Synthesis, structural characterization and enzyme Inhibition studies on 5-(2-Nitrostyryl)-1,3,4-oxadiazole-2-thiol Derivatives

Article abstract of DOI:10.4067/S0717-97072013000400061

In the present work, S-substituted derivatives of 5-(2-nitrostyryl)-1,3,4- oxadiazole-2-thiol (4) were synthesized by successive conversions of 3-(2-nitrophenyl) acrylic acid (1) into its respective ester, hydrazide and 1,3,4-oxadiazole. Finally the targe

Full text of DOI:10.4067/S0717-97072013000400061

J. Chil. Chem. Soc., 58, Nº 4 (2013)
SYNTHESIS, STRUCTURAL CHARACTERIZATION AND ENZYME INHIBITION STUDIES ON
5-(2-NITROSTYRYL)-1,3,4-OXADIAZOLE-2-THIOL DERIVATIVES
MUHAMMAD ATHAR ABBASI*,a, ADNAN AKHTARa, AZIZ-UR-REHMANa, KHADIJA NAFEESAa, SABAHAT
ZAHRA SIDDIQUIa, KHALID MOHAMMED KHANb, MUHAMMAD ASHRAFc, AND SYEDA ABIDA EJAZd
aDepartment of Chemistry, Government College University, Lahore-54000, Pakistan.
bHEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
cDepartment of Biochemistry and Biotechnology; dDepartment of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan.
(Received: November 21, 2013 - Accepted: August 6, 2013)
ABSTRACT
In the present work, S-substituted derivatives of 5-(2-nitrostyryl)-1,3,4-oxadiazole-2-thiol (4) were synthesized by successive conversions of 3-(2-nitrophenyl)
acrylic acid (1) into its respective ester, hydrazide and 1,3,4-oxadiazole. Finally the target compounds were obtained by reaction of 5-(2-nitrostyryl)-1,3,4-
oxadiazole-2-thiol (4) with a series of various electrophiles, (5a-I), in N,N-dimethyl formamide (DMF) in the presence of sodium hydride (NaH). The structural
characterization of these newly synthesized compounds was done by IR, 1H-NMR, HR-MS and EI-MS spectral data. All these compounds were evaluated for their
enzyme inhibitory potentials and found to exhibit broad range spectrum against acetylcholinesterase, butyrylcholinestrase and lipoxygenase enzymes.
Keywords: 3-(2-nitrophenyl)acrylic acid, 5-(2-nitrostyryl)-1,3,4-oxadiazole-2-thiol, 1H-NMR, EI-MS, enzymatic activities.
2.2.
acrylate
Procedure for the preparation of Ethyl-3-(2-nitrophenyl)
1. INTRODUCTION
3-(2-nitrophenyl)acrylic acid (1g; (1)) was taken into a 100 mL round
bottom flask fitted with a reflux condenser, then absolute ethanol (4 mL)
and conc. sulphuric acid (1/2 mL) were added into flask and the reaction
mixture was refluxed for about 1 hour. The reaction was monitored by thin
layer chromatography (TLC). When the reaction was completed, the reaction
mixture was neutralized with concentrated solution of sodium carbonate
(Na2CO ). From this mixture, the desired ester was extracted with ether. This
ethereal3layer was evaporated to afford yellowish brown crystalline substance,
ethyl-3-(2-nitrophenyl)acrylate (2).
1,3,4-oxadiazole is a versatile group of heterocyclic compounds that
have attracted attention for last two decades because of their broad range
biological effectiveness.1 It is derived from furan when the replacement of two
methine (-CH=) is done by two pyridine like nitrogen (-N=) at position 3 and
4.2Aromaticity of oxadiazole ring is reduced because of this replacement due to
which oxadiazole ring bears properties of conjugated diene. The electrophilic
substitutions reactions in oxadiazole ring are very difficult at carbon atom due
to low electron density at carbon atom. Conversely the attack of electrophiles
can occur at nitrogen atom. Nucleophilic attack can occur if substitution
of ring is done with electron releasing groups. Oxadiazole normally do not
undergo nucleophilic attack. However halogen substituted oxadiazole undergo
nucleophilic substitution reactions. Nucleophilic substitution reactions in
oxadiazole are same as occurred at sp2 carbon atom of an aliphatic compound.
Extra heteroatom has inductive effect due to which oxadiazole acts as very
weak base.3 1,3,4-oxadiazole can be used as a skeleton in medicinal chemistry
to synthesize large number of bioactive agents.4 2,5- disubstituted-1,3,4-
Oxadiazole derivatives have possessed broad range of biological activities
such as antifungal,5 antimicrobial,6,7 anti-inflammatory,2,8and antiparasitic.9
HIV replication is inhibited by the use of 2,5-disubstituted derivatives of
1,3,4-oxadiazole.10 Several 2,5-disubstituted-1,3,4-oxadiazole derivatives
have powerful effect against 60 malignant tumor cell lines. Biological effects
demonstrate a very significant anti-tumor activity against leukemia, breast
cancer and colon.11,12 It is extensively used in the treatment of arthritis(jaundice,
rheumatoid and osteoarthritis).13,14 Oxadiazole also exhibit herbicidal,15
pesticidal, analgesic16 and plant growth regulatory activities.17
2.3.
acrylohydrazide
The methanolic solution of ethyl-3-(2-nitrophenyl)acrylate (0.01mol,
Procedure for the preparation of 3-(2-nitrophenyl)
2.21g, (2) was taken in a round bottom flask. Hydrazine hydrate (0.03 mol,
1.47 mL) was added drop wise in this solution with stirring for 3 h at room
temperature. Completion of reaction was checked by TLC. After completion,
distilled water was added and precipitates of 3-(2-nitrophenyl)acrylohydrazide
(3) formed, were filtered and re-washed with water.
2.4.
Procedure for the preparation of 5-(2-nitrostyryl)-1,3,4-
oxadiazole-2-thiol (4)
3-(2-Nitrophenyl)acrylohydrazide. (2.07 g, 0.01mol, 3) was solubilized
in absolute ethanol (12 mL) in 250 mL round bottom flask and potassium
hydroxide (1.68 g, 0.03mol) was added in the reaction mixture. Upon complete
dissolution of potassium hydroxide, carbon disulfide (1.81 mL, 0.03mol) was
added to the solution. This reaction mixture was well stirred and refluxed for 6 h
Initially the color of solution was yellow which turned to brownish yellow with
the progress of reaction. Evolution of hydrogen sulfide gas occurred during this
reaction. After the completion of this reaction, it was diluted with water and
acidified by concentrated hydrochloric acid to pH 2-3. The precipitates formed,
were filtered and washed with water. Re-crystallization was done with ethanol
to afford purified sample of 5-(2-nitrostyryl)-1,3,4-oxadiazole-2-thiol (4).
1.1.1 5-(2-nitrostyryl)-1,3,4-oxadiazole-2-thiol (4)
Literature survey showed that slight modifications in the structure of
1,3,4-oxadiazole can result in quantitative as well as qualitative variations
in the biological activity. So in continuation of our work18,19 to search for the
enzyme inhibitors bearing 1,3,4-oxadiazole nucleus, here we report a series
of S-substituted derivatives of 5-(2-nitrostyryl)-1,3,4-oxadiazole-2-thiol (4)
derived from 3-(2-nitrophenyl)acrylic acid (1) and screened results of enzyme
inhibition activities for all the synthesized compounds.
Yellowish brown solid; m.p. 190-192 °C; yield 79%; IR (KBr, νmax cm–1):
3200 (S-H), 3056 (Ar-H), 1680 (C=N), 1550 (Ar C=C), 1530 (NO2), 840 (C-
N); 1H-NMR (300 MHz, CD3OD): δ 8.06 (dd, J = 8.1, 1.2 Hz, 1H, H-3’), 7.89
(d, J = 16.2 Hz, 1H, H-8’), 7.76 (dd, J = 7.8, 1.8 Hz, 1H, H-6’), 7.74 (br t, J
= 7.8, Hz, 1H, H-4’), 7.61 (ddd, J = 8.4, 8.4, 1.2 Hz, 1H, H-5’), 6.95 (d, J =
16.2 Hz, 1H, H-7’); 13C-NMR (75 MHz, CD3OD): δ 164.5 (C-2), 163.7 (C-5),
149.3 (C-2’), 138.3 (C-7’), 135.5 (C-5’), 134.3 (C-1’), 132.6 (C-4’), 129.6 (C-
6’), 126.9 (C-3’), 110.8 (C-8’); HR-MS: [M]+ 249.2685 (Calcd. for C10H7N3O3;
249.2458); EI-MS: m/z 249 [M]+, 203 [M-NO2]+ , 174 [M-CHNOS]+, 148
[M-C HN2OS]+, 135 [M-C3H N2OS]+, 122 [M-C H N2OS]+.
2. EXPERIMENTAL
2.1. Chemistry
Griffin and George melting point apparatus was used to record the
melting points of the synthesized compounds by open capillary tube and were
uncorrected. Purity was checked on thin layer chromatography (TLC) on pre-
coated silica gel G-25-UV254 plates using different percentage of ethyl acetate
and n-hexane giving single spot. Jasco-320-A spectrophotometer was used
22.5.
General proce2dure for the pr4ep3aration of S-substituted
1
for the IR spectra (wave number in cm-1). H-NMR spectra were recorded in
derivatives (6a-n) of 5-(2-nitrostyryl)-1,3,4-oxadiazole-2-thiol (4)
The calculated amount of 5-(2-nitrostyryl)-1,3,4-oxadiazole-2-thiol (0.2g,
0.0008mol, (4) was taken in a 50 mL round bottom flask and 10 mL of N,N-
dimethyl formamide (DMF) was added in order to dissolve it completely. Then
CD3OD or CDCl3 on a Bruker spectrometers operating at 300 and 400 MHz.
Chemical shifts were recorded in ppm. Mass spectra (HE-MS & EIMS) were
taken on a JMS-HX-110 spectrometer, with complete data system.
e-mail: atrabbasi@yahoo.com
2186

J. Chil. Chem. Soc., 58, Nº 4 (2013)
Lithium hydride (0.0064g, 0.0008mol) was added to the mixture as base. The
reaction mixture was stirred for 30 minutes at room temperature and then
respective alkyl halide (0.0008mol, (5a-n) was added to the reaction mixture
and it was further stirred for 3 h. The progress of reaction was monitored by
TLC test till single spot. Then few drops of 5% solution of sodium hydroxide
were added to covert the unreacted oxadiazole-2-thiol into its salt. Finally,
the addition of distilled water into this solution resulted in the formation of
precipitates of respective S-substituted derivative (6a-n) of parent molecule.
Precipitates were filtered and washed with water and dried to carry out further
studies.
[M-C H N OS].
24.5.26. 25-(2-Nitrostyryl)-2-(3-phenylpropylthio)-1,3,4-oxadiazole (6f)
Yellow amorphous solid; m.p. 78-80 °C; yield 80%; IR (KBr, νmax cm–1):
3051 (Ar-H), 1684 (C=N), 1558 (Ar C=C), 1531 (NO ), 843 (C-N); 1H-NMR
(400 MHz, CDCl3): δ 8.05 (d, J = 8.0 Hz, 1H, H-3’), 72.90 (d, J = 16.0 Hz, 1H,
H-8’), 7.67 (m, 2H, H-4’, H-6’), 7.55 (br t, J = 8.0 Hz, 1H, H-5’), 7.26 (t, J =
6.8 Hz, 3H, H-3”, H-4”, H-5”), 7.19 (br d, J = 6.8 Hz, 2H, H-2”, H-6”), 6.97 (d,
J = 16.0 Hz, 1H, H-7’), 3.28 (t, J = 7.2 Hz, 2H, CH2-9”), 2.79 (t, J = 7.2, 2H,
CH2-7”), 2.17 (quint., J = 7.6 Hz, 2H, CH2-8”); 13C-NMR (75 MHz, CD OD):
δ 165.3 (C-2), 163.1 (C-5), 148.6 (C-2’), 142.4 (C-1”), 138.8 (C-7’), 3135.9
(C-5’), 133.7 (C-1’), 132.7 (C-4’), 131.5 (C-2” & C-6”), 129.9 (C-6’), 129.5
(C-3” & C-5”), 126.7 (C-4”), 126.3 (C-3’), 110.8 (C-8’), 33.9 (C-7”), 30.4 (C-
8”), 27.8 (C-9”); HR-MS: [M]+ 367.4401 (Calcd. for C19H17N3O3S; 367.4216);
EI-MS: m/z 367 [M]+ 321 [M-NO2]+, 276 [M-C7H7]+, 248 [M-C9H11]+, 293
[M-CNOS]+, 267 [M-C2N2OS]+, 254 [M-C HN OS]+, 241 [M-C H2N2OS].
2.5.7. 2-(Benzylthio)-5-(2-nitrostyry3l)-1,23,4-oxadiazole (46g)
2.5.1. 2-(Ethylthio)-5-(2-nitrostyryl)-1,3,4-oxadiazole (6a)
Brownish yellow amorphous solid; m.p. 84-86 °C; yield 84%; IR (KBr,
νmax cm–1): 3049 (Ar-H), 1683 (C=N), 1555 (Ar C=C), 1527 (NO2), 844 (C-N);
1H-NMR (300 MHz, CD3OD): δ 8.06 (dd, J = 8.4, 1.2 Hz, 1H, H-3’), 7.95
(d, J = 16.5 Hz, 1H, H-8’), 7.93 (br d, J = 7.5, Hz, 1H, H-6’), 7.75 (br t, J
= 7.2 Hz, 1H, H-4’), 7.62 (br t, J = 7.5 Hz, 1H, H-5’), 7.14 (d, J = 16.2 Hz,
1H, H-7′), 3.33 (q, J = 7.2 Hz, 2H, CH2-1”), 1.49 (t, J = 7.2 Hz, 3H, CH3-2”);
13C-NMR (75 MHz, CD3OD): δ 164.1 (C-2), 163.3 (C-5), 149.2 (C-2’), 138.1
(C-7’), 135.3 (C-5’), 134.1 (C-1’), 132.2 (C-4’), 129.1 (C-6’), 126.7 (C-3’),
110.3 (C-8’), 23.4 (C-1”), 16.5 (C-2”); HR-MS: [M]+ 277.3067 (Calcd. for
C12H11N3O3S; 277.2991); EI-MS: m/z 277 [M]+, 248 [M-C2H5], 231 [M-NO2]+ ,
203 [M-CNOS]+, 177 [M-C2N OS]+, 164 [M-C HN2OS]+, 151 [M-C4H2N2OS].
2.5.2. 2-(Isopropylthiol)2-5-(2-nitrostyryl3)-1,3,4-oxadiazole (6b)
Brownish yellow amorphous solid; m.p. 128-130 °C; yield 73%; IR (KBr,
νmax cm–1): 3051 (Ar-H), 1685 (C=N), 1559 (Ar C=C), 1532 (NO2), 847 (C-N);
1H-NMR (400 MHz, CDCl3): δ 8.04 (d, J = 8.0 Hz, 1H, H-3’), 7.89 (d, J = 16.0
Hz, 1H, H-8’), 7.67 (br d, J = 7.6 Hz, 2H, H-4’, H-6’), 7.53 (ddd, J = 8.4, 7.8,
1.6 Hz, 1H, H-3’), 7.44 (br d, J = 7.2 Hz, 2H, H-2”, H-6”), 7.28-7.34 (m, 3H,
H-3” to H-5”), 6.96 (d, J = 16.0 Hz, 1H, H-7’), 4.50 (s, 2H, CH2-7”); 13C-NMR
(75 MHz, CD OD): δ 165.7 (C-2), 163.5 (C-5), 148.2 (C-2’), 138.4 (C-7’),
137.5 (C-1”), 3135.6 (C-5’), 133.5 (C-1’), 132.4 (C-4’), 129.7 (C-6’), 129.4
(C-3” & C-5”), 128.5 (C-2” & C-6”), 128.1 (C-4”), 126.5 (C-3’), 110.5 (C-
8’), 32.5 (C-7”); HR-MS [M]+ 339.3827 (Calcd. for C17H13N3O3S; 339.3684);
EI-MS: m/z 339 [M]+, 293 [M-NO2]+, 248 [M-C7H7]+, 265 [M-CNOS]+, 239
[M-C N2OS]+, 226 [M-C HN2OS]+, 213 [M-C4H2N2OS].
Brownish yellow amorphous solid; m.p. 68-70 °C; yield 80%; IR (KBr,
νmax cm–1): 3059 (Ar-H), 1686 (C=N), 1557 (Ar C=C), 1533 (NO2), 847 (C-N);
1H-NMR (300 MHz, CH OD): δ 8.06 (dd, J = 8.1, 1.2 Hz, 1H, H-3’), 7.96 (d,
J = 16.8 Hz, 1H, H-8’), 37.93 (br d, J = 7.2 Hz, 1H, H-6’), 7.76 (br t, J = 7.2
Hz, 1H, H-4’), 7.62 (ddd, J = 8.4, 8.1, 0.9 Hz, 1H, H-5’), 7.14 (d, J = 16.2 Hz,
1H, H-7’), 3.94 (septet, J = 6.9 Hz, 1H, H-1”), 1.51 (d, J = 6.6 Hz, 6H, CH3-2”,
CH -3”); 13C-NMR (75 MHz, CD OD): δ 164.3 (C-2), 163.5 (C-5), 148.9 (C-
2’),3138.3 (C-7’), 135.6 (C-5’), 1333.7 (C-1’), 132.5 (C-4’), 129.5 (C-6’), 126.3
(C-3’), 109.8 (C-8’), 37.2 (C-1”), 23.9 (C-2” & C-3”); HR-MS: M]+ 291.3367
(Calcd. for C13H13N3O3S; 291.3256); EI-MS: m/z 291 [M]+, 245 [M-NO2]+ ,
248 [M-C3H7]+, 217 [M-CNOS]+, 191 [M-C2N2OS]+, 178 [M-C3HN2OS]+, 165
[M-C H N OS].
22-(2-Methylbenzylth3 io)-5-(2-nitrostyryl)-1,3,4-oxadiazole (6h)
Off-white amorphous solid; m.p. 148-150 °C; yield 78%; IR (KBr, νmax
cm–1): 3050 (Ar-H), 1686 (C=N), 1552 (Ar C=C), 1534 (NO2), 846 (C-N);
1H-NMR (400 MHz, CDCl3): δ 8.04 (d, J = 8.0 Hz, 1H, H-3’), 7.91 (d, J =
16.4 Hz, 1H, H-8’), 7.69 (m, 2H, H-4’, H-6’), 7.53 (br t, J = 8.4 Hz, 1H,
H-5’), 7.40 (br d, J = 7.2 Hz, 1H, H-4”), 7.20 (m, 3H, H-3” to H-6”), 6.97 (d,
J = 16.4 Hz, 1H, H-7’), 4.45 (s, 2H, CH -7”), 2.44 (s, 3H, CH3-8”); 13C-NMR
(75 MHz, CD3OD): δ 165.1 (C-2), 1632.5 (C-5), 148.9 (C-2’), 141.4 (C-1”),
138.6 (C-7’), 136.3 (C-2”), 135.6 (C-5’), 134.2 (C-3”), 133.6 (C-1’), 132.5
(C-4’), 129.9 (C-4”), 129.7 (C-6”), 129.6 (C-6’), 127.5 (C-5”), 126.5 (C-3’),
110.5 (C-8’), 28.5 (C-7”), 19.5 (C-8”); HR-MS: [M]+ 353.4121 (Calcd. for
C18H15N3O3S; 353.3951); EI-MS: m/z 353 [M]+, 338 [M-CH3]+, 307 [M-NO2]+ ,
248 [M-C8H9]+, 279 [M-CNOS]+, 253 [M-C2N2OS]+, 240 [M-C3HN2OS]+, 227
[M-C H N OS].
24.5.23. 25-(2-Nitrostyryl)-2-(vinylthio)-1,3,4-oxadiazole (6c)
Off-white amorphous solid; m.p. 92-94 °C; yield 78%; IR (KBr, νmax cm–1):
3056 (Ar-H), 1684 (C=N), 1553 (Ar C=C), 1526 (NO ), 848 (C-N); 1H-NMR
(400 MHz, CDCl ): δ 8.05 (d, J = 8.0 Hz, 1H, H-3’), 72.94 (d, J = 16.4 Hz, 1H,
H-8’), 7.68 (m, 23H, H-4’, H-6’), 7.54 (br t, J = 7.4 Hz, 1H, H-5’), 6.98 (d, J
= 16.4 Hz, 1H, H-7’), 6.88 (dd, J = 16.8, 9.6 Hz, 1H, H-1”), 5.76 (br d, J =
17.2 Hz, 1H, Hb-2”), 5.72 (br d, J = 9.6 Hz, 1H, Ha-2”); 13C-NMR (75 MHz,
CD3OD): δ 164.2 (C-2), 163.7 (C-5), 148.6 (C-2’), 138.8 (C-7’), 135.2 (C-5’),
133.5 (C-1’), 132.9 (C-4’), 131.5 (C-1”), 129.6 (C-6’), 126.6 (C-3’), 116.9 (C-
2”), 109.6 (C-8’); HR-MS: [M]+ 275.2997 (Calcd. for C12H9N3O3S; 275.2831);
EI-MS: m/z 275 [M]+, 248 [M-C2H3]+, 229 [M-NO2]+, 201 [M-CNOS]+, 175
[M-C N OS]+, 162 [M-C3HN OS]+, 149 [M-C4H N2OS].
24.5.28. 22-(4-Fluorobenzylthio)-5-(2-nitrostyryl)-1,3,4-oxadiazole (6i)
Off-white amorphous solid; m.p. 141-143 °C; yield 79%; IR (KBr, νmax
cm–1): 3059 (Ar-H), 1686 (C=N), 1553 (Ar C=C), 1536 (NO2), 842 (C-N);
1H-NMR (400 MHz, CDCl3): δ 8.05 (d, J = 8.0 Hz, 1H, H-3’), 7.90 (d, J = 16.0
Hz, 1H, H-8’), 7.67 (m, 2H, H-4’, H-6’), 7.55 (br t, J = 8.0 Hz, 1H, H-5’), 7.42
(d, J = 8.4 Hz, 2H, H-2”, H-6”), 7.02 (d, J = 8.4 Hz, 2H, H-3”, H-5”), 6.96 (d,
J = 16.0 Hz, 1H, H-7’), 4.45 (s, 2H, CH2-7”); 13C-NMR (75 MHz, CD OD):
δ 165.1 (C-2), 163.3 (C-5), 162.5 (C-4”), 148.8 (C-2’), 138.5 (C-7’), 3135.7
(C-5’), 133.5 (C-1’), 132.4 (C-4’), 132.6 (C-2” & C-6”), 131.7 (C-1”), 129.7
(C-6’), 126.7 (C-3’), 115.6 (C-3” & C-5”), 110.5 (C-8’), 32.5 (C-7”); HR-MS:
[M]+ 357.3692 (Calcd. for C17H12FN3O3S; 357.3588); EI-MS: m/z 357 [M]+,
311 [M-NO2]+, 248 [M-C7H6F]+, 283 [M-CNOS]+, 257 [M-C2N2OS]+, 244
[M-C HN2OS]+, 231 [M-C4H2N2OS].
22.5.24. 2-(Allylthio)-5-(22-Nitrostyryl)-1,3,42-oxadiazole (6d)
Yellow amorphous solid; m.p. 75-77 °C; yield 81%; IR (KBr, νmax cm–1):
3056 (Ar-H), 1688 (C=N), 1554 (Ar C=C), 1539 (NO ), 846 (C-N); 1H-NMR
(300 MHz, CD OD): δ 8.06 (dd, J = 8.1, 1.2 Hz, 1H, 2H-3’), 7.96 (d, J = 16.2
Hz, 1H, H-8’),37.92 (br d, J = 7.5 Hz, 1H, H-6’), 7.75 (br t, J = 7.8 Hz, 1H,
H-4’), 7.62 (ddd, J = 8.7, 8.1, 1.5 Hz, 1H, H-5’), 7.14 (d, J = 16.2 Hz, 1H,
H-7’), 6.04 (m, 1H, H-2”), 5.39 (dd, J = 17.1, 1.2 Hz, 1H, Hb-3”), 5.20 (dd,
J = 9.9, 0.6 Hz, 1H, Ha-3”), 3.93 (d, J = 7.2 Hz, 2H, CH -1”); 13C-NMR (75
MHz, CD3OD): δ 164.6 (C-2), 163.9 (C-5), 148.4 (C-2’), 2138.7 (C-7’), 135.2
(C-5’), 133.4 (C-1’), 132.1 (C-4’), 129.8 (C-6’), 127.2 (C-2”), 126.7 (C-3’),
119.8 (C-3”), 110.8 (C-8’), 36.9 (C-1”); HR-MS: [M]+ 289.3286 (Calcd. for
C13H11N3O3S; 289.3097; EI-MS: m/z 289 [M]+, 248 [M-C3H5]+, 243 [M-NO2]+ ,
215 [M-CNOS]+, 189 [M-C2N2OS]+, 176 [M-C3HN2OS]+, 163 [M-C H2N OS].
2.5.5. 5-(2-Nitrostyryl)-2-(phenylethylthio)-1,3,4-oxadiazole4(6e)2
Off-white amorphous powder; m.p. 85-87 °C; yield 70%; IR (KBr, νmax
cm–1): 3056 (Ar-H), 1680 (C=N), 1550 (Ar C=C), 1532 (NO2), 844 (C-N);
1H-NMR (400 MHz, CDCl3): δ 8.05 (d, J = 8.0 Hz, 1H, H-3’), 7.91 (d, J = 16.0
Hz, 1H, H-8’), 7.69 (m, 2H, H-4’, H-6’), 7.54 (br t, J = 8.4 Hz, 1H, H-5’), 7.30
(br d, J = 7.2 Hz, 2H, H-2”, H-6”), 7.24 (m, 3H, H-3” to H-5”), 6.97 (d, J =
16.4 Hz, 1H, H-7’), 3.52 (t, J = 7.2 Hz, 2H, CH2-8”), 3.14 (t, J = 7.12 Hz, 2H,
CH -7”); 13C-NMR (75 MHz, CD3OD): δ 165.6 (C-2), 163.5 (C-5), 148.9 (C-
2’),2140.5 (C-1”), 138.5 (C-7’), 135.6 (C-5’), 133.7 (C-1’), 132.6 (C-4’), 131.7
(C-3” & C-5”), 129.9 (C-6’), 129.5 (C-2” & C-6”), 127.4 (C-4”), 126.5 (C-3’),
110.8 (C-8’), 35.3 (C-7”), 28.5 (C-8”); HR-MS: [M]+ 353.4072 (Calcd. for
C18H15N3O3S; 353.3951); EI-MS: m/z 353 [M]+, 307 [M-NO2]+ , 262 [M-C7H7]+,
248 [M-C8H9]+, 279 [M-CNOS]+, 253 [M-C2N2OS]+, 240 [M-C3HN2OS]+, 227
23.5.9. 2-(2-Chlorobenzylthio)-5-(2-nitrostyryl)-1,3,4-oxadiazole (6j)
Off-white amorphous solid; m.p. 144-146 °C; yield 72%; IR (KBr, νmax
cm–1): 3051 (Ar-H), 1683 (C=N), 1556 (Ar C=C), 1530 (NO2), 848 (C-N), 560
1
(C-Cl); H-NMR (400 MHz, CDCl3): δ 8.04 (d, J = 8.4 Hz, 1H, H-3’), 7.90
(d, J = 16.0 Hz, 1H, H-8’), 7.67 (m, 2H, H-4’, H-6’), 7.59 (dd, J = 8.8, 2.4,
Hz, 1H, H-3”), 7.52 (ddd, J = 8.4, 8.4, 2.0 Hz, 1H, H-5’), 7.40 (dd, J = 9.2,
2.4 Hz, 1H, H-6”), 7.24 (m, 2H, H-4”, H-5”), 6.95 (d, J = 16.0 Hz, 1H, H-7’),
4.61 (s, 2H, CH2-7”); 13C-NMR (75 MHz, CD3OD): δ 165.3 (C-2), 163.4 (C-
5), 148.7 (C-2’), 138.3 (C-7’), 135.9 (C-5’), 135.8 (C-1”), 133.9 (C-1’), 132.4
(C-4’), 130.8 (C-5”), 130.6 (C-3”), 129.3 (C-6’), 128.9 (C-4”), 128.3 (C-6”),
127.4 (C-2”), 126.7 (C-3’), 110.7 (C-8’), 31.4 (C-7”); HR-MS: [M]+ 373.8314
(Calcd. for C17H12ClN3O3S; 373.8134); EI-MS: m/z 374 [M]+, 328 [M-NO2]+,
249 [M-C H Cl]+, 299 [M-CNOS]+, 273 [M-C2N2OS]+, 260 [M-C3HN2OS]+,
247 [M-C47H26N2OS].
2.5.10. 2-(3-Chlorobenzylthio)-5-(2-nitrostyryl)-1,3,4-oxadiazole (6k)
Light brownish yellow solid; m.p. 120-122 °C; yield 75%; IR (KBr, νmax
cm–1): 3056 (Ar-H), 1687 (C=N), 1552 (Ar C=C), 1539 (NO2), 845 (C-N), 563
2187

J. Chil. Chem. Soc., 58, Nº 4 (2013)
(C-Cl); 1H-NMR (400 MHz, CDCl ): δ 8.05 (d, J = 8.0 Hz, 1H, H-3’), 7.90 (d, J
= 16.0 Hz, 1H, H-8’), 7.67 (m, 2H,3H-4’, H-6’), 7.54 (ddd, J = 8.4, 7.4, 1.6 Hz,
1H, H-5’), 7.44 (br s, 1H, H-2”), 7.34 (m, 1H,H-4”), 7.27 (m, 2H, H-5”, H-6”),
6.95 (d, J = 16.0 Hz, 1H, H-7’), 4.56 (s, 2H, CH2-7”); 13C-NMR (75 MHz,
CD3OD): δ 165.5 (C-2), 163.6 (C-5), 148.6 (C-2’), 139.6 (C-1”), 138.5 (C-7’),
136.4 (C-3”), 135.6 (C-5’), 133.6 (C-1’), 132.2 (C-4’), 130.8 (C-4”), 130.3
(C-5”), 129.5 (C-6’), 128.4 (C-2”), 126.5 (C-3’), 124.5 (C-6”), 110.3 (C-8’),
33.5 (C-7”); HR-MS: [M]+ 373.8314 (Calcd. for C17H12ClN3O3S; 373.8134);
EI-MS: m/z 374 [M]+, 328 [M-NO2]+, 249 [M-C7H6Cl]+, 299 [M-CNOS]+, 273
[M-C N OS]+, 260 [M-C3HN OS]+, 247 [M-C H2N2OS].
10 minutes at 25 °C. 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.
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).
2.5.2112. 2-(4-Chlorobenz2ylthio)-5-(2-nitr4ostyryl)-1,3,4-oxadiazole (6l)
Cream yellow solid; m.p. 128-130 °C; yield 76%; IR (KBr, ν cm–1): 3053
(Ar-H), 1681 (C=N), 1558 (Ar C=C), 1534 (NO2), 847 (C-Nm),ax566 (C-Cl);
1H-NMR ((400 MHz, CDCl3): δ 8.05 (d, J = 8.0 Hz, 1H, H-3’), 7.90 (d, J
= 16.4 Hz, 1H, H-8’), 7.68 (m, 2H, H-4’, H-6’), 7.54 (ddd, J = 8.4, 7.4, 1.6
Hz, 1H, H-3”), 7.39 (d, J = 8.0 Hz, 2H, H-2”, H-6”), 7.30 (d, J = 8.4 Hz, 2H,
H-3”, H-5”), 6.95 (d, J = 16.0 Hz, 1H, H-7’), 4.45 (s, 2H, CH2-7”); 13C-NMR
(75 MHz, CD OD): δ 165.4 (C-2), 163.7 (C-5), 148.6 (C-2’), 138.3 (C-7’),
136.1 (C-4”), 3135.6 (C-5’), 135.2 (C-1”), 133.7 (C-1’), 132.5 (C-4’), 131.3
(C-3” & C-5”), 129.6 (C-6’), 126.9 (C-2” & C-6”), 126.4 (C-3’), 110.8 (C-8’),
32.7 (C-7”); HR-MS: [M]+ 373.8314 (Calcd. for C17H12ClN3O3S; 373.8134);
EI-MS: m/z 374 [M]+, 328 [M-NO2]+ , 249 [M-C7H6Cl]+, 300 [M-CNOS]+, 274
[M-C N OS]+, 261 [M-C3HN OS]+, 248 [M-C H2N2OS].
3. RESULTS AND DISCUSSION
3.1. Chemistry
3-(2-nitrophenyl)acrylic acid (1) was converted to corresponding ester 2,
hydrazide 3 and 1,3,4-oxadiazole 4, successively. Finally 6a-n derivatives of
4 were synthesized by reacting it with different electrophiles 5a-n (scheme
1). Structures of the synthesized compounds were elucidated and confirmed
1
by IR, H-NMR, 13C-NMR, HR-MS and EI-MS techniques. For compound
4, the IR absorption band of aromatic C-H str. appeared at 3056 and a band
at 1530 cm-1 indicated the presence of a nitro group while the peak at 1680
cm-1 (C=N) was typical for an oxadiazole ring. Its molecular formula was
confirmed through HR-MS showing molecular ion peak at m/z 249.2685,
corresponding to C H7N3O3 (Calcd. for 249.2458). The EI-MS spectral data
was also in complet1e0 agreement for the proposed structure of 5-(2-nitrostyryl)-
1,3,4-oxadiazole-2-thiol (4) which was finally confirmed through its 1H-NMR
spectrum. The peaks at δ 8.06 (dd, J = 8.1, 1.2 Hz, 1H, H-3’), 7.76 (dd, J = 7.8,
1.8 Hz, 1H, H-6’), 7.74 (br t, J = 7.8, Hz, 1H, H-4’), and 7.61 (ddd, J = 8.4,
8.4, 1.2 Hz, 1H, H-5’) for aromatic protons and their splitting pattern confirmed
that the ring was ortho-substituted, while the larger coupling constants for
two olefinic protons at δ 7.89 (d, J = 16.2 Hz, 1H, H-8’), and 6.95 (d, J =
16.2 Hz, 1H, H-7’) disclosed their trans disposition. The structure was also
thorough supported by its 13C-NMR spectrum. Similarly all other synthesized
derivatives were characterized by aforesaid spectral techniques and screened
for enzyme inhibitory activities to check their possible therapeutic potential
for the treatment of various ailments associated with the over expression of
studied enzymes.
2.5.2122. 2-(4-Bromobenz2ylthio)-5-(2-nitr4ostyryl)-1,3,4-oxadiazole (6m)
Pale yellow amorphous solid; m.p. 110-112 °C; yield 78%; IR (KBr, νmax
cm–1): 3058 (Ar-H), 1688 (C=N), 1553 (Ar C=C), 1537 (NO2), 844 (C-N), 510
1
(C-Br); H-NMR (400 MHz, CDCl3): δ 8.05 (d, J = 8.0 Hz, 1H, H-3’), 7.90
(d, J = 16.0 Hz, 1H, H-8’), 7.68 (m, 1H, H-6’, H-4’), 7.54 (br t, J = 8.4 Hz,
1H, H-5’), 7.45 (d, J = 8.4 Hz, 2H, H-2”, H-6”), 7.33 (d, J = 8.0 Hz, 2H,
H-3”, H-5”), 6.95 (d, J = 16.4 Hz, 1H, H-7’), 4.44 (s, 2H, CH2-7”); 13C-NMR
(75 MHz, CD3OD): δ 165.2 (C-2), 163.4 (C-5), 148.1 (C-2’), 138.7 (C-7’),
136.2 (C-1”), 135.4 (C-5’), 133.8 (C-1’), 133.4 (C-3” & C-5”), 132.9 (C-4’),
131.4 (C-2” & C-6”), 129.1 (C-6’), 126.7 (C-3’), 124.2 (C-4”), 110.2 (C-8’),
32.4 (C-7”); HR-MS: [M]+ 418.2823 (Calcd. for C17H12BrN3O3S; 418.2644);
EI-MS: m/z 418 [M]+, 372 [M-NO2]+ , 248 [M-C7H6Br]+, 344 [M-CNOS]+, 318
[M-C N OS]+, 305 [M-C HN2OS]+, 292 [M-C4H N2OS].
2.5.2132. 2-(3-Nitrobe3nzylthio)-5-(2-nitrosty2ryl)-1,3,4-oxadiazole (6n)
Off-white amorphous solid; m.p. 82-84 °C; yield 78%; IR (KBr, νmax cm–1):
3050 (Ar-H), 1685 (C=N), 1552 (Ar C=C), 1538 (NO2), 843 (C-N); 1H-NMR
(400 MHz, CDCl3): δ 8.34 (br s, 1H, H-2”), 8.15 (br d, J = 8.0 Hz, 1H, H-4”),
8.05 (d, J = 8.0 Hz, 1H, H-3’), 7.91 (d, J = 16.4 Hz, 1H, H-8’), 7.84 (br d, J =
7.6 Hz, 1H, H-6”), 7.68 (m, 2H, H-4’, H-6’), 7.51-7.56 (m, 2H, H-3”, H-5”),
6.94 (d, J = 16.4 Hz, 1H, H-7’), 4.56 (s, 2H, CH -7”); 13C-NMR (75 MHz,
CD3OD): δ 165.1 (C-2), 163.9 (C-5), 149.6 (C-3”), 2148.5 (C-2’), 138.9 (C-7’),
138.2 (C-1”), 135.1 (C-5’), 133.2 (C-1’), 132.6 (C-6”), 132.4 (C-4’), 131.3
(C-5”), 129.2 (C-6’), 126.8 (C-4”), 126.3 (C-3’), 124.4 (C-2”), 110.7 (C-8’),
34.5 (C-7”); HR-MS: [M]+ 384.3847 (Calcd. for C17H12N4O5S; 384.3659); EI-
MS: m/z 384 [M]+, 338 [M-NO2]+, 292 [M-N2O4]+, 248 [M-C7H6NO2]+, 310
[M-CNOS]+, 284 [M-C N2OS]+, 271 [M-C3HN2OS]+, 258 [M-C4H2N2OS].
3. Enzyme Inhibit2ion Assays
3.2. Enzyme Inhibition Activity:
3.2.1. AChE inhibitory potential
All the synthesized compounds were screened against acetylcholinesterase
and these molecules exhibited weak to moderate inhibitory potential against
this enzyme (table 1). Among these, the parent molecule 4 exhibited relatively
greater inhibitory potential having IC value of 68.91±0.22 µM relative to
eserine, a reference standard, having a5v0 alue of 0.04±0.001 µM. Moreover, it
was also inferred that the substitution of various alkyl/aralkyl groups at the
sulfur atom of the parent molecule 4 was not an effective tool to enhance the
biological activity of such molecules against this enzyme.
3.2.2. BChE inhibitory activity
Butyrylcholinesterase inhibition potential was also analyzed for all these
derivatives. Results (table 1) revealed that a number of derivatives posses
moderate inhibitory potential against this enzyme. The molecule 6n showed
better activity with IC50 of 61.21±0.12 µM which could be attributed to the
substitution of 3-nitrobenzyl group in this molecule. The compounds 6m, 6c,
6i and 6k demonstrated an endurable potential with IC50 values 73.21±0.17,
74.51±0.11, 80.31±0.21and 99.61±0.15 µM, respectively which depicted the
impact of substitution of various moieties on the parent molecule.
3.2.3. LOX inhibition potential
1.1.
Assays
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
The AChE and BChE inhibition activities were performed according to
the reported method20 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. 10 µL test compound (0.5 mM well-1) was added, followed
by the addition of 10 µL (0.005 unit well-1) enzyme. 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), 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
A few of the synthesized compounds indicated very temperate potential
against lipoxygenase (table 1). The molecule 6m exhibited better potential
with IC50 value of 28.11±0.11 µM. The compounds 6b, 6d and 6g also bared a
moderate inhibitory potential with IC values of 99.61±0.18, 85.75±0.11 and
76.75±0.14 respectively, relative to ba5i0calein having the IC50 value of 22.4±1.3
µM.
IC50 values were calculated using EZ–Fit Enzyme kinetics software
(Perrella Scientific Inc. Amherst, USA).
3.2.4. Structure-activity relationship
Overall, the heterocycle 6m was found to exhibit inhibitory activity against
all the three enzymes namely, acetylcholinesterase, butyrylcholinesterase and
lipoxygenase, but inhibition activity against lipoxygenase is appreciable,
while some compounds were active against butyrylcholinesterase while some
others revealed inhibitory potential against lipoxygenase. The 6m derivative
possesses a p-bromobenzyl substitution on parent oxadiazole ring and
mesomeric and inductive effect on parent moiety collectively could influence
1.2. Lipoxygenase (LOX) Assay
Lipoxygenase activity was assayed according to the reported methods21-23
with slight modifications. Atotal 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
2188

J. Chil. Chem. Soc., 58, Nº 4 (2013)
enzyme inhibition activity against all the three enzymes. However, the better
inhibition potential by parent 4 against acetylcholinesterase enzyme revealed
that the substitution of such groups was not a useful means to synthesize potent
inhibitors of this enzyme. In compound 6n, the substitution of m-nitrobenzyl
group makes it a better entity as butyrylcholinesterase inhibitor. However,
the compounds bearing olefinic group and m- and p-substituted halogenated
aromatic ring also rendered moderate inhibition against this enzyme.
1,3,4-oxadiazole are well supported by spectroscopic analysis. The enzyme
inhibition data help to conclude that the compounds reveal moderate activity
against butyrylcholinesterase enzyme, and sound inhibitory potential against
acetylcholinesterase and lipoxygenase enzymes, as it was evident from their
IC50 values relative to the standard used. Hence, on the basis of aforesaid
results, these synthesized derivatives provide an overall indispensable basis
to introduce potent drugs. It is concluded from the described research work
that halogenated aromatic substituted derivatives of 1,3,4-oxadiazoles seem
valuable drug candidates for the treatment of Alzheimer and inflammatory
diseases.
4. CONCLUSION
The anticipated structures of the synthesized derivatives of
Scheme-1: Outline for the synthesis of S-substituted derivatives, 6a-n, of 5-(2-nitrophenyl)-1,3,4-oxadiazole-2-thiol (4)
2189

J. Chil. Chem. Soc., 58, Nº 4 (2013)
Table-1: Enzyme inhibition activity of S-substituted derivatives, 6a-n, of 5-(2-nitrophenyl)-1,3,4-oxadiazole-2-thiol (4).
AChE
BChE
LOX
S.
Code
Inhibition (%)
Conc./well
(0.5 mM)
Inhibition (%)
Conc./well
(0.5 mM)
Inhibition (%)
Conc./well
(0.5 mM)
IC50
µM
IC50
µM
IC50
µM
4
6a
6b
6c
6d
6e
6f
89.51±0.56
72.34±0.37
66.56± 0.68
61.24±0.55
57.41±0.68
-
68.91±0.22
135.21±0.31
254.61±0.18
301.21±0.19
> 400
71.31±0.62
69.39±0.14
68.11± 0.34
86.51±0.31
22.41±0.88
-
115.41±0.22
132.61±0.12
138.71±0.18
74.51±0.11
-
52.91±0.51
63.23±0.33
69.33± 0.61
27.91±0.14
73.65±0.32
-
> 300
201.31±0.23
99.61±0.18
-
85.75±0.11
-
-
-
62.13±0.22
54.88±0.52
53.71±0.85
53.41±0.61
47.34±0.88
32.54±0.64
56.96±0.11
75.89±0.23
52.51±0.21
289.61±0.19
> 400
70.97±0.91
33.83±0.45
47.89±0.82
87.54±0.61
65.94±0.66
76.23±0.11
71.77±0.21
86.29±0.22
90.78±0.24
114.21±0.14
-
61.92±0.87
71.51±0.58
63.95±0.18
59.01±0.15
31.69±0.71
45.93±0.44
37.79±0.21
98.69±0.22
54.51±0.27
201.91±0.14
6g
6h
6i
76.75±0.14
> 400
-
196.11±0.21
> 400
80.31±0.21
152.31±0.10
99.61±0.15
109.61±0.23
73.21±0.17
61.21±0.12
> 400
6j
-
-
6k
6l
-
-
-
> 400
6m
6n
101.21±0.17
> 400
28.11±0.11
> 400
Eserine
91.29±1.17
Eserine
82.82±1.09
Baicalein
93.79±1.27
Control
0.04±0.001
0.85±0.0001
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 = Acetyl cholinesterase.
BChE = Butyrylcholinesterase.
LOX = Lipoxygenase.
12. Nagaraj, K.C. Chaluvaraju, M.S. Niranjan, S. Kiran, Int. J. Pharm. Pharm.
Sci. 3(3), 9, (2011)
5. REFERENCES
13. S. Cao, X.H. Qian, G. Song, B. Chai, Z. Jiang, J. Agric. Food Chem. 51,
152, (2003)
1. F.A. Omar, N.M. Mahfouz, M.A. Rahman, Eur. J. Med. Chem. 31, 819,
(1996)
14. S.N. Pandeya, A textbook of medicinal chemistry, S.G. publishers,
Varanasi p. 353, (2001)
2. B.N. Goswami, J.C.S. Kataky, J.N. Baruash, J. Heterocyclic Chem. 21,
205, (1984)
15. B.N. Goswami, J.C. Kataky, M.M. Datta, Ind. J. Chem. 23B, 796, (1984)
16. R.S. Varma, V. Bajpai, Ind. J. heterocycl. Chem. 8, 281, (1999)
17. T.P. Dabhi, V.H. Shah, A.R. Parikh, Indian Drugs 54, 98, (1992)
18. Aziz-ur-Rehman, S.Z. Siddiqui, M.A. Abbasi, N. Abbas, K.M. Khan, M.
Shahid, Y. Mahmood, M.N. Akhtar, and N.H. Lajis, Int. J. Pharm. Phram.
Sci. 4(2), 676, (2012)
3. R. Alan, F.R. Katritzky, W. Charles, Comprehensive Heterocyclic
Chemistry II, 1st ed. UK: pergamon (1996)
4. S.J. Dolman, F. Gosselin, P.D. Oshea, I.W. Davis, J. Org. Chem. 71, 9548,
(2006)
5. B.S. Holla, K.N. Poojary, B. Kalluraya, P.V. Gowda, Indian J. Heterocy.
Chem. 5, 273, (1996)
19. Aziz-ur-Rehman, K. Nafeesa, M.A. Abbasi, H. Khalid, K.M. Khan, M.
Ashraf, I. Ahmad and S.A. Ejaz, Asian J. Pharma. Hea. Sci. 2(3), 370,
(2012)
20. G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, Bio.
Pharm. 7, 88, (1961)
6. K.Y. Matsumoto, Y. Yasuda, T. Tanimoto, K. Matsumoto, T. Yoshida, J.
Shoji, J. Antibiot. 42, 1465, (1998)
7. S.S. Shafi, T.R. Radhakrishnan, Indian J. Heterocy. Chem. 5, 133, (1995)
8. A. Hasan, N.F. Thomas, S. Gapil, Molecules 16, 1297, (2011)
9. M.T. Omar, Arch. Pharm. Res. 20, 602, (1997)
10. T.M. Tan, Y.Chen, K.H. Kong, J. Bai, S.G. Li-Lim, H. Ang, Y. Lam.
Antivir. Res. 71, 7, (2006)
21. A.L. Tappel, Arch. Biochem. Biophys. 44(2), 378, (1953)
22. A.T. Evans, Bio Pharm. 36, 2035, (1987)
23. S. Baylac, P. Racine, Int. J. Aromatherap. 13, 138, (2003)
11. S. Wagle, A.A. Vasudeva, N.K. Suchetha, Indian J. Chem. 47B, 439,
(2008)
2190