, 1H, J = 6.2, J = 3.3 Hz, H-600), 7.24 (d, 1H, J = 7.7 Hz, H-200),
7.18 (m, 2H, Ar–H), 6.92 (s, 2H, H-20/60), 5.4 (s, 2H, Ha), 3.60 (t,
2H, J = 6.7 Hz, Hb, CH2), 3.98 (s, 1H, NH), 3. 81 (s, 9H, OCH3), 3.05
Yield: 2.01 g (62%); mp: 144 °C; Rf = 0.69 (ethyl acetate/hexane/
chloroform = 1:1:0.5); FTIR (KBr)
mmax: 3409 (NH), 1635 (C@N),
1331 (C@S), 1026 (C–O); 1H NMR (400 MHz, CDCl3): d 7.74 (d,
2H, J = 8.1 Hz, H-20/60), 7.51 (t, 1H, J = 7.7 Hz, H-500), 7.37–7.42

S. A. Shahzad et al. / Bioorg. Med. Chem. 22 (2014) 1008–1015
1013
(m, 2H, H-600/400), 7.30 (d, 2H, J = 8.1 Hz, H-30/50), 6.83 (bt, 1H, J = 6.8
Hz, NH), 5.60 (br d, 2H, J = 6.8 Hz, CH2), 2.38 (s, 3H, CH3), 2.34 (s, 3H,
CH3), 2.32 (s, 3H, CH3); EI MS: m/z (rel abund.%): 191 (48), 132
(100), 120 (6), 119 (24), 117 (11), 105 (7) (14), 104 (8), 102 (3),
91 (41), 76 (3), 65 (13); Anal. Calcd for: C18H19N3OS (325.43): C,
66.43; H, 5.88; N, 12.91. Found: C, 66.48; H, 5.84; N, 12.95.
(C@C), 1437 (C@S), 1266 and 1199 (C–O–C), 783; 1H NMR
(400 MHz, CDCl3): d 7.83 (dd, 1H, J = 7.6 Hz, 1.3 Hz, H-60), 7.76
(d, 1H, J = 7.2 Hz, H-40,), 7.53 (t, 1H, J = 7.2, Hz, H-30), 7.47 (m, 1H,
H-50); 7.36 (d, 2H, J = 7.7 Hz, H-200/600), 7.53 (d, 2H, J = 7.7 Hz, H-
300/500), 5.53 (s, 2H, CH2), 5.33 (br s, 1H, NH); EI MS: m/z (rel
abund.%): 257 (100), 255 (99), 197 (92), 195 (93), 186 (30), 185
(64), 184 (33), 183 (78), 181 (13), 172 (12), 170 (10), 157 (41),
155 (44), 76 (19), 50 (13); Anal. Calcd for C15H11Br2N3OS
(441.14): C, 40.84; H, 2.51; N, 9.53 Found: C, 40.84; H, 2.51; N,
9.53.
4.3.12. 3-[300,400-Dichlorophenylaminomethyl]-5-(40-methyl
phenyl)-1,3,4-oxadiazoline-2-thione (3l)
Yield:2.59 g (71%);mp: 137–139 °C; Rf = 0.59(ethyl acetate/hex-
ane/chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1622 (C@N), 1599
(C@C), 1448 (C@S), 1239 and 1166 (C–O–C), 766; 1HNMR (CDCl3):
d 7.78–7.05 (m, 3H, H-400/500/600), 7.36 (d, 2H, J = 8.9 Hz, H-20/60),
6.60 (d, 2H, J = 8.9 Hz, H-30/50), 5.67 (bt, 1H, NH), 5.00 (br d, 1H,
CH2), 2.4 (s, 3H, CH3); EI MS: m/z (rel abund.%): 191 (39), 178 (16),
176 (15), 174 (48), 164 (6), 162 (7), 160 (21), 141 (10), 139 (33),
132 (100), 119 (16), 117 (7), 104 (11), 102 (5), 91 (45), 90 (2) 65
(22), 91 (50), 76 (3); Anal. Calcd for: C16H13Cl2N3OS (366.26): C,
52.47; H, 3.58; N, 11.47. Found: C, 52.49; H, 3.54; N, 11.50.
4.3.17. 3-(2-Methoxyphenylaminomethyl)-5-(2-bromophenyl)-
1,3,4-oxadiazoline-2-thione (3q)
Yield: 3.66 g (94%); mp: 112 °C; Rf = 0.70 (ethyl acetate/hexane/
chloroform = 1:1:0.5); FTIR (KBr)
mmax: 3219 (NH), 1649 (C@N),
1063 (C–O–C); 1H NMR (500 MHz, CD3OD): d 7.82 (dd, 1H, J = 7.7,
1.6 Hz, H-60), 7.77 (d, 1H, J = 7.5 Hz, H-40), 7.52 (t, 1H, J = 7.5,
J = 7.5 Hz, H-30), 7.41 (m, IH, H-50); 7.31–7.36 (m, 2H, H-600/400),
7.63 (d, 1H, J = 7.9 Hz, H-300), 7.56 (t, 1H, J = 7.9 Hz, H-500), 5.60 (s,
2H, CH2), 5.51 (br s, 1H, NH), 3.91 (s, 3H, OCH3); EI MS: m/z (rel
abund.%): 257 (100), 255 (99), 197 (92), 195 (93), 185 (64), 183
(78), 181 (13), 157 (21), 155 (24), 76 (19), 50 (13); 136 (24), 122
(29), 121 (34), 107 (2), 92 (6) 91 (5); Anal. Calcd for C16H14BrN3O2S
(392.27): C, 48.99; H, 3.60; N, 10.71. Found: C, 48.95; H, 3.65; N,
10.75.
4.3.13. 3-[300,400-Dimethylphenylaminomethyl]-5-(40-methyl
phenyl)-1,3,4-oxadiazoline-2-thione (3m)
Yield: 2.40 g (74%); mp: 158 °C; Rf = 0.67 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1619 (C@N), 1596
(C@C), 1435 (C@S), 1250 and 1190 (C–O–C), 760; 1H NMR
(300 MHz, CD3COCD3): d 7.72 (d, 2H, J = 8.1 Hz, H-20/60), 7.22 (d, 2H,
J = 8.1 Hz, H-30/50), 7.12 (s, 1H, H-200), 7.39 (d, 1H, J = 7.7 Hz, H-500),
7.15 (d, 1H, J = 7.7 Hz, H-600), 5.71 (bt, 1H, NH), 5.55 (br d, 2H, CH2),
2.39 (s, CH3), 2.31 (s, 6H, 2CH3); EI MS: m/z (rel abund.%): 191 (44),
134 (4), 132 (100), 120 (7), 119 (20), 117 (12), 105 (11), 104 (15),
102 (6), 91 (6), 76 (5), 65 (19); Anal. Calcd for C18H19N3OS (325.42):
C, 66.43; H, 5.88; N, 12.91. Found C, 66.48; H, 5.84; N, 12.94.
4.3.18. 3-[200-Methylphenylaminomethyl]-5-{40-chlorophenyl}-
1,3,4-oxadiazoline-2-thione (3r)
Yield: 2.41 g (73%); mp: 140 °C; Rf = 0.60 ((ethyl acetate/hex-
ane/chloroform = 1:1:0.5); IR
(mmax,
KBr, cmꢁ1): 1641 (C@N),
1590 (C@C), 1455 (C@S), 1272 and 1161 (C–O–C), 785; 1H NMR
(500 MHz, DMSO-d6): d 7.88 (d, 2H, J = 8.6 Hz, H-20/60), 7.65 (d,
2H, J = 8.6 Hz, H-30/50), 7.31-7.44 (m, 4H, H-600/500/400/300), 5.51 (br
s, 1H, NH), 5.40 (t, 2H, CH2), 2.35 (s, 3H, CH3); EI MS: m/z (rel
abund.%): 213 (19), 211 (62), 179 (6), 153 (15), 151 (47), 141 (4),
139 (32), 137 (100), 120 (44), 117 (11), 106 (29), 102 (16), 90
(21), 76 (13); Anal. Calcd for C16H14ClN3OS (331.82): C, 57.91; H,
4.25; N, 12.66. Found C, 57.95; H, 4.20; N, 12.69.
4.3.14. 3-[300-Methoxyphenylaminomethyl]-5-(40-methyl
phenyl)-1,3,4-oxadiazoline-2-thione (3n)
Yield: 2.48 g (76%); mp: 140–142 °C; Rf = 0.61 (ethyl acetate/
hexane/chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1633 (C@N),
1597 (C@C), 1430 (C@S), 1244 and 1180 (C–O–C), 763; 1HNMR
(400 MHz, CDCl3): d 7.72 (d, 2H, J = 8.5 Hz, H-20/60), 7.22 (d, 2H,
J = 8.5 Hz, H-30/50), 7.19-7.15 (m, 2H, H-400/600), 7.18 (s, 1H, H-200),
7.39 (t, 1H, J = 7.8 Hz, H-500), 5.71 (t, 1H, J = 6.6 Hz, NH), 5.55 (d,
2H, J = 6.6 Hz, CH2), 3.46 (s, 3H, OCH3), 2.39 (s, 3H, CH3); EI MS:
m/z (rel abund.%): 191 (45), 136 (41, 132 (100), 122 (12), 121
(29), 119 (16), 117 (6), 107 (23), 104 (14), 102 (9), 91 (47), 65
(28), 76 (3), 65 (13)); Anal. Calcd for C17H17N3O2S (327.40): C,
62.36; H, 5.23; N, 12.83. Found: C, 62.39; H, 5.22; N, 12.81.
4.3.19. 3-(4-Bromophenylaminomethyl)-5-(4-chlorophenyl)-
1,3,4-oxadiazoline-2-thione (3s)
Yield: 2.01 g (51%); mp: 143 °C; Rf = 0.63 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1651 (C@N), 1586
(C@C), 1445 (C@S), 1263 and 1180 (C–O–C), 750; 1H NMR
(500 MHz, DMSO-d6): d 8.09 (d, 2H, J = 7.4 Hz, H-20/60), 7.75 (d,
2H, J = 7.4 Hz, H-30/50), 7.55 (d, 2H, J = 7.9 Hz, H-200/600), 7.66 (d,
2H, J = 7.9 Hz, H-300/500), 6.01 (bt, 1H, NH), 5.53 (d, 2H, J = 7.2 Hz,
CH2); EI MS: m/z (rel abund.%): 213 (33), 211 (100), 179 (5), 186
(59), 184 (62), 172 (100), 170 (98), 167 (6), 165 (8), 153 (27),
151 (82), 141 (4), 139 (14), 137 (14), 117 (5), 102 (11), 76 (17);
Anal. Calcd for C15H11Cl Br N3OS (396.69): C, 45.42; H, 2.79; N,
10.59. Found: C, 45.46; H, 2.74; N, 10.63.
4.3.15. 3-[200-Chlorophenylaminomethyl]-5-(2’-bromophenyl)-
1,3,4-oxadiazoline-2-thione (3o)
Yield: 3.62 g (93%); mp: 143 °C; Rf = 0.71 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1609 (C@N), 1574
(C@C), 1411 (C@S), 1250 and 1166 (C–O–C), 740; 1H NMR
(500 MHz, CDCl3 + CD3OD): d 7.85(dd, 1H, J = 7.9, 1.9 Hz, H-60),
7.79 (d, 1H, J = 6.5 Hz, H-3’’), 7.78 (d, 1H, J = 7.5 Hz, H-30), 7.52 (t,
1H, J = 7.5, 7.5 Hz, H-40), 7.44 (m, 1H, H-50), 7.42–7.37 (m, 3H,
H-4’’/5’’/6’’), 6.01 (s, 2H, CH2), 5.57 (br s, 1H, NH); EI MS: m/z (rel
abund.%): 257 (60), 255 (59), 197 (47), 195 (46), 185 (9), 183
(15), 181 (6), 157 (7), 155 (8), 142 (34), 140 (11), 128 (65), 126
(22), 113 (18), 111 (7), 105 (45), 91 (53), 76 (27), 50 (8); C15H11-
BrClN3OS (396.69): Anal. Calcd for C, 45.42; H, 2.79; N, 10.59.
Found: C, 45.40; H, 2.76; N, 10.55.
4.3.20. 3-[2-Chlorophenylaminomethyl]-5-{4-chlorophenyl}-
1,3,4-oxadiazoline-2-thione (3t)
Yield: 2.88 g (82%); mp: 156 °C; Rf = 0.62 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1643 (C@N), 1592
(C@C), 1440 (C@S), 1239 and 1169 (C–O–C), 767; 1H NMR
(500 MHz, DMSO-d6): d 7.78 (d, 2H, J = 8.0 Hz, H-20/60), 7.61 (d,
2H, J = 8.0 Hz, H-30/50), 7.56–7.30 (d, 4H, J = 7.8 Hz, H-600/ 500/400/
300), 5.57 (bt, 1H, NH), 5.22 (bt, 2H, J = 7.0 Hz, CH2); EI MS: m/z
(rel abund.%): 213 (35), 211 (100), 179 (3), 153 (30), 151 (82),
142 (48), 141 (4), 140 (15), 139 (14), 137 (14), 126 (2), 117 (5),
111 (4), 109 (14), 105 (17), 102 (8), 76 (17); Anal. Calcd for C15H11-
Cl2N3OS (352.23): C, 51,15; H, 3.15; N, 11.93. Found: C, 51,19; H,
3.11; N, 11.89.
4.3.16. 3-[400-Bromophenylaminomethyl]-5-(2’-bromophenyl)-
1,3,4-oxadiazoline-2-thione (3p)
Yield: 2.99 g (68%); mp: 157 °C; Rf = 0.65 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1619 (C@N), 1590

1014
S. A. Shahzad et al. / Bioorg. Med. Chem. 22 (2014) 1008–1015
4.3.21. 3-[2-Methoxyphenylaminomethyl]-5-{4-chlorophenyl}-
1,3,4-oxadiazoline-2-thione (3u)
References and notes
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Yield: 3.01 g (87%); mp: 149 °C; Rf = 0.66 (ethyl acetate/hexane/
chloroform = 1:1:0.5); IR (m
max, KBr, cmꢁ1): 1608 (C@N), 1581
(C@C), 1427 (C@S), 1252 and 1176 (C–O–C), 742; 1H NMR
(500 MHz, DMSO-d6): d 7.71 (d, 2H, J = 8.5 Hz, H-20/60), 7.65 (d,
2H, J = 8.5 Hz, H-30/50), 7.41–7.46(m, 2H, H-600/400), 7.31–7.37 (m,
2H, H-300/500), 5.58 (br s, 1H, J = 7.0 Hz, NH), 5.30 (bt, 2H, J = 7.0
Hz, CH2), 3.91 (s, 3H, OCH3);i EI MS: m/z (rel abund.%): 213 (35),
211 (100), 179 (3), 153 (26), 151 (82), 141 (4), 139 (14), 137
(14), 117 (5), 102 (8), 76(7) 136 (9), 122 (21), 107 (13), 91 (11),
76 (41); Anal. Calcd for C16H14.ClN2OS (347.82): C, 55.25; H, 4.06;
N, 12.08. Found: C, 55.29; H, 4.00; N, 12.1.
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4.4. Thymidine phosphorylase inhibition
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TP/PD-ECGF (E. coli TP (Sigma T6632)) activity was determined
by measuring the absorbance at 290 nm spectrophotometrically.
The original method reported by Krenitsky (Krenitsky et al.,
1979)36 was modified. Briefly, in 96 wells, flat bottom, microplate
with each well capacity 200
pared which contained 145
l
l
l, reaction mixture of 200
l of potassium phosphate buffer (pH
ll was pre-
7.4), 20
substrate, 30
tion 0.05 and 0.002 U, respectively, were incubated with 5
l
l of 1.5 mM Thymidine 50 mono phosphate solution as
ll of enzyme (E. coli TP (Sigma T6632) at concentra-
ll of
test materials for 10 min at 25 °C in temperature controlled incu-
bator before taking readings by microplate reader (SpectraMax
Plus384, USA) at 290 nm. The wells containing reaction mixture de-
void of substrate were blank and the mean OD of these blank wells
was substracted from wells containing reaction mixture with
substrate. The readings were taken continuously after 10, 20, and
30 min by microplate reader. All assays were performed in triplicate.
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Corina online tool46 was applied to creating three-dimensional
structure of compounds. Gasteiger–Marsili charges were assigned
by Sybyl-X 1.147 following check of atom types and protonation
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the mol2 format.
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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 Hermes 1.5.48 The presence of water molecules
within 5 Å from ONP was also taken into account. They were set
as toggle.
30. Naveena, C. S.; Boja, P.; Kumari, N. S. Eur. J. Med. Chem. 2010, 45, 4708.
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Docking was performed using Gold 5.1 program.49 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 PyMOL and Maestro.50,51
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Acknowledgments
The authors are grateful to the Higher Education Commission
(HEC) Pakistan for providing financial support under TTS program.
Molecular modeling studies were financially supported by Polish
National Center of Science, Postdoctoral Research Grant No.
DEC-2012/04/S/NZ2/00116. We also acknowledge Prof Dr M.I.
Choudhary and Prof. Dr. K.M. Khan for their help and support
during this research.
43. El Omari, K.; Bronckaers, A.; Liekens, S.; Perez-Perez, M. J.; Balzarini, J.;
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48. Hermes 1.5, The Cambridge Crystallographic Data Centre: Cambridge, UK,
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49. Gold 5.1, The Cambridge Crystallographic Data Centre: Cambridge, UK, 2011.
50. PyMOL 0.99rc6, DeLano Scientific LLC: Palo Alto, CA, USA, 2006.
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