Oxadiazoles and thiadiazoles: Novel α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.07.032

Oxadiazoles and thiadiazoles 1-37 were synthesized and evaluated for the first time for their α-glucosidase inhibitory activities. As a result, fifteen of them 1, 4, 5, 7, 8, 13, 17, 23, 25, 30, 32, 33, 35, 36 and 37 were identified as potent inhibitors of the enzyme. Kinetic studies of the most active compounds (oxadiazoles 1, 23 and 25, and thiadiazoles 35 and 37) were carried out to determine their mode of inhibition and dissociation constants K_i. The most potent compound of the oxadiazole series (compound 23) was found to be a non-competitive inhibitor (K_i= 4.36 ± 0.017 μM), while most potent thiadiazole 35 was identified as a competitive inhibitor (K_i= 6.0 ± 0.059 μM). The selectivity and toxicity of these compounds were also studied by evaluating their potential against other enzymes, such as carbonic anhydrase-II and phosphodiesterase-I. Cytotoxicity was evaluated against rat fibroblast 3T3 cell line. Interestingly, these compounds were found to be inactive against other enzymes, exhibiting their selectivity towards α-glucosidase. Inhibition of α-glucosidase is an effective strategy for controlling post-prandial hyperglycemia in diabetic patients. α-Glucosidase inhibitors can also be used as anti-obesity and anti-viral drugs. Our study identifies two novel series of potent α-glucosidase inhibitors for further investigation.

Full text of DOI:10.1016/j.bmc.2014.07.032

Bioorganic & Medicinal Chemistry 22 (2014) 5454–5465
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Oxadiazoles and thiadiazoles: Novel a-glucosidase inhibitors
Hamdy Kashtoh a, Shafqat Hussain b, Ajmal Khan b, Syed Muhammad Saad b, Jalaluddin A. J. Khan a,
c
a,b,
Khalid Mohammed Khan b, , Shahnaz Perveen , M. Iqbal Choudhary
⇑
⇑
a Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21412, Saudi Arabia
b H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
c PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxadiazoles and thiadiazoles 1–37 were synthesized and evaluated for the first time for their a-glucosi-
Received 24 June 2014
Revised 17 July 2014
Accepted 18 July 2014
Available online 31 July 2014
dase inhibitory activities. As a result, fifteen of them 1, 4, 5, 7, 8, 13, 17, 23, 25, 30, 32, 33, 35, 36 and 37
were identified as potent inhibitors of the enzyme. Kinetic studies of the most active compounds (oxadi-
azoles 1, 23 and 25, and thiadiazoles 35 and 37) were carried out to determine their mode of inhibition
and dissociation constants Ki. The most potent compound of the oxadiazole series (compound 23) was
found to be a non-competitive inhibitor (Ki = 4.36 0.017
lM), while most potent thiadiazole 35 was
Keywords:
Oxadiazoles
Thiadiazoles
identified as a competitive inhibitor (Ki = 6.0 0.059 M). The selectivity and toxicity of these compounds
l
were also studied by evaluating their potential against other enzymes, such as carbonic anhydrase-II and
phosphodiesterase-I. Cytotoxicity was evaluated against rat fibroblast 3T3 cell line. Interestingly, these
a-Glucosidase inhibition
compounds were found to be inactive against other enzymes, exhibiting their selectivity towards
cosidase. Inhibition of -glucosidase is an effective strategy for controlling post-prandial hyperglycemia
in diabetic patients. -Glucosidase inhibitors can also be used as anti-obesity and anti-viral drugs. Our
study identifies two novel series of potent -glucosidase inhibitors for further investigation.
Ó 2014 Elsevier Ltd. All rights reserved.
a-glu-
Post-prandial hyperglycemia
Diabetes
a
a
a
1. Introduction
1,3,4-oxadiazole is less aromatic than the preceding isomers.12,13
All nitrogen and sulfur containing heterocycles, especially 2-mer-
capto-1,3,4-oxadiazole and its 1,3,4-thadiazole analogues, posses
a wide range of biological activities. This includes antimicrobial,14
anti-inflammatory,15 anti-HIV,16,17 antiparasitic,18 fungicidal,19,20
anticonvulsant,21 antialfatoxigenic,22 as well as pyrophosphatases,
phosphodiesterases,23 and urease inhibitory properties.5,24,25 Dur-
ing the last decades, many 1,3,4-oxadiazole and 1,3,4-thiadiazole
analogues were developed for applications in agriculture, such as
herbicides and as chitinase inhibitors.26,27 These studies have been
largely focused on incorporating hydrazide moiety into substituted
oxadiazoles and thiadiazoles, because of its versatile biological
action, especially antidiabetic properties.28–30
Heterocyclic compounds have applications in diverse fields,
such as pharmaceuticals, agriculture and industries.1 Many
approaches have been reported in the literature for the high yield-
ing synthesis of heterocyclic compounds.2–4 Nitrogen containing
heterocyclic molecules, such as 1,3,4-oxadiazoles and 1,3,4-thi-
adiazoles, contain two nitrogen atoms in a five-membered ring.5
Due to their hetero-atomic composition (nitrogen, carbon and sul-
fur/oxygen), they exhibit interesting biological properties.6,7 1,3,4-
Oxadiazoles and 1,3,4-thiadiazole bearing O/S atom, respectively,
are analogues of each other.8,9 Heterocyclic ring of both oxadiaz-
oles, and thiadiazoles are derived from furan/thiophene, respec-
tively, in which two methine groups (@CH) are replaced with
nitrogen atoms.10,11
Diabetes mellitus is a chronic metabolic disorder, characterized
by a high level of glucose in blood.31 Type-2 diabetes mellitus is
effectively managed by preventing the absorption of carbohydrates
after a meal, thus controlling the post-prandial hyperglycemia,
which is an independent risk factor for cardiovascular diseases,
Oxadiazoles and thiadiazoles may have different arrange-
ment of two nitrogen atoms as isomeric forms which exhibit
different physico-chemical properties, such as N–N isomer is ther-
modynamically more stable than 1,3,4-thiadiazoles, however,
as well as associated with diabetic complications.32
a
-Glucosidase
(EC 3.2.1.20) is a typical exo-type glycosidase enzyme that cata-
lyzes the releases of -glucosides from the non-reducing end of
the carbohydrates.33 It is the key enzyme involved in intestinal glu-
cose absorption. -Glucosidase inhibitors are effective in reducing
the post-prandial glucose levels. By suppressing the absorption of
a
⇑
Corresponding authors. Tel.: +92 21 34824910; fax: +92 21 34819018 (K.M.K.),
tel.: +92 21 34824924 5; fax: +92-21 4819018 9 (M.I.C.).
E-mail addresses: hassaan2@super.net.pk, khalid.khan@iccs.edu (K.M. Khan),
hej@cyber.net.pk (M.I. Choudhary).
a
http://dx.doi.org/10.1016/j.bmc.2014.07.032
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
5455
M)
Table 1
-Glucosidase inhibitory activity of compounds 1–37
a
Compound
R1
R2
IC50 SEM (l
Oxadiazoles
N
N
O
S
R1
O
R2
OH
Cl
1
24.1 1.1
Cl
Cl
Cl
Cl
Cl
OMe
2
3
4
5
NA
OMe
NA
F
49.1 0.2
81.6 0.8
MeO
6
7
NA
Cl
Cl
74.2 1.3
OH
Cl
8
9
111.8 0.6
NA
Br
10
11
NA
NA
N
O
O
F
12
13
NA
OMe
183.5 0.7
(continued on next page)

5456
H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
Table 1 (continued)
Compound
R1
R2
IC50 SEM (lM)
Cl
Cl
14
15
16
NA
Cl
NA
NA
Br
N
N
17
124.9 1.2
OH
N
N
18
19
NA
NA
Cl
NO2
N
N
20
21
22
NA
NA
NA
F
OMe
MeO
O
O
23
11.8 0.07
OMe
24
NA

H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
5457
M)
Table 1 (continued)
Compound
R1
R2
IC50 SEM (l
OH
O
25
17.9 1.2
Cl
Cl
O
26
27
NA
NA
O
Cl
Thiadiazoles
N
N
S
S
S
O
O
R2
R1
OCH3
NO2
OCH3
NO2
28
29
NA
NA
30
31
51.8 0.02
Cl
Br
Cl
Br
NA
32
33
34
165.5 1.3
24.6 0.6
NA
F
F
Cl
Cl
Cl
Cl
35
36
10.8 0.03
94.3 1.2
MeO
MeO
(continued on next page)

5458
H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
Table 1 (continued)
Compound
R1
R2
IC50 SEM (lM)
OH
OH
37
20.4
1
Std.
(Acarbose)
—
937 1.6
NA, not active; SEM, standard error mean.
B
A
0.08
0.04
0
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.04
-0.08
-15
-10
-5
0
5
10
15
-10 -8 -6 -4 -2
1/Substrate (4-Nitrophenyl -D-glucopyranoside)
0
2
4
6
8
10
(Inhibitor compound 23)
0.6
C
0.4
0.2
0
-0.2
-0.4
-10 -8 -6 -4 -2
0
2
4
6
8
10
(Inhibitor compound 23)
Figure 1. The inhibition of
a-glucosidase by compound 23 (A) is the Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) versus reciprocal of substrate in the
absence (4), and in presence of 6
l
M (j), 8 M (h), 10 M (d) and 12 M (s) of compound 23. The figure (B) is the secondary replot of Lineweaver–Burk plot between the
l
l
l
slopes of each line on Lineweaver–Burk plot versus different concentrations of compound 23. (C) is the Dixon plot of reciprocal of rate of reaction (velocities) versus different
concentrations of compound 23.
glucose, they can be effective in the treatment and management of
hyperglycemia and hyperlipidemia. Hyperglycemia in diabetes
mellitus is associated with numerous complications, such as ath-
erosclerosis, cardiac dysfunction, retinopathy, neuropathy,
nephropathy, etc.34,35 Hyperglycemia also induces abnormal glyca-
tion of different proteins, which leads to chronic dysfunctions.
Therefore, managing glucose concentration is a key strategy to
discover safe and effective inhibitors of this key enzyme for the
control of diabetic disorders.
We describe here the synthesis and characterization of oxadiaz-
ole and thiadiazole derivatives 1–37. Both types of compounds
were first time evaluated for their
by employing a medium throughput biochemical mechanism-
based assay. The selectivity towards -glucosidase enzyme and
a-glucosidase inhibitory activity
a
reduce diabetes related disorders.
a
-Glucosidase inhibitors, such
cytotoxicity of these compounds were also evaluated.
as acarbose, miglitol and voglibose, are widely used since the early
1990s for the treatment of patients with type-2 diabetes as oral
drugs. However, they are known to cause various side effects, such
as flatulence, diarrhea and abdominal discomfort.36 Unfortunately,
all three of them also have low efficacy against enzymes with high
IC50 values. Due to the vital role of this enzyme in hyperglycemia
and side effects of the existing drugs, there is an urgent need to
2. Results and discussion
2.1. Chemistry
2-Mercapto oxadiazoles and thiadiazoles were synthesized in
two steps by using various hydrazides. The resulting heterocyclic

H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
5459
2.2. Bioactivity
Table 2
The mode of inhibition of compounds 23, 1, 35, and 37
Compounds 1–37 were evaluated for their inhibitory potential
against the -glucosidase enzyme (Table 1). Compounds 1–37
belong to two series, oxadiazoles 1–27 and thiadiazoles 28–37. In
oxadiazoles, compounds 1, 4, 5, 7, 8, 13, 17, 23, and 25 were found
to be active, while the rest of the compounds showed less than 50%
inhibition. Compounds 1, 4, 23, and 25 posses IC50 values in the
Compounds
Ki (
l
M)
Type of inhibition
a
Oxadiazole (1)
12.00 0.0012
4.36 0.017
11.20 0.056
6.00 0.059
14.30 0.085
890.00 0.012
Non-competitive inhibition
Non-competitive inhibition
Non-competitive inhibition
Competitive inhibition
Non-competitive inhibition
Competitive inhibition
Oxadiazole (23)
Oxadiazole (25)
Thiadiazole (35)
Thiadiazole (37)
Standard (Acarbose)
range of 11.8–49.1
bose (IC50 = 937.0 1.6
found to be more active as well, with the IC50 values between
74.2 and 183.5 M.
In thiadiazoles, compounds 35 and 37 were found to be
lM, more active than the standard drug acar-
lM). Compounds 5, 7, 8, 13 and 17 were
compounds were then reacted with different substituted 2-bromo-
acetophenone. Both steps of the reaction were carried out in etha-
nol and the resulting products were obtained in high yields.
l
significantly active with IC50 values 10.8 0.03 and 20.4
1 lM,
A
B
0.4
0.04
0.2
0
0.02
0
-0.02
-0.04
-0.2
-0.4
-10 -8 -6 -4 -2
0
2
4
6
8
10
-10 -8 -6 -4 -2
0
2
4
6
8
10
1/Substrate (4-Nitrophenyl Ǘ -D-glucopyranoside)
(Inhibitor compound 35)
C
0.4
0.2
0
-0.2
-0.4
-10 -8 -6 -4 -2
0
2
4
6
8
10
(Inhibitor compound 35)
Figure 2. The inhibition of
a
-glucosidase by compound 35 (A) is the Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) versus reciprocal of substrate in the
M (j), 8 M (h) and 10 M (d) of compound 35. The figure (B) is the secondary replot of Lineweaver–Burk plot between the slopes of each
absence (4), and in presence of 6
l
l
l
line on Lineweaver–Burk plot versus different concentrations of compound 35. (C) is the Dixon plot of reciprocal of rate of reaction (velocities) versus different concentrations
of compound 35.
Table 3
The carbonic anhydrase-II and phosphodiesterase-I inhibition, and cytotoxicity of compounds 1, 23, 25, 35 and 37
Compounds
Carbonic anhydrase-II
% Inhibition IC50 SEM (
Phosphodiesterase-I
% Inhibition IC50 SEM (
Cytotoxicity (3T3 cell line)
l
M)
lM)
IC50 SEM (lM)
1
23
25
35
37
9.0
33.0
48.2
33.8
42.9
NA
NA
NA
NA
NA
26.0
8.5
1.8
12.3
4.6
NA
NA
NA
NA
NA
>30
>30
>30
>30
>30
NA, not active; SEM, standard error mean.

5460
H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
respectively. Compounds 30, 32, 33 and 36 were more active than
the standard with IC50 values between 24.6 and 165.5 M.
donating effect of bromine. The ortho substituted compound 36
was found to be significantly active with IC50 = 94.3 1.2 M. Com-
pound 30 posses biphenyl rings at both R1 and R2 positions and
l
l
The limited structure–activity relationship (SAR) study indi-
cated that compounds were active mainly due to the oxadiazole
and thiadiazole moieties and the carbonyl oxygen which may
interact with the active site of enzyme by hydrogen bonding. The
substitution on aromatic ring at both sides also influenced the
activity.
In oxadiazoles, compound 23 was found to be most active with
an IC50 of 11.8 0.07 lM. This compound has a para methoxy phe-
nyl at R2 and benzyloxy phenyl at R1 positions. The excellent activ-
ity of this compound may be due to its ability to engage in forming
exhibited an IC50 value of 51.8 0.02 lM.
2.3. Kinetic studies
To investigate the inhibition mechanism, kinetics studies on the
most active compounds were performed. Oxadiazoles 1, 23 and 25
and thiadiazoles 35 and 37 were selected for this purpose. From
the kinetic studies, it was clear that the compounds 1, 23, and 25
(oxadiazoles) and 37 (thiadiazole) are non-competitive inhibitors
a
p
-interaction with aromatic amino acid residues at the active site
of enzyme. In oxadiazoles, compound 25 was found to be the sec-
ond most active compound with IC50 = 17.9 1.2 M. This decrease
with Ki values between 4.36 and 14.3 lM (Table 2). The type of
inhibition was determined by Lineweaver–Burk plots. The recipro-
cal of the rate of the reaction were plotted against the reciprocal of
substrate concentrations to monitor the effect of the inhibitor on
both Km and Vmax. Figure 1 shows that the Km of the enzyme was
not affected by varying concentrations of compound 23, while
the Vmax of the enzyme decrease. This indicated a non-competitive
inhibition. Compound 35 (thiadiazole) was found to be a purely
l
in the activity may be due the hydroxyl substitution at the meta
position of the phenyl ring (R2). This hydroxyl group may involve
in hydrogen bonding and as a result phenyl ring less available for
p
-interaction with aromatic amino acid residues at the active site
of enzyme.
The activity was further decreased by another substitution of
chloro on the phenyl ring at R1 position, as observed in compound
1 (IC50 = 24.1 1.1 M). When we compared the activity of com-
pounds 1 and 4, compound 4 (IC50 = 49.1 0.2 M) exhibited an
activity that was reduced two-fold relative to that of compound
1 (IC50 = 24.1 1.1 M). Interestingly both compounds have dis-
competitive inhibitor with Ki values 6 0.059
lM. Figure 2 shows
that the Vmax of -glucosidase was not affected by the presence
a
l
of different concentrations of compound 35, while the Km increased
which indicated a purely competitive-type of inhibition. The sec-
ondary re-plots of Lineweaver–Burk plots were plotted to deter-
mine the Ki values (Figs. 1B and 2B). The Ki values were
calculated by plotting the slope of each line in the Lineweaver–
Burk plots against different concentrations of compounds (1, 23,
25, 35 and 37). The Ki values were deduced from Dixon plots by
plotting the reciprocal of the rate of reaction against different con-
centrations of compounds 1, 23, 25, 35 and 37.
l
l
tinctly similar structures, but the low activity of compound 4
may be attributed to the fluoro substitution on the phenyl ring at
R2, instead of a hydroxyl group. When the activities of methoxy-
substituted compounds, such as compounds 2, 3 and 5 were com-
pared, the meta and para substituted compounds (2 and 3), respec-
tively, were found to be inactive, while the ortho substituted
In order to evaluate the selectivity of these compounds against
compound 5 was found to be active (IC50 = 81.6 0.8
dichloro substituted compound 7 was also found to be active with
an IC50 value 74.2 1.3 M. Compound 8 was significantly active
with an IC50 value 111.8 0.6 M. However, compound 8 was
found to be four times less active than 1, though both have dis-
tinctly similar structural features. The only difference is that com-
pound 1 has a chloro substituent on the phenyl ring at R1 position.
This indicates that the chloro substituent on the phenyl ring as R1
position increases the activity. Compound 17 showed an
l
M). The
a-glucosidase, the most potent compounds 1, 23, 25, 35 and 37
were also evaluated for their activity against the phosphodiester-
ase-I and carbonic anhydrase-II enzymes. These compounds
showed no inhibition of either enzyme (Table 3). The cytotoxicity
of these compounds was also assessed by using the 3T3 cell line,
which showed a nontoxic behavior.
In conclusion, the ortho methoxy-substituted oxadiazoles and
thiadiazoles, such as compounds 5 and 23 in oxadiazoles and com-
pound 36 in thiadiazoles, were found to be most active in these
series. The hydroxyl or fluoro substitution at the meta position in
both series increases the activities, as in compounds 1, 4, 8, 25,
33 and 37. Most of the compounds were found to more active than
the standard drug, acarbose. This opens up new outlooks for fur-
ther studies on these compounds as potential drug candidates.
l
l
IC50 = 124.9 1.2
ring as R1.
lM. It contains a biphenyl ring as R2 and pyridyl
In thiadiazoles, compound 35 was the most active with an IC50
value 10.8 0.03 M. The potent activity of 35 may also be due to
the -interaction of the phenyl ring at R1 and at R2 positions with
l
p
the active site residues of the enzyme. Thiadiazole 37 was found to
be the second most active compound with an IC50 value
20.4 1.0 lM. This compound has hydroxyl substituents at meta
3. Experimental
positions on both phenyl moieties as R1 and R2. An excellent activ-
ity was exhibited by compounds with fluoro groups at the meta
position on both phenyl rings at R1 and R2 positions, as in com-
pound 33 (IC50 = 24.6 0.6 lM). The para substituted compounds,
such as 28, 29, 31 and 34 were found to be inactive. In case of
bromo substitution at the para position, a good activity of com-
pounds was observed in compound 32 (IC50 = 165.5 1.3 lM).
The good activity of compound 32 may be due the more electron
3.1. In vitro a-glucosidase inhibition assay
a
-Glucosidase inhibitory activity was evaluated by using 0.1 M
phosphate buffer (pH 6.8) at 37 °C.37 The enzyme (0.2 U/mL) in
phosphate buffered saline, incubated with various concentrations
of test compounds at 37 °C for 15 min. The substrate, p-nitro-
phenyl-a-D-glucopyranoside (0.7 mM), was added and the change
in absorbance at 400 nm was monitored up to 30 min by
O
O
R2
N N
O
O
S
a
H H
N N
N N
O
O
b
Br
R1
NH
SH
R1
S
NH2
R1
R1
SH
c
R2
a . CS2, Ethanol, reflux, b. HCl 10% , ice bath, c. (Et)3N, Ethanol
Scheme 1. Synthesis of oxadiazoles 1–27.

H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
5461
O
N N
S
R
N N
S
S
S
S
S
a
b
H H
N N
Br
NH2NH2. H2O
SH
O
O
HS
HS
SH
R2
R1
a. CS2, Ethanol, reflux, b. H2SO4 10% , ice bath, c. (Et)3N, Ethanol
Scheme 2. Synthesis of thiadiazoles 28–37.
multiplate spectrophotometer and the compound was replaced by
DMSO (7.5% final) as control. Acarbose was used as the standard
inhibitor and all reactions were performed in triplicate. The per-
cent inhibition was calculated by using the following formula:
% Inhibition = 100 ꢀ (OD test well/OD control) ꢁ 100.
well and further incubated for 4 h. After this, MTT solution was
aspirated and cells were PBS-washed once, and 100 L of DMSO
l
was added to dissolve the blue insoluble MTT formazan produced
by the action of mitochondrial dehydrogenase. The plate was agi-
tated at room temperature for 15 min and then read at 540 nm
by using microplate readers. The percentage of viable cells was cal-
culated as the relative ratio of optical densities.
3.2. Carbonic anhydrases-II inhibition assay
In this assay, 4-nitrophenyl acetate (4-NPA), a colorless com-
pound, was hydrolyzed to 4-nitrophenol and CO2. The reaction
was followed by measuring the formation of 4-nitrophenol, a yel-
low colored compound. The reaction was performed at 25 °C in
buffer containing HEPES and Tris–HCl at a total concentration of
20 mM and pH of 7.4 for each sample. The reaction mixture con-
3.5. Statistical analysis
The EZ-Fit Enzyme Kinetics Program (Perrella Scientific Inc.,
Amherst, USA) was employed to calculate the IC50 values. All
graphs were plotted by using GraFit program (1999).41 Correlation
coefficients, intercepts, slopes, and their standard errors were cal-
culated by the linear regression analysis by using the same pro-
gram. Each point in the graphs represents the mean of the three
experiments.41
tained 140
aqueous solution of purified bovine erythrocyte CA-II (0.1 mg/mL
of deionized water for 96-well), 20 L of test compound in DMSO
(10% final concentration), 20 L of substrate 4-PNA at a concentra-
lL of the HEPES–tris solution, 20 lL of freshly prepared
l
l
tion of 0.7 mM diluted in ethanol.
3.6. Synthetic method for oxadiazoles 1–27
The reaction was initiated by addition of 4-NPA after 15 min
incubation of test compound, and each compound was tested 3-
times at different concentrations. In this assay, the reaction was
performed using 96-well plates. The plate was placed in a spectro-
photometer and the amount of product formed was monitored at a
1 min interval for 30 min at 400 nm.38
In the first step, sodium hydroxide (0.2 g) was added to a stirred
mixture of substituted phenyl hydrazide (2.0 mmol) with carbon
disulfide (2.0 mmol) in ethanolic condition. This reaction mixture
was refluxed for 6–7 h and monitored by TLC until the reaction
was complete. After completion of reaction, the mixture was
allowed to cool in an ice bath and was neutralized by a 10%
solution of hydrochloric acid, white precipitates of 2-mercapto-
oxadiazole were produced. In the next step, synthetic 2-mercap-
tooxadiazole (1.0 mmol) was added a few drops of triethylamine
as a base, and stirred for 15 min, then added (1.0 mmol) substi-
tuted 2-bromoacetophenones, while the reaction mixture was
refluxed for appropriate time. White precipitates were produced
after a few minutes. It took 3 h for the completion of reaction
(TLC analysis) and white precipitates were separated as S-substi-
tuted oxadiazoles, filtered, and washed with cold ethanol. The pure
product was obtained in high yield (Scheme 1). The structures of
all synthetic compounds were deduced by 1H NMR and EI-MS
spectroscopy. All compounds gave satisfactory CHN analyses.
3.3. Phosphodiesterase-1 inhibition assay
In this assay, the activity against snake venom phosphodiester-
ase-I (Sigma P-4631) (EC 3.1.4.1) was evaluated by using the
reported method39. Tris–HCl buffer 33 mM (pH 8.8), 30 mM Mg-
acetate as a co-factor was added with 0.000742 U of enzyme phos-
phodiesterase I using a 96-well flat-bottomed plate and 0.33 mM
bis(p-nitrophenyl) phosphate (Sigma N-3002) as a substrate. EDTA
(E. Merck) was used as positive controls. After 30 min of incuba-
tion, the enzyme activity was monitored at 37 °C on a microtitre
plate reader spectrophotometer, by following the release of
p-nitrophenol from p-nitrophenyl phosphate at 410 nm. All the
reactions were performed in triplicate, and the initial rates were
measured as the rates of changes in the OD/min (optical density/
min) and used in subsequent calculations.
3.7. Synthetic method for thiadiazoles 28–37
In the first step of this reaction (0.2 g), sodium hydroxide was
added to a stirred mixture of hydrazine hydrate (2.0 mmol) with
carbon disulfide (4.0 mmol) in ethanol and the reaction mixture
was refluxed for 6–7 h. The reaction was monitored by TLC till
the disappearance of starting material. After completion of reac-
tion, the mixture was allowed to cool in an ice bath and neutralized
by 10% solution of sulfuric acid, white precipitate of 1,3,4-thiadia-
zole-2,5-dithiol was produced. In the subsequent step, synthetic
1,3,4-thiadiazole-2,5-dithiol (1.0 mmol), was added few drops of
triethylamine as a base, and stirred for 15 min, substituted 2-bro-
moacetophenone (2.0 mmol) was added. The reaction mixture
was refluxed for appropriate time. White precipitates were pro-
duced after few minutes but reaction was completed in 3 h (TLC
analysis). White precipitate was separated as S-disubstituted
3.4. Cytotoxicity evaluation of compounds on 3T3 cells
The experiment was performed according to the method
described by Dimas et al.40 Rat fibroblast 3T3 cells were used in
this assay. Briefly the 3T3-adherent cells (2 ꢁ 105 cells/mL) were
cultured in a 96-well plate overnight in CO2 environment at
37 °C. Supernatant was removed and 50
pounds (100–12.5 g/mL) and 150 L complete medium DMEM
supplemented with 5% (v/v) fetal bovine serum, penicillin (100
units/mL) and streptomycin (100 g/mL) were added to each well.
After the incubation, the culture medium was aspirated carefully
and 50 L of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazo-
lium bromide (MTT) solution (2 mg/mL PBS) was added to each
lL of serially diluted com-
l
l
l
l

5462
H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
1,3,4-thiadiazole-2,5-dithiol, filtered and washed with ethanol. The
pure product was obtained in high yield (Scheme 2). The structures
of all synthetic compounds were deduced by 1H NMR and EI-MS
spectroscopy. All compounds exhibited satisfactory CHN analyses.
(75), 153 (60); Anal. Calcd C22H15ClN2O2S, C, 64.94; H, 3.72; N,
6.88; Found: C, 64.96; H, 3.61; N, 6.74.
3.7.7. 1-(3,4-Dichlorophenyl)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)
sulfanyl]-1-ethanone (7)
3.7.1. 2-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)-1-(3-
hydroxyphenyl)ethanone (1)
Yield: 0.48 g (88%); 1H NMR (300 MHz, DMSO-d6): d 8.50 (s, 1H,
H-20), 7.92 (m, 4H, H-2/3/4/5), 7.58 (d, 2H, J6 ,5 /5 6 = 7.2 Hz, H-50/60),
5.16 (s, 2H, –CH2–); HREI-MS Calcd 363.9840 Found 363.9831. EI-
MS m/z (rel. abund.%): 366 (M++2, 5), 364 (M+, 14), 312 (100), 270
(32), 121 (100), 77 (85); Anal. Calcd: C16H10Cl2N2O2S, C 57.50; H,
3.54; N, 13.41; Found: C, 57.47; H, 3.58; N, 13.45.
0
0
0
0
Yield: 0.51 g (89%); 1H NMR (300 MHz, DMSO-d6): d 9.91 (s, 1H,
O-H), 7.94 (d, 2H, J2,3/6,5 = 8.4 Hz, H-2/6), 7.64 (d, 2H,
J3,2/5,6 = 8.7 Hz, H-3/H-5), 7.52 (d, 1H, J6 ,5 = 7.5 Hz, H-60), 7.3(m,
0
0
2H, H-20/H-50), 7.09 (dd, 1H, J4 ,5 /4 ,2 = 6.0 Hz, 1.8 Hz, H-40), 5.15
(s, 2H, –CH2–); HREI-MS Calcd 346.0179, Found 346.0196. EI-MS
m/z (rel. abund.%): 348 (M++2, 9), 346 (M+, 30), 142 (42), 134
(29), 121 (100), 93 (60) Anal. Calcd C16H11ClN2O3S, C, 55.41; H,
3.04; N, 8.08; Found: C, 55.38; H, 3.07; N, 8.04.
0
0
0
0
3.7.8. 1-(3-Hydroxyphenyl)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)
sulfanyl]-1-ethanone (8)
Yield: 0.5 g (89%); 1H NMR (300 MHz, DMSO-d6): d 9.88 (s, 1H,
O–H), 7.93 (d, 2H, J6,5/2,3 = 6.6 Hz, H-2/6), 7.57 (m, 4H, H-20/H-3/H-
4/H-5), 7.37 (t, 2H J4/3,5,5 /4 ,6 = 8.1 Hz, H-4/H-50), 7.09 (d, 1H
0
0
0
3.7.2. 2-{[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-1-
(3-methoxyphenyl)-1-ethanone (2)
J4 ,5 = 9 Hz, H-40), 5.12 (s, 2H, –CH2–); HREI-MS Calcd 312.0569
Found 312.0559. EI-MS m/z (rel. abund.%): 312 (M+, 65), 134
(32), 120 (100), 105 (47), 93 (60); Anal. Calcd C16H12N2O3S, C,
61.53; H, 3.87; N, 8.97; Found: C, 61.48; H, 3.83; N, 8.99.
0
0
Yield: 0.56 g (90%); 1H NMR (300 MHz, DMSO-d6): d 7.95 (d, 2H,
J2,3/6,5 = 8.7 Hz, H-2/H-6), 7.64 (m, 3H, H-40/H-50/H-60), 7.51 (dd, 1H,
J2 ,4 = 2.0 Hz, J2 ,6 = 2.2 Hz, H-20), 5.16 (s, 2H, –CH2–), 3.82 (s, 3H,
–OCH3); HREI-MS 360.0335 Found 360.0338, EI-MS m/z (rel.
abund.%): 362 (M++2, 27), 361 (M++1, 11), 360 (M+, 70), 318 (27),
212 (13), 135 (100), 107 (98), 77 (47); Anal. Calcd C17H13ClN2O3S,
C, 56.59; H, 3.63; N, 7.76; Found: C, 56.63; H, 3.58; N, 7.73.
0
0
0
0
3.7.9. 1-(3-Chlorophenyl)-2-(5-phenyl-1,3,4-oxadiazol-2-ylthio)
ethanone (9)
Yield: 0.48 g (92%); 1H NMR (300 MHz, DMSO-d6): d 8.08 (t, 1H,
J2 /4 ,6 = 1.2 Hz, H-20), 8.04 (d, 1H, J6 ,5 = 4.5 Hz, H-60), 7.93 (d, 2H,
J2,3/6,5 = 5.1 Hz, H-2/H-6), 7.78 (m, 1H, H-50), 7.62 (m, 4H, H-3/H-
4/H-5/H-40), 5.17 (s, 2H, –CH2–); HREI-MS Calcd 330.0230 Found
330.0227. EI-MS m/z (rel. abund.%): 330 (M+, 34), 288 (23), 138
(100), 111 (50), 103 (77), 77 (58); Anal. Calcd C16H11ClN2O2S, C,
58.09; H, 3.35; N, 8.47; Found: C, 58.06; H, 3.21; N, 8.32.
0
0
0
0
0
3.7.3. 2-{[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-1-
(4-methoxyphenyl)-1-ethanone (3)
Yield: 0.45 g (86%); 1H NMR (300 MHz, DMSO-d6): d 8.02 (d, 2H,
J2,3/6,5 = 9.0 Hz, H-2/H-6), 7.93 (d, 2H, J3,2/5,6 = 8.7 Hz, H-3/H-5), 7.43
(d, 2H, J2 ,3 /6 ,5 = 8.7 Hz, H-20/H-60), 6.96 (d, J3 ,2 /5 ,6 = 8.7 Hz, H-30/
H-50), 4.93 (s, 2H, –CH2–), 3.88 (s, 3H, –OCH3); HREI-MS Calcd
360.0179, Found 360.0196. EI-MS m/z (rel. abund.%): 362 (M++2,
16), 361 (M++1, 12), 360 (M+, 54), 134 (100), 136 (100), 77 (53);
Anal. Calcd C17H13ClN2O3S, C, 56.59; H, 3.63; N, 7.76; Found: C,
56.55; H, 3.59; N, 7.73.
0
0
0
0
0
0
0
0
3.7.10. 1-(4-Bromophenyl)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)
sulfanyl]-1-ethanone (10)
Yield: 0.46 g (87%); 1H NMR (300 MHz, DMSO-d6): d 8.01 (d, 2H,
J3 ,2 /5 ,6 = 8.7 Hz, H-30/H-50), 7.80 (d, 2H, J2 ,3 /6 ,5 = 6.2 Hz, H-20/H-
60), 7.57 (m, 3H, H-3/H-4/H-5), 5.15 (s, 2H, –CH2–) HREI-MS Calcd
373.9725 Found 373.9712. EI-MS m/z (rel. abund.%): 376 (M++2, 9),
374 (M+, 11), 334 (7), 185 (100), 183 (96), 77 (15); Anal. Calcd
0
0
0
0
0
0
0
0
3.7.4. (2-{[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-1-
(3-fluorophenyl)-1-ethanone (4)
Yield: 0.44 g (86%); 1H NMR (300 MHz, DMSO-d6): d 7.92 (d, 2H,
J2,3/6,5 = 8.4 Hz, H-2/H-6), 7.83 (d, 1H, J3,2 = 7.5 Hz, H-3), 7.71 (d, 1H,
J5,6 = 9.3 Hz, H-5), 7.49 (m, 3H, H-40/H-50/H-60), 7.34 (m, 1H, H-2),
4.91 (s, 2H, –CH2–), HREI-MS Calcd 348.0136 Found 348.0131, EI-
MS m/z (rel. abund.%): 350 (M++2, 33), 349 (M++1, 15), 348 (M+,
84), 305 (62), 179 (40), 138 (68), 122 (100); Anal. Calcd
C16H11BrN2O2S, C, 51.21; H, 2.95; N, 7.47; Found: C, 51.25; H,
2.98; N, 7.36.
3.7.11. 3-(2-{[5-(3-Pyridinyl)-1,3,4-oxadiazol-2-yl]sulfanyl}
acetyl)-2H-chromen-2-one (11)
Yield: 0.43 g (88%); 1H NMR (300 MHz, DMSO-d6): d 9.12 (d, 1H,
J2/4 = 1.5 Hz, H-2), 8.85 (s, 1H, ACH@), 8.77 (d, 1-H, J4/5 = 1.2 Hz, H-
C16H10ClFN2O2S, C, 55.10; H, 2.89; N, 8.03; Found: C, 55.14; H,
4), 8.31 = 8.1 Hz, H-6), 7.62 (d, 1H, J6 /5 = 7.5 Hz, H-60), 7.54 (m, 3H,
H-20/H-40/H-50), 7.45 (dd, 1-H, J5,6/5,4 = 2.4 Hz, J = 5.7 Hz, H-5), 5.07
(s, 2H, –CH2–), HREI-MS Calcd 365.0470 Found 365.0487. EI-MS m/
z (rel. abund.%): 365 (M+, 13), 192 (12), 174 (42), 173 (100), 119
(21); Anal. Calcd C18H11N3O4S, C, 59.98; H, 3.03; N, 11.50; Found:
C, 60.05; H, 2.97; N, 12.57.
0
0
2.91; N, 7.98.
3.7.5. 2-{[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-1-
(2-methoxyphenyl)-1-ethanone (5)
Yield: 0.47 g (91%); 1H NMR (300 MHz, DMSO-d6): d 7.93 (d, 2H,
J2,3/6,5 = 8.7 Hz, H-2/H-6), 7.65 (m, 4H, H-20/H-30/H-40/H-50), 7.24 (d,
1H, J5/2 = 8.4 Hz, H-5), 7.10 (d, 1H, J3/5 = 7.2 Hz, H-3), 4.94 (s, 2H, –
CH2–), 3.94 (s, 3H, –OCH3); HREI-MS Calcd 360.0335 Found
360.0330, EI-MS m/z (rel. abund.%): 362 (M++2, 8), 360 (M+, 28),
318 (19), 211 (11), 134 (100), 110 (33); Anal. Calcd C17H13ClN2O3S,
C, 56.59; H, 3.63; N, 7.76; Found: C, 56.57; H, 3.62; N, 7.74.
3.7.12. 1-(3-Fluorophenyl)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)
sulfanyl]-1-ethanone (12)
Yield: 0.46 g (86%); 1H NMR (300 MHz, DMSO-d6): d 7.92 (m,
3H, H-40/H-50/H-60), 7.84 (s, 1H, H-20), 7.64 (m, 5H, H-2/H-3/H-4/
H-5/H-6), 5.1 (s, 2H, –CH2–); EI-MS m/z (rel. abund.%): HREI-MS
Calcd 314.0525 Found 314.0511, 314 (M+, 38), 272 (39), 145 (23),
124 (29), 123 (100); Anal. Calcd C16H11FN2O2S, C, 61.14; H, 3.34;
N, 8.91; Found: C, 61.O5; H, 3.31; N, 8.87.
3.7.6. 1-[1,10-Biphenyl]-4-yl-2-{[5-(4-chlorophenyl)-1,3,4-oxadi
azol-2-yl]sulfanyl}-1-ethanone (6)
Yield: 0.52 g (90%); 1H NMR (300 MHz, DMSO-d6): d 8.11 (d, 2H,
J2,3/6,5 = 8.4 Hz, H-2/H-6), 7.93 (d, 2H, J3,2/5,6 = 8.7 Hz, H-3/H-5), 7.73
(d, J2 ,3/6,5 = 8.4 H-20/H-60), 7.62(d, 2H, J3 ,2 /5 ,6 = 6.9 Hz H-30/H-50),
7.44 (m, 5H, H-200/H-300/H-400/H-500/H-600), 4.99 (s, 2H, –CH2–);
HREI-MS Calcd 406. 0543 Found 406.0539. EI-MS m/z (rel.
abund.%): 408 (M++2, 4), 406 (M+, 9), 182 (84), 180 (100), 152
3.7.13. 1-(3-Methoxyphenyl)-2-[(5-phenyl-1,3,4-oxadiazol-2-
yl)sulfanyl]-1-ethanone (13)
0
0
0
0
0
Yield: 0.42 g (83%); 1H NMR (300 MHz, CDCl3): d 7.97 (d, 2H,
J6,5/2,3 = 6.0 Hz, H-2/H-6), 7.62 (d, 1H, J6 ,5 = 7.5 Hz, H-60), 7.49 (m,
0
0

H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
5463
4H, H-20/H-3/H-4/H-50), 7.41 (t, 2H, J4/3,5 = 8.1 Hz, H-4/H-5), 7.18
EI-MS m/z (rel. abund.%): 333 (M++2, 13), 332 (M++1, 8), 331 (M+,
(dd, 1H, J4 ,5 = 5.9 Hz, H-40, J4 ,2 = 2.4 Hz, H-40), 4.95 (s, 2H,
–CH2–), 3.86 (s, 3H, –OCH3); HREI-MS Calcd 326.0725 Found
326.0738. EI-MS m/z (rel. abund.%): 326 (M+, 10), 148 (35), 135
(100), 107 (40), 77 (44); Anal. Calcd C17H14N2O3S, C, 62.56; H,
4.32; N, 8.58; Found: C, 62.54; H, 4.29; N, 8.55.
29), 298 (11), 139 (100), 113 (38), 111 (78); Anal. Calcd
0
0
0
0
C16H11ClN2O2S, C, 58.09; H, 3.35; N, 8.47; Found: C, 57.98; H,
3.32; N, 8.43.
3.7.20. 1-(4-Nitrophenyl)-2-{[5-(3-pyridinyl)-1,3,4-oxadiazol-2-
yl]sulfanyl}-1-ethanone (20)
3.7.14. 1-(4-Chlorophenyl)-2-{[5-(4-chlorophenyl)-1,3,4-oxadia
zol-2-yl]sulfanyl}-1-ethanone (14)
Yield: 0.49 g (91%); 1H NMR (300 MHz, DMSO-d6): d 9. 9.11 (d,
1H, J2,4 = 1.8 Hz, H-2), 8.78 (d, 1H, J4,5 = 3.6 Hz, H-4), 8.39 (d, 2H,
Yield: 0.53 g (92%); 1H NMR (300 MHz, DMSO-d6): d 8.85 (d,
J = 7.2 Hz, H-2/H-6), 7.96 (d, J = 7.2 Hz, H-3/H-5, J = 7.2 Hz, H-60),
J2 ,3 /6 ,5 = 8.7 Hz, H-20/H-60), 8.31 (m, 1H, H-6), 8.30 (d, 2H,
0
0
0
0
J3 ,2 /5 ,6 = 8.7 Hz, H-30/H-50), 7.61 (dd, 1H, J5,6 = 4.8 Hz, J5,4 = 5.1,
H-5), 5.26 (s, 2H, –CH2), HREI-MS 342.0423 Found 342.0404,
EI-MS m/z (rel. abund.%): 342 (M+, 5), 300 (12), 179 (26), 150
(100), 120 (16), 104 (31), 78 (26); Anal. Calcd: C15H10N4O4S, C,
52.63; H, 2.94; N, 16.37; Found: C, 52.61; H, 2.88; N, 16.35.
0
0
0
0
7.58 (m, 2H, H-3/H-5), 7.52 (d, 1H, J3 ,2 = 8.4 Hz, H-30), 7.45 (m,
1H, H-50), 5.05 (s, 2H, –CH2–); HREI-MS Calcd 363.9832, Found
63.9826, EI-MS m/z (rel. abund.%): 366 (M++2, 12), 365 (M++1, 7),
364 (M+, 46), 202 (23), 187 (25), 173 (100), 145 (52); Anal. Calcd
0
0
C16H10Cl2N2O2S, C, 58.09; H, 3.35; N, 8.47; Found: C, 58.05; H,
3.29; N, 8.45.
3.7.21. 1-(3-Fluorophenyl)-2-{[5-(3-pyridinyl)-1,3,4-oxadiazol-
2-yl]sulfanyl}-1-ethanone (21)
3.7.15. (1-(4-Chlorophenyl)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)
sulfanyl]-1-ethanone (15)
Yield: 0.47 g (88%); 1H NMR (300 MHz, DMSO-d6): d 9.11 (d, 1H,
J2,4 = 1.5 Hz, H-2), 8.78 (dd, 1H, J2 ,4 = 1.2 Hz, J2 ,6 = 1.4, H-20), 8.32
(d, 1H, J4,5 = 8.1 Hz, H-4), 7.92 (d, 1H, J6,5 = 7.5 Hz, H-6), 7.86 (d,
0
0
0
0
Yield: 0.46 g (89%); 1H NMR (300 MHz, DMSO-d6): d 8.00 (d, 4H,
J = 8.4 Hz, H-20/H-30/H-50/H-60), 7.49 (m, 5H, H-2/H-3/H-4/H-5/H-
6), 4.91 (s, 2H, –CH2–); HREI-MS Calcd 330.0230 Found 330.0228,
EI-MS m/z (rel. abund.%): 332 (M++2, 13), 331 (M++1, 7), 330 (M+,
34), 288 (16), 145 (12), 141 (91), 139 (100) Anal. Calcd C16H11ClN2-
O2S, C, 58.09; H, 3.35; N, 8.47; Found: C, 58.05; H, 3.32; N, 8.45.
1H, J6 ,5 = 9.3 Hz, H-60), 7.62 (m, 3H, H-40/H-5/H-50), 5.19 (s, 2H,
–CH2–), HREI-MS Calcd 315.0478, Found 315.0487, EI-MS m/z
(rel. abund.%): 315 (M+, 58), 273 (44), 146 (16), 124 (59), 123
(100), 95 (95); Anal. Calcd: C15H10FN3O2S, C, 57.14; H, 3.20; N,
13.33; Found: C, 57.11; H, 3.17; N, 13.30.
0
0
3.7.16. 1-(4-Bromophenyl)-2-{[5-(3-pyridinyl)-1,3,4-oxadiazol-
2-yl]sulfanyl}-1-ethanone (16)
3.7.22. 1-(4-Methoxyphenyl)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)
sulfanyl]-1-ethanone (22)
Yield: 0.47 g (91%); 1H NMR (300 MHz, DMSO-d6): d 9.22 (s, 1H,
Yield: 0.50 g (91%); 1H NMR (300 MHz, DMSO-d6): d 8.04 (d, 2H,
H-2), 8.75 (d, 1H, J4,5 = 3.9 Hz, H-4), 8.31 (d, 1H, J6 ,5 = 7.8 Hz, H-60),
J2,3/6,5 = 8.7 Hz, H-2/H-6), 7.92 (d, 2H, J2 ,3 /6 ,5 = 9.0 Hz, H-20/H-60),
0
0
0
0
0
0
7.91(d, 2H, J2,3/6,5 = 9.3 Hz, H-2/H-6), 7.66 (d, 2H, J3 ,2 /5 ,6 = 8.4 Hz,
H-30/H-50), 7.48 (dd, 1H, J5,4 = 4.8 Hz, J4,6 = 5 Hz, H-5), 4.92 (s, 2H,
–CH2–), HREI-MS Calcd 376.0356, Found 346.0364, EI-MS m/z
(rel. abund.%): 377 (M++2, 4), 375 (M+, 8), 190 (26), 183 (100),
153 (18); Anal. Calcd: C15H10BrN3O2S, C, 47.89; H, 2.68; N, 11.17;
Found: C, 47.83; H, 2.65; N, 11.12.
7.57 (m, 3H, H-3/H-4/H-5), 7.09 (d, 2H, J3 ,2 /5 ,6 = 9 Hz, H-30/H-50),
5.11 (s, 2H, –CH2–), 3.85 (s, 3H, –OCH3); HREI-MS Calcd:
326.0751, Found 326.0758. EI-MS m/z (rel. abund.%): 326 (M+,
34), 178 (09), 139 (83), 135 (100), 107 (41); Anal. Calcd C17H14N2-
O3S, C, 62.56; H, 4.32; N, 8.58; Found: C, 62.54; H, 4.30; N, 8.55.
0
0
0
0
0
0
0
0
3.7.23. 2-({5-[4-(Benzyloxy)phenyl]-1,3,4-oxadiazol-2-yl}
sulfanyl)-1-(2-methoxyphenyl)-1-ethanone (23)
3.7.17. 1-[1,10-Biphenyl]-4-yl-2-{[5-(3-pyridinyl)-1,3,4-
oxadiazol-2-yl]sulfanyl}-1-ethanone (17)
Yield: 0.47 g (89%); 1H NMR (300 MHz, DMSO-d6): d 7.86 (d, 2H,
Yield: 0.52 g (90%); 1H NMR (300 MHz, DMSO-d6): d 9.25 (s, 1H,
H-2), 8.77 (s, 1H, H-4), 8.39 (d, 1H, J6/5 = 7.8 Hz, H-6), 8.12 (d, 2H,
J2,3/6,5 = 7.2 Hz, H-2/H-6), 7.65 (d, 1H, J6 ,5 = 7.5 Hz, H-60), 7.45 (m,
0
0
7H, H-40/H-50/H-200/H-300/H-400/H-500/H-600), 7.28 (dd, 1H J3 ,4 = 6.0 -
0
0
J2 3 /6 5 = 8.4 Hz, H-20/H-60), 7.74 (d, 2H, J3 2 /5 6 = 8.1 Hz, H-30/H-
Hz, J3 ,5 = 2.1 Hz, H-30), 7.20 (d, 2H, J3,4/5,6 = 8.7 Hz, H-3/H-5), 5.19
(s, 2H, –CH2–), 5.13 (s, 2H, –CH2–); 3.82 (s, 3H, –OCH3); HREI-MS
Calcd: 432.1156, Found, 432.1061, EI-MS m/z (rel. abund.%): 432
0
0
0
0
0
0
0
0
0
0
50), 7.65 (d, 2H, J2 3 /6 5 = 7.2 Hz, H-200/H-600), 7.45 (m, 4H, H-4/H-
300/H-400/H-500), 5.02 (s, 2H, –CH2–); HREI-MS Calcd 373.0885 Found
373.0891. EI-MS m/z (rel. abund.%): 373 (M+, 14), 181 (100), 153
(89), 152 (85), 78 (15); Anal. Calcd C22H16N2O2S, C, 70.95; H,
4.33; N, 7.52; Found: C, 70.88; H, 4.28; N, 7.49.
00 00
00 00
(M+, 28), 135 (65), 107 (19), 90 (100), 77 (20); Anal. Calcd C24H20
-
N2O4S, C, 66.65; H, 4.66; N, 6.48; Found: C, 66.51; H, 4.62; N, 6.43.
3.7.24. 2-({5-[4-(Benzyloxy)phenyl]-1,3,4-oxadiazol-2-yl}
sulfanyl)-1-(4-methoxyphenyl)-1-ethanone (24)
3.7.18. 1-(3-Hydroxyphenyl)-2-{[5-(3-pyridinyl)-1,3,4-
oxadiazol-2-yl]sulfanyl}-1-ethanone (18)
Yield: 0.48 g (88%); 1H NMR (300 MHz, DMSO-d6): d 8.04 (d, 2H,
Yield: 0.51 g (88%); 1H NMR (300 MHz, DMSO-d6): d 9.89 (s 1H,
O–H), 9.11(d, 1H, J2,4 = 1.5, H-2), 8.77 (dd, 1H, J4/5 = 6 Hz,
J4/2 = 1.2 Hz, H-4), 8.32 (d, 1H, J6/5 = 8.1 Hz, H-6), 7.62 (m, 1H,
J2,3/6,5 = 8.5 Hz, H-2/H-6), 7.86 (d, 2H, J2 ,3 /6 ,5 = 9 Hz, H-20/H-60),
0
0
0
0
7.39 (m, 5H, H-200/H-300/H-400/H-500/H-600), 7.19 (d, 2H,
J3 ,2 /5 ,6 = 7.8 Hz, H-30/H-50), 7.06 (d, 2H, J3,2/5,6 = 9 Hz, H-3/H-5),
5.19 (s, 2H, –CH2–), 5.09 (s, 2H, –CH2–), 3.82 (s, 3H, –OCH3); HREI-
MS Calcd 432.1089, Found 432.1078. EI-MS m/z (rel. abund.%): 432
(M+, 30), 136 (10), 135 (100), 91 (100), 77 (8); Anal. Calcd C24H20N2-
O4S, C, 66.65; H, 4.66; N, 6.48; Found: C, 66.67; H, 4.64; N, 6.45.
0
0
0
0
H-40), 7.52 (d, 1H, J6 /5 = 7.8 Hz, H-60), 7.37 (m, 2H, H-5/H-50), 7.1
0
0
(dd, 1H, J2 ,4 = 3.4 Hz, J2 ,6 = 1.8 Hz, H-20), 5.15 (s, 2H, –CH2–); HREI-
MS Calcd 313.0521, Found 313.0512, EI-MS m/z (rel. abund.%): 313
(M+, 17), 179 (10), 121 (100), 93 (15); Anal. Calcd C15H11N3O3S, C,
57.50; H, 3.54; N, 13.41; Found: C, 57.45; H, 3.43; N, 13.36.
0
0
0
0
3.7.25. 2-({5-[4-(Benzyloxy)phenyl]-1,3,4-oxadiazol-2-yl}
sulfanyl)-1-(3-hydroxyphenyl)-1-ethanone (25)
3.7.19. 1-(4-Chlorophenyl)-2-{[5-(3-pyridinyl)-1,3,4-oxadiazol-
2-yl]sulfanyl}-1-ethanone (19)
Yield: 0.46 g (87%); 1H NMR (300 MHz, DMSO-d6): d 9.89 (s, 1H,
O–H), 7.87 (d, 2H, J2,4/6,5 = 8.7 Hz, H-2/H-6), 7.45 (m, 8H, H-20/H-30/
H-40/H-50/H-60/H-400/H-500/H-600), 7.19 (d, 2H, J3,2/5,6 = 8.7 Hz, H-3/H-
Yield: 0.48 g (87%); 1H NMR (300 MHz, DMSO-d6): d 9.11 (s, 1H,
H-2), 8.78 (d, 1H, J4,5 = 3.6 Hz, H-4), 8.32 (d, 1H, J6/5 = 8.1 Hz, H-6),
8.0 (d, 2H, J2 3 /6 5 = 8.7 Hz, H-20/H-60), 7.62 (m, 3H, H-5/H-30/H-50),
5), 7.0 (d, 1H, J2 ,4 = 1.8 Hz, H-20), 5.19 (s, 2H, –CH2–), 5.0 (s, 2H,
–CH2–), HREI-MS Calcd 418.0987, Found 418.0979, EI-MS m/z
0
0
0
0
0
0
5.19 (s, 2H, –CH2–); HREI-MS Calcd 330.0230 Found 330.022.

5464
H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
(rel. abund.%): 418 (M+, 20), 121 (18), 91 (100), 65 (12); Anal.
Calcd: C23H18N2O4S, C, 66.01; H, 4.34; N, 6.69; Found; C, 65.98;
H, 4.32; N, 6.64.
3.7.32. 1-(4-Bromophenyl)-2-[(5-{[2-(4-bromophenyl)-2-
oxoethyl]sulfanyl}-1,3,4-thiadiazol-2-yl)sulfanyl]-1-ethanone
(32)
Yield: 0.55 g (89%); 1H NMR (300 MHz, DMSO-d6): d 7.95 (d, 4H,
J = 8.7 Hz, H-2/H-6/H-20/H-60), 7.76 (d, 4H, J3,2/5,6 = 8.7 Hz, H-3/H-5/
H-30/H-50), 5.03 (s, 4H, 2ꢁ –CH2–); HREI-MS Calcd 541.0429 Found
541.0441. EI-MS m/z (rel. abund.%): 547 (M++4, 5), 546 (M++2, 19),
544 (M+, 31), 360 (18), 185 (100), 90 (26); Anal. Calcd C20H18N2O4-
S3, C, 39.72; H, 2.22; N, 5.15; Found: C, 39.68; H, 2.19; N, 5.08.
3.7.26. 3-(2-{[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}
acetyl)-2H-chromen-2-one (26)
Yield: 0.45 g (86%); 1H NMR (300 MHz, DMSO-d6): d 8.85 (s, 1H,
ACH@), 7.99 (m, 3H, H-30, H-40/H-50), 7.78 (d, 1H, J2 ,3 = 7.2 Hz, H-
20), 7.77 (t, 1H, H-6), 7.65 (d, 2H, J3,2/5,6 = 8.7 Hz, H-3/H-5), 7.49 (m,
2H, H-2/H-6), 5.05 (s, 2H, –CH2–); HREI-MS Calcd 398.0179, Found
398.0196. EI-MS m/z (rel. abund.%): 400 (M++2, 11), 399 (M++1, 5),
398 (M+, 26), 225 (29), 186 (23), 173 (100), 135 (38); Anal. Calcd
0
0
3.7.33. 1-(3-Fluorophenyl)-2-[(5-{[2-(3-fluorophenyl)-2-
oxoethyl]sulfanyl}-1,3,4-thiadiazol-2-yl)sulfanyl]-1-ethanone
(33)
C19H11ClN2O4S, C, 57.22; H, 2.78; N, 7.02; Found: C, 57.19; H,
2.74; N, 6.98.
Yield: 0.46 g (91%); 1H NMR (300 MHz, DMSO-d6): d 7.80 (d, 2H,
J6,5 = 7.5 Hz, 2ꢁ H-6), 7.68 (d, 2H, J2,F = 8.7 Hz, 2ꢁ H-2), 7.47 (dd,
2H, J = 7.8 Hz, J = 8.0, H-4/H-40), 7.31 (m, 2H, 2ꢁ H-5), 5.0 (s, 4H,
2ꢁ –CH2–); HREI-MS 422.0029 Found 422.0018, EI-MS m/z (rel.
abund.%): 422 (M+, 16), 347 (29), 123 (100), 95 (54), 59 (25); Anal.
Calcd C18H12F2N2O3S2, C, 53.19; H, 2.98; N, 6.89; Found: C, 53.17;
H, 2.95; N, 6.87.
3.7.27. 1-(3-Chlorophenyl)-2-{[5-(4-chlorophenyl)-1,3,4-oxadi
azol-2-yl]sulfanyl}-1-ethanone (27)
Yield: 0.53 g (92%); 1H NMR (300 MHz, DMSO-d6): d 8.85 (s, 1H,
H-2), 7.96 (m, 2H, H-2/H-6), 7.78 (1, 2H, J6 ,5 = 7.2 Hz, H-60), 7.58
0
0
(m, 2H, H-3/H-5), 7.52 (d, 1H, J4 ,5 = 8.4 Hz, H-40), 7.45 (t, 1H,
0
0
J5 /4 ,6 = 7.5 Hz, H-50), 5.05 (s, 2H, –CH2–); HREI-MS Calcd
363.9856 Found 363.9851, EI-MS m/z (rel. abund.%): 366 (M++2,
12), 365 (M++1, 7), 364 (M+, 46), 202 (23), 187 (25), 173 (100),
145 (52); Anal. Calcd C16H11ClN2O2S, C, 58.09; H, 3.35; N, 8.47;
Found: C, 58.05; H, 3.29; N, 8.45.
0
0
0
3.7.34. 1-(3,4-Dichlorophenyl)-2-[(5-{[2-(3,4-dichlorophenyl)-
2-oxoethyl]sulfanyl}-1,3,4-thiadiazol-2-yl)sulfanyl]-1-ethanone
(34)
Yield: 0.52 g (90%); 1H NMR (300 MHz, DMSO-d6): d 8.24 (s, 2H,
H-2/H-20), 7.96 (d, 2H, J = 8.4 Hz, H-6/H-60), 7.83 (d, 2H, J = 8.1 Hz,
H-5/H-50), 5.04 (s, 4H, 2ꢁ –CH2–); HREI-MS Calcd 521.8659, Found
521.8668, EI-MS m/z (rel. abund.%): 526 (M++2, 3), 324 (M+, 5), 176
(11), 173 (100), 144 (31), 74 (10); Anal. Calcd C18H10Cl4N2O2S3, C,
41.24; H, 1.92; N, 5.34; Found: C, 41.21; H, 1.88; N, 5.32.
3.7.28. 1-(4-Methoxyphenyl)-2-[(5-{[2-(4-methoxyphenyl)-2-
oxoethyl]sulfanyl}-1,3,4-thiadiazol-2-yl)sulfanyl]-1-ethanone
(28)
Yield: 0.48 g (87%); 1H NMR (300 MHz, DMSO-d6): d 8.01 (d, 4H,
J = 11.4 Hz, H-2/H-20/H-4/H-40), 7.07 (d, 4H, J = 8.7 Hz, H-3/H-30/H-
5/H-50), 5.00 (s, 4H, 2ꢁ –CH2–), 3.84 (s, 6H, 2ꢁ –OCH3); HREI-MS
(C20H18N2O4S3), Calcd 446.0429, EI-MS m/z (rel. abund.%): 446
(M+, 5), 136 (46), 135 (100), 121 (20); Anal. Calcd C20H18N2O4S3,
C, 53.79; H, 4.06; N, 6.27; Found: C, 53.82; H, 3.99; N, 6.24.
3.7.35. 2-({5-[(2-Oxo-2-phenylethyl)sulfanyl]-1,3,4-thiadiazol-
2-yl}sulfanyl)-1-phenyl-1-ethanone (35)
Yield: 0.51 g (87%); 1H NMR (300 MHz, DMSO-d6): d 8.0 (d, 4H,
J2,3/6,5 = 7.2 Hz, H-2/H-6), 7.0 (t, 2H, J = 7.2 Hz, H-4/H-40), 7.0 (m,
4H, J = 7.5 Hz, H-3/H-30/H-5/H-50), 5.0 (s, 4H, 2ꢁ –CH2–); HREI-
MS 386.0217 Found 386.0214, EI-MS m/z (rel. abund.%): 386 (M+,
7), 106 (13), 105 (100), 77 (59), 51 (17); Anal. Calcd C18H14N2O2S3:
C, 55.93; H, 3.65; N, 7.25; Found: C, 55.91; H, 3.62; N, 7.22.
3.7.29. 2,20-(1,3,4-Thiadiazole-2,5-diyl)bis(sulfanediyl)bis(1-(4-
nitrophenyl)ethanone) (29)
Yield: 0.52 g (91%); 1H NMR (300 MHz, DMSO-d6): d 8.71 (s,
2H, H-2/H-20), 8.46 (m, 4H, H-3/H-30/H-6/H-60), 7.85 (d, 2H, J5,6/
= 8.1, H-5/H-50), 5.1 (s, 4H, 2ꢁ –CH2–); HREI-MS Calcd
3.7.36. 1-(2-Methoxyphenyl)-2-[(5-{[2-(2-methoxyphenyl)-2-
oxoethyl]sulfanyl}-1,3,4-thiadiazol-2-yl)sulfanyl]-1-ethanone
(36)
50,60
475.0417 Found 475.0424, EI-MS m/z (rel. abund.%): 476 (M+,
25), 326 (21), 313 (72), 150 (100), 120 (25); Anal. Calcd C18H12N4-
O6S2, C, 46.95; H, 2.63; N, 12.17; Found: C, 46.92; H, 2.61; N,
12.14.
Yield: 0.52 g (91%); 1H NMR (300 MHz, DMSO-d6): d 7.67 (m,
4H, H-5/H-50/H-6/H-60), 7.21 (d, 2H, J = 8.4 Hz, H-3/H-30), 7.02 (t,
2H, J = 7.5 Hz, H-4/H-40), 4.82 (s, 4H, 2ꢁ –CH2–), 3.91 (s, 6H, 2ꢁ
–OCH3); HREI-MS Calcd 446.0429 Found 446.0421, EI-MS m/z
(rel. abund.%): 446 (M+, 26), 297 (18), 134 (100), 91 (24); Anal.
Calcd C20H18N2O4S3, C, 53.79; H, 4.06; N, 6.27; Found: C, 53.76;
H, 3.99; N, 6.21.
3.7.30. 1-[1,10-Biphenyl]-4-yl-2-({5-[(2-[1,10-biphenyl]-4-yl-2-
oxoethyl)sulfanyl]-1,3,4-thiadiazol-2-yl}sulfanyl)-1-ethanone
(30)
Yield: 0.45 g (86%); 1H NMR (300 MHz, DMSO-d6): d 8.10 (d, 4H,
J = 8.1 Hz, H-2/H-20/H-4/H-40), 7.85 (d, 4H, J3,2/5,6 = 8.1 Hz, H-3/H-
30/H-5/H-50), 7.75 (d, 4H, J = 7.2 Hz, H-2/H-20/H-6/H-60), 7.48 (m,
6H, 2ꢁ H-300/H-400/H-500), 5.14 (s, 4H, 2ꢁ –CH2–), HREI-MS
538.0843 Found 538.0842. EI-MS m/z (rel. abund.%): 538 (M+, 3),
344 (19), 181 (100), 152 (57); Anal. Calcd C30H22N2O2S3, C, 66.89;
H, 4.12; N, 5.20; Found: C, 66.87; H, 4.07; N, 5.18.
3.7.37. 2,20-(1,3,4-Thiadiazole-2,5-diyl)bis(sulfanediyl)bis(1-(3-
hydroxyphenyl)ethanone) (37)
Yield: 0.47 g (87%); 1H NMR (300 MHz, DMSO-d6): d 9.86 (s, 2H,
2ꢁ –OH), 7.47 (d, 2H, J = 7.8 Hz, H-6/H-60), 7.34 (t, 4H, J5/4,6 = 8.0,
H-4/H-40/H-5/H-50), 7.05 (d, 2H, J = 7.8 Hz, H-2/H-20), 5.07 (s, 4H,
2ꢁ –CH2–); HREI-MS Calcd 418.0254 Found 418.0261, EI-MS m/z
(rel. abund.%): 418 (M+, 6), 284 (20), 136 (48), 121 (100), 93
(97); Anal. Calcd C18H14N2O4S3, C, 51.66; H, 3.37; N, 6.69; Found:
C, 51.60; H, 3.35; N, 6.67.
3.7.31. 1-(4-Chlorophenyl)-2-[(5-{[2-(4-chlorophenyl)-2-oxoethyl]
sulfanyl}-1,3,4-thiadiazol-2-yl)sulfanyl]-1-ethanone (31)
Yield: 0.46 g (87%); 1H NMR (300 MHz, DMSO-d6): d 8.0 (d, 4H,
J2,3/6,5 = 8.4 Hz, 2ꢁ H-2/H-6), 7.65 (d, 4H, J = 8.7 Hz, H-3/H-30/H-5/
H-50), 5.01 (s, 4H, 2ꢁ –CH2–); HREI-MS Calcd 453.9438 Found
453.9431. EI-MS m/z (rel. abund.%): 458 (M+4, 4), 456 (M++2, 9),
454 (M+, 22), 360, 315 (18), 302 (15), 141 (100), 111 (80), 75
(36); Anal. Calcd C18H12Cl2N2O2S3, C, 47.47; H, 2.66; N, 6.15;
Found: C, 47.43; H, 2.59; N, 6.09.
Acknowledgments
This work was supported by the Higher Education Commission
(HEC) Pakistan, Project No. 20-1910 under the National Research
Program for Universities. One of us (H.K.) acknowledges the

H. Kashtoh et al. / Bioorg. Med. Chem. 22 (2014) 5454–5465
5465
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