Synthesis, characterization and pharmacological evaluation of n-substituted derivatives of 5-(4-nitrophenyl)-1,3,4-oxadiazole-2yl-2-sulphanyl acetamide

Article abstract of DOI:10.14233/ajchem.2013.14333

A series of new N-substituted 5-(4-nitrophenyl)-1,3,4-oxadiazole-2-yl-2- sulphanyl acetamides was synthesized and enzyme inhibiting activity was screened for all these chemical entities. Structural characterization of all these compounds was made by IR, EI-MS and 1H NMR. All derivatives demonstrated different extent of activity for lipoxygenase (LOX), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Compounds 6j exhibited excellent lipoxygenase inhibitory potential but against acetylcholinesterase the inhibitory potential is not appreciable and it is inactive against butyrylcholinesterase. Similarly compound 6e have good inhibitory potential against lipoxygenase with IC50 value 50.41 ± 0.12 ?M while for acetylcholinesterase IC50 value is 174.21 ± 0.11 ?M and 6e does not show any inhibitory potential for butyrylcholinesterase. Compound 6o showed good inhibition against acetylcholinesterase and butyrylcholinesterase but for lipoxygenase its inhibitory activity is the least.

Full text of DOI:10.14233/ajchem.2013.14333

Asian Journal of Chemistry; Vol. 25, No. 11 (2013), 6231-6236
http://dx.doi.org/10.14233/ajchem.2013.14333
Synthesis, Characterization and Pharmacological Evaluation of N-Substituted
Derivatives of 5-(4-Nitrophenyl)-1,3,4-oxadiazole-2yl-2"-sulphanyl Acetamide
1
1,*
1
1
SAMREEN GUL , AZIZ-UR-REHMAN , MUHAMMAD ATHAR ABBASI , KHADIJA NAFEESA ,
2
3
3
4
ABDUL MALIK , MUHAMMAD ASHRAF , TAYABA ISMAIL and IRSHAD AHMAD
1Department of Chemistry, Government College University, Lahore-54000, Pakistan
2HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
3Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan
4Department of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan
*Corresponding author: Tel: +92 42 111000010; Ext. 449; E-mail: azizryk@yahoo.com
(Received: 19 July 2012;
Accepted: 2 May 2013)
AJC-13443
A series of new N-substituted 5-(4-nitrophenyl)-1,3,4-oxadiazole-2-yl-2"-sulphanyl acetamides was synthesized and enzyme inhibiting
activity was screened for all these chemical entities. Structural characterization of all these compounds was made by IR, EI-MS and 1H
NMR. All derivatives demonstrated different extent of activity for lipoxygenase (LOX), acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE). Compounds 6j exhibited excellent lipoxygenase inhibitory potential but against acetylcholinesterase the
inhibitory potential is not appreciable and it is inactive against butyrylcholinesterase. Similarly compound 6e have good inhibitory
potential against lipoxygenase with IC50 value 50.41 0.12 µM while for acetylcholinesterase IC50 value is 174.21 0.11 µM and 6e does
not show any inhibitory potential for butyrylcholinesterase. Compound 6o showed good inhibition against acetylcholinesterase and
butyrylcholinesterase but for lipoxygenase its inhibitory activity is the least.
Key Words: 4-Nitrobenzoic acids, Oxadiazoles, Cholinesterase, Lipoxygenase, 1H NMR and EI-MS.
to their antiinflammatory, antituberculosis, anticonvulsant,
antianalgesic, antidepressant and anti HIV activities5-7. The
significance of these compounds lies in the fact that they can
be successfully utilized as antibacterial, analgesic, antiinflam-
matory, anticancer, anti HIV agent, antitubercular and insecti-
cidal agents8-10. Literature survey revealed that minor modifi-
cation in the structure of substituted 1,3,4-oxadiazole can lead
to quantitative as well as qualitative changes in the biological
activity. Biologically and pharmacologically most important
class of oxadiazoles is 1,3,4-oxadiazole-2-thiol. In view of
the published information and in the continuation of our
previous work11, we now report the synthesis of N-substituted
5-(4-nitrophenyl)-1,3,4-oxadiazole-2-yl- 2"-sulphanyl acetamides
and screening out their biological activities.
INTRODUCTION
Most of the drugs used for the treatment of Alzheimer's
disease (AD) are acetylcholinesterase inhibitors. Acetylcho-
linesterase causes the hydrolysis of acetyl choline in the
synaptic cleft, so acetyl cholinesterase inhibitors ensure the
availability of acetylcholine in the synaptic cleft by decreasing
the activity of acetylcholinesterase and help to enhance the
cholinergic neurotransmission1. Lipoxygenase enzymes contain
iron in their structural frame work and are involved in
dioxygenation of lipids, consisting of poly unsaturated fatty
acids. Lipoxygenases perform a key role in the synthesis of
leukotrienes, which are responsible for pathophysiology of
different allergic diseases. Lipoxygenase inhibitors are required
to cure these allergic and inflammatory diseases2. Butyryl
cholinesterase is a sister enzyme of acetyl cholinesterase. It is
also known as pseudocholinesterase3. Butyrylcholinesterase
is toxicologically and pharmacologically very important4.
The aim of our present research work was to synthesize
new and pharmacologically active compounds to meet the
needs of pharmacy due to increased resistance against present
antibacterial, antiviral, fungicidal and pesticidal drugs.
Oxadiazole analogues are pharmacologically very active due
EXPERIMENTAL
Chemicals were purchased from Sigma Aldrich and Alfa
Aesar (Germany). By using open capillary tube method melting
points were taken on Griffin and George melting point apparatus.
Melting points were uncorrected. By using thin layer chroma-
tography and n-hexane and ethyl acetate as mobile phase
purity of the synthesized compounds was detected at 364 nm.
By using KBr, IR peaks were recorded on a Jasco-320-A

6232 Gul et al.
Asian J. Chem.
spectrophotometer. 1H NMR signals were recorded at 500 MHz
in mixture of CDCl3 and CD3OD using Bruker spectrometers,
chemical shift values are mentioned in ppm unit. EIMS signals
are recorded by utilizing a JMS-HX-110 spectrometer.
Synthesis of ethyl-4-nitro benzoate (2): 4-Nitrobenzoic
acid (1) (5g, 0.0295 mol.) was refluxed with absolute ethanol
(20 mL) and conc. H2SO4 (2.5 mL) in round bottom flask fitted
with condenser for 1 h. Thin layer chromatography was used
to check the completion of reaction. Reaction mixture was
poured in 50 mL distilled water in separating funnel, after the
indication of completion of reaction by TLC. Diethyl ether
(45 mL) was used to extract the product by vigorous shaking
in separating funnel and addition of conc. sodium carbonate
afforded the neutralization of reaction mixture. Two layers
were separated by allowing the solution to stand for some time.
To avoid contaminants upper organic layer containing ethyl-
4-nitrobenzoate was collected from the neck of separating
funnel. Lemon coloured ester was collected by evaporating
diethyl ether.
1653 (C=N, str. of oxadiazole ring), 1275, 1053 (C-O-C bond
str.), 641 (C-S bond str.); 1H NMR (MeOD, 300 MHz): δ (ppm)
8.23 (d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.16 (d, J = 9.0 Hz,
2H, H-2' and H-6'), 7.10 (d, J = 7.8 Hz, 1H, H-6"'), 7.06 (br.t,
J = 7.8 Hz, 1H, H-5"'), 6.97 (d, J = 8.4 Hz, 1H, H-3"'), 6.86
(ddd, J = 8.4, 1.5 Hz, 1H, H-4"'), 4.31 (s, 2H, H-2"), 3.85 (s,
3H, OCH3-2"'); EIMS (m/z): 386 (17 %)[M+], 264 (21 %),
164 (100 %), 150 (37 %), 148 (71 %), 122 (56 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(3-methoxyphenyl)acetamide (6b): Creamy white colour;
yield: 60 %; m.p. 130-132 ºC; m.f.: C17H14N4O5S; m.w. 386;
IR (KBr, νmax, cm-1): 3343 (N-H, stretching), 3079 (C-H, str.
of aromatic ring), 1663 (C=O str.), 1557 (C=C, aromatic str.),
1671 (C=N, str. of oxadiazole ring), 1281, 1061 (C-O-C bond
str.), 637 (C-S bond str.); 1H NMR (MeOD, 300 MHz): δ (ppm)
8.24 (d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.17 (d, J = 9.0 Hz,
2H, H-2' and H-6'), 7.29 (d, J = 1.8 Hz, 1H, H-2"'), 7.21 (t, J =
8.4 Hz, 1H, H-5"'), 7.08 (dd, J = 8.4, 1.8 Hz, 1H, H-6"'), 6.70
(dd, J = 8.4, 1.8 Hz, 1H, H-4"'), 4.28 (s, 2H, H-2"), 3.77 (s,
3H, OCH3-3"'); EIMS (m/z): 386 (20 %) [M+], 264 (24 %),
164 (100 %), 150 (41 %), 148 (68 %), 122 (49 %).
Preparation of 4-nitro benzohydrazide (3): Synthesis
of 4-nitrobenzohydrazide was afforded by allowing ethyl 4-
nitrobenzoate (2 g, 0.01 mol), on complete dissolution, to react
with hydrazine hydrate (7.90 mL, 0.03 mol.) in methanol (15
mL) by vigorous stirring at room temperature for 3 h in a
round bottom flask. Thin layer chromatography was used to
check the completion of reaction. Precipitates of product were
quenched by addition of distilled water and filtered washed.
Recrystallization was brought about by using methanol.
Synthesis of 5-(4-nitrophenyl)-1,3,4-oxadiazole-2-thiol
(4): Compound (3) (4 g, 0.022 mol) to carbon disulphide (1.32
mL, 0.022 mol) in the presence of potassium hydroxide (2.46
g, 0.044 mol) and ethanol (40 mL) as solvent in 250 mL round
bottom flask, reaction assembly was set to reflux reaction
contents for 5 h with continuous stirring. By using TLC reaction
coordinates were monitored after every hour. On completion
reaction mixture was acidified and pH was set 2.0-3.0 then
treated with distilled water to quench the synthesized product
that was filtered and washed with distilled water. Recrystalli-
zation was done by using methanol.
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(2-methylphenyl)acetamide (6c): Lemon yellow colour;
yield: 75 %; m.p. 42-44 ºC; m.f.: C17H14N4O4S; m.w. 370; IR
(KBr, νmax, cm-1): 3325 (N-H, stretching), 3033 (C-H, str. of
aromatic ring), 1610 (C=O str.), 1521 (C=C, aromatic str.),
1630 (C=N, str. of oxadiazole ring), 1235, 1065 (C-O-C bond
str.), 618 (C-S bond str.); 1H NMR (DMSO, 500 MHz): δ (ppm)
14.95 (br. s, 1H, -NH), 8.24 (d, J = 9.0 Hz, 2H, H-3' and H-5'),
8.12 (d, J = 9.0 Hz, 2H, H-2' and H-6'), 7.37 (d, J = 7.5 Hz,
1H, H-6"'), 7.21 (d, J = 7.5 Hz, 1H, H-3"'), 7.15 (br.t, J = 7.5
Hz, 1H, H-5"'), 7.09 (ddd, J = 6.5, 1.0 Hz, 1H, H-4"'), 4.41 (s,
2H, H-2"), 2.19 (s, 3H, CH3-2"'); EIMS (m/z): 370 (20 %)[M+],
222 (41 %), 148 (100 %), 122 (35 %), 91 (75 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(3-methylphenyl)acetamide (6d): Lemon yellow colour;
yield: 70 %; m.p. 42-44 ºC; m.f.: C17H14N4O4S; m.w. 370; IR
(KBr, νmax, cm-1): 3341 (N-H, stretching), 3075 (C-H, str. of
aromatic ring), 1669 (C=O str.), 1563 (C=C, aromatic str.),
1667 (C=N, str. of oxadiazole ring), 1279, 1058 (C-O-C bond
str.), 631 (C-S bond str.); 1H NMR (MeOD, 300 MHz): δ (ppm)
8.38 (d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.21 (d, J = 9.0 Hz,
2H, H-2' and H-6'), 8.37 (d, J = 3.0 Hz, 1H, H-2"'), 8.12 (d,
J = 8.0 Hz, 1H, H-6"'), 7.17 (t, J = 8.0 Hz, 1H, H-5"'), 6.88 (d,
J = 8.0 Hz, 1H, H-4"'), 4.37 (s, 2H, H-2"), 2.26 (s, 3H, CH3-
3"'); EIMS (m/z): 370 (10 %)[M+], 222 (32 %), 148 (75 %),
122 (39 %), 91 (100 %).
General procedure for the synthesis of S-substituted
5-(4-nitrophenyl)-1,3,4-oxadiazole derivatives (6a-o):
Compound (4), (0.1 g, 0.0045 mol.) was allowed to react with
equimolar ratios of aliphatic/ aromatic substituted bromo-
acetamide (5a-o) by dissolving the first in DMF (5-10 mL) by
stirring at room temperature in 100 mL round bottom flask in
the presence of NaH (0.002 g), that acts as a strong base. The
time span for different aliphatic/aromatic substituted
bromoacetamide varies from 2-3 h. Thin layer chromatography by
using ethyl acetate and n-hexane as a mobile phase was carried
out to check the reaction completion. Distilled water was added
in the reaction mixture to separate the precipitates. Precipitates
were washed, dried and subjected for spectral analysis.
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(4-methylphenyl)acetamide (6e): Off-white colour; yield:
67 %; m.p. 42-44 ºC; m.f.: C17H14N4O4S; m.w. 370; IR (KBr,
νmax, cm-1): 3345 (N-H, stretching), 3060 (C-H, str. of aromatic
ring), 1630 (C=O str.), 1537 (C=C, aromatic str.), 1645 (C=N,
str. of oxadiazole ring), 1255, 1090 (C-O-C bond str.), 625
(C-S bond str.); 1H NMR (MeOD, 300 MHz): δ (ppm) 8.23
(d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.17 (d, J = 9.0 Hz, 2H,
H-2' and H-6'), 7.44 (d, J = 7.1 Hz, 2H, H-2"' and H-6"'), 7.14
(d, J = 7.1 Hz, 2H, H-3"' and H-5"'), 4.27 (s, 2H, H-2"), 2.29
(s, 3H, CH3-4"'); EIMS (m/z): 370 (12 %)[M+], 222 (17 %),
148 (100%), 122 (37 %), 91 (71 %).
Characterization of the synthesized compounds
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(2-methoxyphenyl)acetamide (6a): Lemon yellow colour;
yield: 73 %; m.p. 110-112 ºC; m.f.: C17H14N4O5S; m.w. 386;
IR (KBr, νmax, cm-1): 3327 (N-H, stretching), 3067 (C-H, str.
of aromatic ring), 1649 (C=O str.), 1547 (C=C, aromatic str.),

Vol. 25, No. 11 (2013)
Synthesis of N-Substituted Derivatives of 5-(4-Nitrophenyl)-1,3,4-oxadiazole-2yl-2"-sulphanylAcetamide 6233
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(2,4-dimethylphenyl)acetamide (6f): Lemon yellow
colour; yield: 63 %; m.p. 160-162 ºC; m.f.: C18H16N4O4S; m.w.
384; IR (KBr, νmax, cm-1): 3339 (N-H, stretching), 3085 (C-H,
str. of aromatic ring), 1655 (C=O str.), 1560 (C=C, aromatic
str.), 1667 (C=N, str. of oxadiazole ring), 1287, 1065 (C-O-C
bond str.), 637 (C-S bond str.); 1H NMR (MeOD, 300 MHz):
δ (ppm) 8.26 (d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.17 (d, J =
9.0 Hz, 2H, H-2' and H-6'), 7.23 (d, J = 8.1 Hz, 1H, H-6"'),
7.03 (s, 1H, H-3"'), 6.99 (d, J = 8.1 Hz, 1H, H-5"'), 4.30 (s,
2H, H-2"), 2.27 (s, 3H, CH3-2"'), 2.20 (s, 3H, CH3-4"'); EIMS
(m/z): 384 (12 %) [M+], 264 (17 %), 148 (100 %), 122 (37 %),
120 (71 %).
2' and H-6'), 7.96 (s, 2H, H-2"' to H-6"'), 7.18 (s, 1H, H-4"'),
4.26 (s, 2H, H-2"), 2.26 (s, 6H, CH3-3"' and CH3-5"'); EIMS
(m/z): 384 (18 %) [M+], 264 (21 %), 148 (100 %), 122 (31 %),
120 (73 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-benzylacetamide (6k): Lemon yellow colour; yield: 67 %;
m.p. 150-152 ºC; m.f.: C17H14N4O4S; m.w. 370; IR (KBr, νmax
,
cm-1): 3343 (N-H, stretching), 3053 (C-H, str. of aromatic ring),
1663 (C=O str.), 1573 (C=C, aromatic str.), 1677 (C=N, str. of
oxadiazole ring), 1273, 1058 (C-O-C bond str.), 653 (C-S bond
1
str.); H NMR (MeOD, 300 MHz): δ (ppm) 8.41 (d, J = 9.0
Hz, 2H, H-3' and H-5'), 8.16 (d, J = 9.0 Hz, 2H, H-2' and H-
6'), 7.29-7.06 (m, 5H, H-2"' to H-6"'), 4.28 (s, 2H, H-2"), 3.76
(s, 2H, H-7"'); EIMS (m/z): 370 (15 %) [M+], 264 (24 %), 148
(100 %), 134 (45 %), 122 (69 %), 106 (47 %), 91 (73 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(2-phenylethyl)acetamide (6l): Light yellow colour; yield:
59 %; m.p. 156-158 ºC; m.f.: C18H16N4O4S; m.w. 384; IR (KBr,
νmax, cm-1): 3351 (N-H, stretching), 3047 (C-H, str. of aromatic
ring), 1660 (C=O str.), 1576 (C=C, aromatic str.), 1671 (C=N,
str. of oxadiazole ring), 1279, 1069 (C-O-C bond str.), 647
(C-S bond str.); 1H NMR (MeOD, 300 MHz): δ (ppm) 8.41
(d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.24 (d, J = 9.0 Hz, 2H, H-
2' and H-6'), 7.27-7.12 (m, 5H, H-2"' to H-6"'), 4.03 (s, 2H,
H-2"), 3.44 (t, J = 7.2 Hz, 2H, H-8"'), 2.72 (t, J = 7.2 Hz, 2H,
H-7"'); EIMS (m/z): 384 (19 %) [M+], 264 (21 %), 162 (81 %),
148 (100 %), 122 (67 %), 120 (43 %), 105 (67 %), 91 (77 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(2-hydroxyphenyl)acetamide (6m): Coffee colour; yield:
68 %; m.p. 90-92 ºC; m.f.: C16H12N4O5S; m.w. 372; IR (KBr,
νmax, cm-1): 3351 (N-H, stretching), 3047 (C-H, str. of aromatic
ring), 1660 (C=O str.), 1576 (C=C, aromatic str.), 1671 (C=N,
str. of oxadiazole ring), 1279, 1069 (C-O-C bond str.), 647
(C-S bond str.); 1H NMR (MeOD, 300 MHz): δ (ppm) 8.41
(d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.18 (d, J = 9.0 Hz, 2H, H-
2' and H-6'), 7.80 (dd, J = 8.1, 1.5 Hz, 1H, H-6"'), 7.00 (ddd,
J = 8.1, 1.5 Hz, 1H, H-4"'), 6.85 (dd, J = 8.1, 1.5 Hz, 1H, H-
3"'), 6.79 (ddd, J = 8.1, 1.5 Hz, 1H, H-5"'), 4.33 (s, 2H, H-2");
EIMS (m/z): 372 (11 %) [M+], 264 (23 %), 222 (76 %), 150
(45 %), 148 (79 %), 136 (56 %), 108 (91 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(2,5-dimethylphenyl)acetamide (6g): Mustard colour;
yield: 65 %; m.p. 180-182 ºC; m.f.: C18H16N4O4S; m.w. 384;
IR (KBr, νmax, cm-1): 3315 (N-H, stretching), 3065 (C-H, str.
of aromatic ring), 1676 (C=O str.), 1547 (C=C, aromatic str.),
1640 (C=N, str. of oxadiazole ring), 1265, 1043 (C-O-C bond
str.), 642 (C-S bond str.); 1H NMR (MeOD, 300 MHz): δ (ppm)
8.25 (d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.15 (d, J = 9.0 Hz,
2H, H-2' and H-6'), 7.18 (d, J = 1.5 Hz, 1H, H-6"'), 7.08 (dd,
J = 8.1, 1.5 Hz, 1H, H-4"'), 6.92 (d, J = 8.1 Hz, 1H, H-3"'),
4.30 (s, 2H, H-2"), 2.18 (s, 3H, CH3-2"'), 2.16 (s, 3H, CH3-
5"'); EIMS (m/z): 384 (17 %) [M+], 264 (22 %), 148 (100 %),
122 (31 %), 120 (67 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(2,6-dimethylphenyl)acetamide (6h): Creamy white
colour; yield: 66 %; m.p. 160-162 ºC; m.f.: C18H16N4O4S; m.w.
384; IR (KBr, νmax, cm-1): 3315 (N-H, stretching), 3067 (C-H,
str. of aromatic ring), 1676 (C=O str.), 1538 (C=C, aromatic
str.), 1658 (C=N, str. of oxadiazole ring), 1283, 1047 (C-O-C
bond str.), 643 (C-S bond str.); 1H NMR (MeOD, 300 MHz):
δ (ppm) 8.27 (d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.17 (d, J =
9.0 Hz, 2H, H-2' and H-6'), 7.08-7.06 (m, 3H, H-3"' to H-5"'),
4.35 (s, 2H, H-2"), 2.20 (s, 6H, CH3-2"' and CH3-6"'); EIMS
(m/z): 384 (16 %)[M+], 264 (19 %), 148 (100 %), 122 (41 %),
120 (73 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(3,4-dimethylphenyl)acetamide (6i): Buff colour; yield:
71 %; m.p. 145-147 ºC; m.f.: C18H16N4O4S; m.w. 384; IR (KBr,
nmax, cm-1): 3313 (N-H, stretching), 3076 (C-H, str. of aromatic
ring), 1648 (C=O str.), 1567 (C=C, aromatic str.), 1673 (C=N,
str. of oxadiazole ring), 1279, 1059 (C-O-C bond str.), 642
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(2,4-dinitrophenyl)acetamide (6n): Lemon yellow colour;
yield: 61 %; m.p. 160-162 ºC; m.f.: C16H10N6O8S; m.w. 446;
IR (KBr, νmax, cm-1): 3356 (N-H, stretching), 3041 (C-H, str.
of aromatic ring), 1657 (C=O str.), 1569 (C=C, aromatic str.),
1659 (C=N, str. of oxadiazole ring), 1283, 1071 (C-O-C bond
str.), 635 (C-S bond str.); 1H NMR (MeOD, 300 MHz): δ (ppm)
8.98 (d, J = 2.7 Hz, 1H, H-3"'), 8.82 (d, J = 9.6 Hz, 1H, H-6"'),
8.69 (d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.53 (dd, J = 9.6, 2.4
Hz, 1H, H-5"'),8.39 (d, J = 9.0 Hz, 2H, H-2' and H-6'), 4.16 (s,
2H, H-2"); EIMS (m/z): 446 (17 %) [M+], 264 (21 %), 224
(100 %), 210 (37 %), 182 (71 %), 148 (41 %), 122 (56 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-cyclohexylacetamide (6o): Buff colour; yield: 58 %; m.p.
140-142 ºC; m.f.: C16H18N4O4S; m.w. 362; IR (KBr, νmax, cm-1):
3357 (N-H, stretching), 3035 (C-H, str. of aromatic ring), 1653
(C=O str.), 1583 (C=C, aromatic str.), 1657 (C=N, str. of
oxadiazole ring), 1263, 1057 (C-O-C bond str.), 653 (C-S bond
1
(C-S bond str.); H NMR (MeOD, 300 MHz): δ (ppm) 8.23
(d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.17 (d, J = 9.0 Hz, 2H, H-
2' and H-6'), 7.29 (d, J = 2.4 Hz, 1H, H-2"'), 7.26 (dd, J = 8.1,
2.4 Hz, 1H, H-6"'), 7.04 (d, J = 8.1 Hz, 1H, H-5"'), 4.26 (s,
2H, H-2"), 2.23 (s, 3H, CH3-4"'), 2.21 (s, 3H, CH3-3"'); EIMS
(m/z): 384 (15 %) [M+], 264 (27 %), 148 (100 %), 122 (41
%), 120 (69 %).
2-[{5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-yl}sulphanyl]-
N-(3,5-dimethylphenyl)acetamide (6j):Yellow colour; yield:
69 %; m.p. 163-165 ºC; m.f.: C18H16N4O4S; m.w. 384; IR (KBr,
νmax, cm-1): 3317 (N-H, stretching), 3063 (C-H, str. of aromatic
ring), 1654 (C=O str.), 1558 (C=C, aromatic str.), 1661 (C=N,
str. of oxadiazole ring), 1291, 1069 (C-O-C bond str.), 633
1
(C-S bond str.); H NMR (MeOD, 300 MHz): δ (ppm) 8.40
(d, J = 9.0 Hz, 2H, H-3' and H-5'), 8.23 (d, J = 9.0 Hz, 2H, H-
1
str.); H NMR (MeOD, 300 MHz): δ (ppm) 8.25 (d, J = 9.0

6234 Gul et al.
Asian J. Chem.
Hz, 2H, H-3' and H-5'), 8.18 (d, J = 9.0 Hz, 2H, H-2' and H-
6'), 4.06 (s, 2H, H-2"), 3.61 (m, 1H, H-1"'), 1.87-1.71 (m, 4H,
H-2"' and H-6"'), 1.64-1.60 (m, 2H, H-4"'), 1.36-1.17 (m, 4H,
H-3"' and H-5"'); EIMS (m/z): 362 (17 %) [M+], 264 (26 %),
148 (100 %), 140 (49 %), 126 (71 %), 122 (46 %), 98 (77 %),
83 (75 %).
AChE and BChE inhibitors. The need of hours is to introduce
pharmacologically active drugs to help pharmacy against the
increasing resistance for existing drugs. Keeping that objective
in mind, we synthesized N-substituted 5-(4-nitrophenyl)-1,3,4-
oxadiazole-2-yl-2"-sulphanyl acetamides in excellent yield and
having good biological activities. The synthesis was carried
out through the intermolecular cyclization of 4-nitrobenzoic
acid hydrazide to 5-(4-nitrophenyl)-1,3,4-oxadiazole-2-thiol
and finally a series of N-substituted 5-(4-nitrophenyl)-1,3,4-
oxadiazole-2-yl-2"-sulphanyl acetamides (6a-o) after the
reaction with N-aryl/alkyl-substituted 2-bromoacetamide (5a-
o) in DMF using sodium hydride as a base as represented in
Scheme-I. Complete conversion was achieved within 2-4 h by
simple stirring at room temperature. The product was isolated
by adding cold water in the reaction mixture and subsequently,
it was taken out through solvent extraction method by chloro-
form/ethyl acetate.All the derivatives (6a-o) were characterized
Enzyme inhibition essays
Acetylcholinesterase and butyrylcholinesterase assay:
The AChE and BChE inhibition activity was performed
according to the method12 with slight modifications. Total
volume of the reaction mixture was 100 µL. It contained 60 µL
Na2HPO4 buffer with concentration of 50 mM and pH 7.7.
Ten µL test compound (0.5 mM well-1) was added, followed
by the addition of 10 µL (0.005 unit well-1) enzyme (Sigma,
USA). The contents were mixed and pre-read at 405 nm. Then
contents were pre-incubated for 10 min at 37 ºC. The reaction
was initiated by the addition of 10 µL of 0.5 mM well-1 substrate
(acetylthiocholine iodide or butyrylthiocholine chloride),
followed by the addition of 10 µL DTNB (0.5 mM well-1).
After 15 min of incubation at 37 ºC absorbance was measured
at 405 nm using 96-well plate reader Synergy HT, Biotek,
USA. All experiments were carried out with their respective
controls in triplicate. Eserine (0.5 mM well-1) was used as a
positive control. The percent inhibition was calculated by the
help of following equation
1
by IR, H NMR and EI-MS analysis. IR spectra revealed
characteristic peaks at 3357-3313 cm-1 for N-H str. 1676-1610
for C=O str. of amide group, 1677-1630 for C=N str. of oxadiazole
1
ring. H NMR data gave peaks for -NH at 14.95 ppm and
-CH2 of acetamide group appeared at 4.03-4.41 ppm as singlet
having integration of two protons. Compound (6a) was
obtained as lemon yellow colour solid with m.f. C17H14N4O5S,
that is confirmed by EI-MS and molecular ion peak appeared
at 386. In IR spectrum characteristic peaks appeared at 3327
(N-H, stretching), 3067 (C-H, str. of aromatic ring), 1649 (C=O
str.), 1547 (C=C, aromatic str.), 1653 (C=N, str. of oxadiazole
ring), 1275, 1053 (C-O-C bond str.), 641 (C-S bond str.), 1267
cm-1 (C=S, stretching) confirming the presence of oxadiazole
ring. In 1H NMR spectrum signals of aromatic proton appeared
at δ 8.23 (br.d, J = 9.0 Hz, 2H, H-3' and H-5') and 8.16 (br.d,
J = 9.0 Hz, 2H, H-2' and H-6') typical for para di-substituted
aromatic ring other aromatic ring showed peaks at 7.10 (d, J =
7.8 Hz, 1H, H-6"'), 7.06 (br.t, J = 7.8 Hz, 1H, H-5"'), 6.97 (d,
J = 8.4 Hz, 1H, H-3"'), 6.86 (ddd, J = 8.4, 1.5 Hz, 1H, H-4"')
for four different protons of ortho substituted ring. In 1H NMR
spectrum signals appeared in the aliphatic region at 4.31 (s,
2H, H-2"), 3.85 (s, 3H, OCH3-2"') which specified -CH2 group
of acetamide moiety and methoxy group present on the
benzene ring. On the basis of these spectral evidences, the
structure was determined as 2-[{5-(4-nitrophenyl)-1,3,4-
oxadiazole-2-yl}sulphanyl]-N-(2-methoxyphenyl)acetamide.
Similarly on the basis of spectral evidences from IR, EI-MS
and 1H NMR (Table-1), the structures of other derivatives of
2,5-disubstituted 1,3,4-oxadiazole were elucidated as described
in experimental section.
Control − Test
Inhibition (%) =
×100
Control
IC50 values were calculated using EZ-Fit Enzyme kinetics
software (Perrella Scientific Inc. Amherst, USA).
Lipoxygenase assay: Lipoxygenase (LOX) activity was
assayed addording to the reported method13-15 with slight
modifications. A total volume of 200 µL lipoxygenase assay
mixture contained 150 µL sodium phosphate buffer (100 mM,
pH 8.0), 10 µL test compound and 15 µL purified lipoxygenase
enzyme (600 units well-1, Sigma Inc.). The contents were mixed
and pre-read at 234 nm and preincubated for 10 min at 25 ºC.
The reaction was initiated by addition of 25 µL substrate
solution. The change in absorbance was observed after 6 min
at 234 nm using 96-well plate reader Synergy HT, Biotek,
USA. All reactions were performed in triplicates. The positive
and negative controls were included in the assay. Baicalin (0.5
mM well-1) was used as a positive control. The percentage
inhibition (%) was calculated by formula given below.
Control − Test
Inhibition (%) =
×100
Control
Pharmacology:All the compounds were screened against
acetylcholinesterase (AChE), butyrylcholinesterase (BChE)
and lipoxygenase (LOX) enzymes and results are mentioned
in Table-1. Compounds 2-[{5-(4-nitrophenyl)-1,3,4-
oxadiazole-2-yl}sulphanyl]-N-(3,5-dimethyl phenyl)acetamide
(6j) exhibit excellent inhibitory potential against lipoxygenase
with IC50 value 46.51 0.02 µmol/L relative to Baicalein, a
reference standard with IC50 value of 22.4 1.3 µmol/L but
against acetylcholinesterase the inhibitory potential is not
appreciable and it is inactive against butyrylcholinesterase.
Compound 2-[{5-(4-nitrophenyl)-1,3,4-oxadiazole-2-yl}-
where Control = total enzyme activity without inhibitor, Test
= activity in the presence of test compound.
IC50 values was calculated using EZ-Fit Enzyme Kinetics
software (Perrella Scientific Inc. Amherst, USA).
Statistical analysis: All the measurements were done in
triplicate and statistical analysis was performed by Microsoft
Excel 2003. Results are presented as mean sem.
RESULTS AND DISCUSSION
The aim of the present research work was to synthesize
new compounds that have great potential as lipoxygenase,

Vol. 25, No. 11 (2013)
Synthesis of N-Substituted Derivatives of 5-(4-Nitrophenyl)-1,3,4-oxadiazole-2yl-2"-sulphanylAcetamide 6235
N
N
O
O
C
O
C
CS2, KOH
H2SO4/EtOH
MeOH, 0-5oC
C
O
OH
OCH2CH3
SH
NH NH2
Reflux, 4 hrs
NH2-NH2 (80%)
O2N
O2N
O2N
O2N
Ester
2
p-Nitrobenzoic acid
1
p-Nitrobenzoic acid hydrazide
3
5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-thiol
4
N
N
4
5
3
2
O
5a-o
2''
CH2
1
O
R
C CH2 Br
1''
R
3'
S
Stirr & heat
3 hr, RT
5'
1'
DMF, LiH
O2N
O
S-substituted-5-(4-Nitrophenyl)-1,3,4-
oxadiazole-2-thiol derivatives
6a-o
Comp.
-R
Comp.
-R
Comp.
-R
Comp.
-R
H3CO
CH3
HO
2'''
2'''
6'''
2'''
6'''
2'''
6'''
HN
CH
3
6a
6e
6i
6j
6m
4'''
HN
HN
CH3
HN
4'''
4'''
4'''
6'''
CH3
OCH3
H3C
O2N
2'''
2'''
6'''
2'''
6'''
2'''
HN
6b
6c
6f
4'''
6n
6o
HN
CH3
HN
NO2
HN
4'''
4'''
4'''
6'''
6'''
CH3
H3C
H3C
2'''
6'''
2'''
6'''
2'''
6'''
7'''
H2C
2'''
6'''
HN
HN
HN
6g
4'''
6k
6l
4'''
4'''
HN
4'''
CH3
H3C
CH
3
2'''
2'''
7'''
H2C
8'''
HN H2C
2'''
HN
4'''
4'''
–
6d
6h
–
HN
4'''
6'''
6'''
6'''
H3C
Scheme-I: Synthesis of N-substituted derivatives of 5-(4-nitrophenyl)-1,3,4-oxadiazole-2yl-2"-sulfanyl acetamide
TABLE-1
BIOACTIVITY STUDIES OF N-SUBSTITUTED DERIVATIVES OF
5-(4-NITROPHENYL)-1,3,4-OXADIAZOLE-2YL-2"-SULFANYL ACETAMIDE
AChE
BChE
Inhibition (%)
LOX
Inhibition (%)
Sample
code
Inhibition (%) conc./well
(0.5 mM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
–
conc./well (0.5 mM)
28.62 0.51
34.92 0.37
62.41 0.47
40.19 0.22
36.88 0.48
46.23 0.54
53.21 0.17
73.06 0.55
51.76 0.22
18.18 0.11
57.23 0.91
67.56 0.08
51.03 0.39
18.90 0.25
94.51 0.22
Eserine 82.82 1.09
conc./well (0.5 mM)
41.76 0.58
60.85 0.66
64.83 0.58
54.07 0.25
95.83 0.22
70.16 0.11
39.59 0.87
68.61 0.69
69.51 0.81
96.91 0.69
45.25 0.18
14.15 0.19
38.18 0.56
58.43 0.15
51.26 0.35
Baicalein 93.79 1.27
6a
6b
55.11 0.64
76.09 0.82
66.79 0.15
65.33 0.72
67.52 0.63
79.74 0.09
77.55 0.14
77.37 0.59
68.80 0.37
57.12 0.17
67.88 0.15
74.45 0.11
40.33 0.55
53.65 0.31
91.06 0.23
Eserine 91.29 1.17
<400
–
147.81 0.15
198.51 0.01
205.31 0.11
174.21 0.11
112.11 0.05
134.21 0.12
128.11 0.14
134.71 0.11
<400
–
248.51 0.14
215.41 0.21
<400
6c
281.31 0.11
6d
–
6e
–
50.41 0.12
147.91 0.15
–
6f
–
<400
6g
6h
182.31 0.07
<300
183.25 0.13
98.61 0.11
46.51 0.02
–
6i
6j
–
6k
150.11 0.15
158.21 0.14
–
<400
6l
231.71 0.06
<400
–
6m
6n
–
<400
–
285.21 0.25
<400
6o
98.11 0.14
0.04 0.001
81.21 0.15
0.85 0.0001
Control
22.4 1.3
Note: IC50 values (concentration at which there is 50 % enzyme inhibition) of compounds were calculated using EZ-Fit Enzyme kinetics software
(Perella Scientific Inc. Amherst, USA). AChE = Acetylcholinesterase. BChE = Butyrylcholinesterase. LOX = Lipoxygenase.

6236 Gul et al.
Asian J. Chem.
sulphanyl]-N-cyclohexylacetamide (6o) showed good inhibition
against AChE and BChE but for lipoxygenase its inhibitory
activity is not mentionable and IC50 values forAChE and BChE
are 98.11 0.14 and 81.21 0.15 µmol/L, respectively. Simi-
larly the compound 2-[{5-(4-nitrophenyl)-1,3,4-oxadiazole-
2-yl}sulphanyl]-N-(4-methylphenyl)acetamide (6e) revealed
good inhibitory potential against lipoxygenase enzyme with
IC50 value of 50.41 0.12 µmol/L while for AChE IC50 value
is 174.21 0.11 µmol/L and 6e does not show any inhibitory
potential for BChE.
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