Synthesis, characterization and antioxidant activity of bis (arylsulfonylmethyl/arylaminosulfonylmethylazolyl) pyridines

Article abstract of DOI:10.1002/jhet.4123

A new class of bis(arylsulfonylmethylazolyl)pyridines and bis(arylaminosulfonylmethyl-azolyl)pyridines were synthesized from the synthetic intermediates methyl arylsulfonylacetic acid hydrazide and methyl arylaminosulfonylacetic acid hydrazide adopting a green methodology-ultrasonication. All the synthesized compounds were resulted in higher yield and in shorter reaction times. The spectral parameters such as IR, ¹H NMR, ¹³C NMR, mass and microanalyzes were used to determine the structures of all the synthesized compounds and were assayed for antioxidant activity. The bis(arylaminosulfonylmethylazolyl)pyridines showed higher radical scavenging activity than the bis(arylsulfonylmethylazolyl)pyridines. Besides, unsubstituted, and methyl substituted compounds exhibited greater activity. Among all the tested compounds 8b and 11b were identified as potential antioxidants.

Full text of DOI:10.1002/jhet.4123

Synthesis, characterization and antioxidant activity of bis (arylsulfonylmethyl /
arylaminosulfonylmethylazolyl) pyridines
Anil Kumar Gunthanakkala, Madhu Sekhar Mangali, Padmavathi Venkatapuram, Padmaja
Adivireddy*
Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India,
Tel: +91 877 2289303; Fax: +91 877 2249532, E-mail: adivireddyp@yahoo.co.in
A new class of bis(arylsulfonylmethylazolyl)pyridines and bis(arylaminosulfonylmethyl-
azolyl)pyridines were synthesized from the synthetic intermediates methyl arylsulfonylacetic acid
hydrazide and methyl arylaminosulfonylacetic acid hydrazide adopting a green methodology-
ultrasonication. All the synthesized compounds were resulted in higher yield and in shorter reaction
1
times. The spectral parameters such as IR, H NMR, 13C NMR, mass and microanalyses were used
to determine the structures of all the synthesized compounds and were assayed for antioxidant
activity. The bis(arylaminosulfonylmethylazolyl)pyridines showed higher radical scavenging
activity than the bis(arylsulfonylmethylazolyl)pyridines. Besides, unsubstituted and methyl
substituted compounds exhibited greater activity. Amongst all the tested compounds 8b and 11b
were identified as potential antioxidants.
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/jhet.4123
This article is protected by copyright. All rights reserved.

INTRODUCTION
Antioxidants are one of the prominent disciplines of scientific research as they can protect
organism and cell from damage that are induced by oxidative stress. Scientists in different areas
become interested in new compounds which prevent or reduce the impact of oxidative stress on
cell.[1,2] Therefore, many chemists are searching for new antioxidants which can be structurally
modified from the basic structures and effectively inhibit the radical scavenging activity.
Heterocyclic compounds, particularly azoles, are of great scientific importance since they are used
as key starting compounds for many pharmaceuticals.[3] One such type of compounds are
oxadiazoles, thiadiazoles and triazoles. 1,3,4-Oxadiazoles and their derivatives show a wide
spectrum of biological activities, including antioxidant, antibacterial, anti-inflammatory, antifungal,
and antitubercular etc., Besides, oxadiazole is a privileged scaffold in many drugs viz., raltegravir,
zibotentan, tiodazosin and furamizole.[4] Moreover, 1,3,4-oxadiazoles are very good bioisosteres of
amides and esters, which enhance pharmacological activities by participating in hydrogen bonding
interactions with the receptors.[5] It is reported that 2,5-disubstituted 1,3,4-oxadiazoles are prepared
either by thermal/acid catalyzed reaction of 1,2-diacylhydrazines[6] or by reaction of
semicarbazone/hydrazone promoted by an oxidant[7] or by irradiation of a mixture of acid hydrazide
and carboxylic acid by microwaves.[8] 1,3,4-Thiadiazole exhibit antioxidant, antimicrobial,
10]
anticancer, anti-inflammatory, herbicidal and insecticidal activities.[9,
The reaction of
acylthiosemicarbazides with acid reagents such as trifluoroacetic acid[11] and methanesulfonic
acid[12] is most frequently used procedure for the preparation of 1,3,4-thiadiazoles. Thiadiazole
moiety makes the core structure of most familiar drugs-acetazolamide, megazol, sulfamethazole and
cefazedone.[13] Besides, 1,2,4-triazoles possess antibacterial,[14] antifungal[15] and anti-
inflammatory[16] activities. Some chemotherapeutics like vorozole, letrozole and anastrozole contain
1,2,4-triazole.[17] One of the synthetic methods for the synthesis of triazoles involves the utilization
of N, N’ -dimethylformamide dimethyl acetals.[18] Replacement of –O– by –S– or –NH– in some
heterocycles is reported viz., Bordners[19] synthesis of pyrroles from furan and the conversion of
epoxide to episulfides by the treatment of thiocyanates or thiourea.[20-22] However, reports about the
conversion of 1,3,4-oxadiazoles to 1,3,4-thiadiazoles and 1,2,4-triazoles are relatively less.[23, 24]
Besides, pyridine is a ubiquitous moiety in coenzyme vitamin B6 family and in numerous
alkaloids.[25] Pyridine and its derivatives display antioxidant,[26] antimicrobial,[27] and anti-
inflammatory activities.[28] The sulfone and sulfonamide are important functional groups found in a
wide variety of natural products, drugs and materials.[29] The combination of different
pharmacophores in one molecular frame could lead to a novel entity with increased biological
properties. Hence, the development of green techniques for the preparation of azoles remains an
This article is protected by copyright. All rights reserved.

important task for synthetic community. In the recent years, ultrasound irradiation methodology
offers high yield, shorter reaction time and milder reaction conditions[30] when compared with the
traditional method.[31] Motivated by these observations and our ongoing efforts towards
development of bioactive heterocycles,[32-34] it is planned to prepare hitherto unknown bis
heterocycles with a variety of pharmacophoric units under ultrasonication and to study their
antioxidant activity.
RESULTS AND DISCUSSION
The methyl arylsulfonylacetic acid hydrazide (3) and methyl arylaminosulfonylacetic acid
hydrazide (6) were used as synthons to prepare the target compounds bis(arylsulfonylmethyl
oxadiazolyl / thiadiazolyl / triazolyl)pyridines and bis(arylaminosulfonylmethyl oxadiazolyl /
thiadiazolyl
/
triazolyl)pyridines. Esterification of arylsulfonylacetic acid (1) and
arylaminosulfonylacetic acid (4) with methanol in the presence of concentrated sulphuric acid gave
methyl arylsulfonylacetic acid ester (2) and methyl arylaminosulfonylacetic acid ester (5). The
treatment of compounds 2 and 5 with hydrazine hydrate in the presence of pyridine resulted in 3
and 6 (Scheme 1). The reaction of 2 mmol of compound 3 with 1 mmol of 7 in the presence of
POCl3 under ultrasonication produced 2,6-bis(2-arylsulfonylmethyl-1,3,4-oxadiazol-5-yl)pyridine
(8). The oxadiazole ring present in compounds 8 and 11 were interconverted to thiadiazole and
triazole rings by reaction with appropriate nucleophiles. In fact, the reaction of 8 with thiourea in
THF under ultrasonication yielded 2,6-bis(2-arylsulfonylmethyl-1,3,4-thadiazol-5-yl)pyridine (9).
On the other hand, 2,6-bis(3-arylsulfonylmethyl-4-amino-1,2,4-triazol-5-yl)pyridine (10) was
1
obtained by the treatment of 8 with hydrazine hydrate in n-butanol (Scheme 2). The H NMR
spectra of 8a, 9a and 10a displayed a singlet at δ 5.25, 5.45 and 5.40 due to CH2 protons attached to
C-2 / C-3 respectively. Moreover, a broad singlet appeared at δ 5.62 ppm in 10a was attributed to
NH2 and disappeared on deuteration. Furthermore, 2,6-bis(2-arylaminosulfonylmethyl-1,3,4-
oxadiazol-5-yl)pyridine (11) was synthesized by the cyclocondensation of 2 mmol of compound 6
with 1 mmol of 7 in the presence of POCl3 under ultrasonication. In a similar way, 2,6-bis(2-
(arylaminosulfonylmethyl)-1,3,4-thadiazol-5-yl)pyridine (12) was prepared by the reaction of
compound 11 with thiourea in THF. Likewise, 2,6-(bis(3-arylaminosulfonylmethyl-4-amino-1,2,4-
triazol-5-yl)pyridine (13) was synthesized by the treatment of 11 with hydrazine hydrate in n-
1
butanol (Scheme 3). The H NMR spectra of 11a and 12a displayed a singlet at δ 5.45 and 5.30
This article is protected by copyright. All rights reserved.

1
(CH2) and a broad singlet at 10.30 and 10.32 ppm (NH) in addition to aromatic protons. The H
NMR spectrum of 13a exhibited a singlet at δ 5.04 due to CH2 protons attached to C-3 and two
broad singlets at 5.60 (NH2) and 10.15 ppm (NH). The signals of NH and NH2 disappeared upon
13
addition of D2O. IR, C NMR, mass spectra and microanalyses were also utilized to establish the
structures of all the synthesized compounds.
Scheme 1. Preparation of arylsufonyl/arylaminosufonylacetic acid hydrazide.
Scheme 2. Ultrasound assisted synthesis of bis(arylsufonylmethylazolyl) pyridines.
This article is protected by copyright. All rights reserved.

Scheme 3. Ultrasound assisted synthesis of bis(arylaminosufonylmethylazolyl) pyridines.
This article is protected by copyright. All rights reserved.

ANTIOXIDANT ACTIVITY
Compounds 8-13 were assayed for antioxidant activity by three methods (DPPH, NO, and
H2O2) at 50, 75 and 100 µM concentrations (Tables 1, 2 & 3) using ascorbic acid as the standard
drug. The results indicated that compounds 8a, 8b, 10b, 11a, 11b, 13a and 13b displayed good
activity. The compounds 9c, 10c, and 12c showed low activity whereas the remaining compounds
exhibited moderate activity. The structure-activity relationship of the tested compounds indicated
that the compounds containing oxadiazole units (8 and 11) showed higher antioxidant activity than
those containing thiadiazole (9 and 12) and triazole (10 and 13) moieties. It was observed that
compounds having triazole displayed slightly higher radical scavenging activity than thiadiazole.
The sulfonamide bis heterocycles (11-13) displayed comparatively higher antioxidant activity than
the sulfone bis heterocycles (8-10). This may be due to the presence of increasing number of
chromophoric groups. Moreover, electron donating methyl substituent on the phenyl ring enhanced
the radical scavenging activity when compared with the electron withdrawing chloro substituted
compounds. In fact, the radical scavenging activity increases with increase in concentration. In all
three methods, the IC50 values of 8b and 11b are 32.95, 31.64, 32.42 and 32.01, 29.90, 31.34.
Amongst all the tested compounds 8b and 11b exhibited excellent antioxidant activity.
CONCLUSION
In
conclusion,
some
new
bis(arylsulfonylmethylazolyl)pyridines
and
bis(arylaminosulfonylmethylazolyl)pyridines were synthesized from the synthetic intermediates,
methyl arylsulfonylacetic acid hydrazide and methyl arylaminosulfonylacetic acid hydrazide
adopting a green approach – ultrasonication and resulted in high yield in shorter reaction time. IR,
NMR, mass and elemental analyses were used to elucidate the structures of all the synthesized
compounds and were also tested for antioxidant activity. The compounds having oxadiazole ring
displayed greater antioxidant activity than those having thiadiazole and triazole units. It was also
noticed that compounds bearing sulfonamide moiety displayed higher radical scavenging activity
than those with sulfone. This may be due to the presence of more chromophore units. Moreover,
unsubstituted and methyl substituted compounds showed higher radical scavenging activity than the
chloro substituted compounds. Amongst all the tested compounds, 8b and 11b were found to be
potential antioxidant agents.
EXPERIMENTAL
This article is protected by copyright. All rights reserved.

General.
Melting points were determined in open capillaries on a Mel-Temp apparatus
and are uncorrected. The homogeneity of the compounds was determined by TLC (silica gel H,
BDH, hexane/ethyl acetate, 3:1). The IR spectra were run on a Thermo Nicolet IR 200 FT-IR
1
13
spectrometer as KBr pellets and the wave numbers were reported in cm−1. The H NMR and C
NMR spectra were recorded in DMSO-d6 on a Jeol JNM spectrometer operating at λ-400 and λ-100
MHz. High-resolution mass spectra were run on Micro mass Q-TOF spectrometer using
electrospray ionization. All chemical shifts were given in δ (ppm) using TMS as an internal
standard. The elemental analyses were carried out on a Perkin-Elmer 240C elemental analyzer. The
temperature was measured by flexible probe throughout the reaction. Ultrasound irradiation was
performed in a Bandelin Sonorex RK 102H ultrasonic bath operating at frequency of 35 KHz. The
synthetic intermediates methyl arylsulfonylacetic acid hydrazide (3) and methyl
arylaminosulfonylacetic acid hydrazide (6) were synthesized as per the literature procedures. [32, 35]
General procedure for the synthesis of 2,6-bis(2-arylsulfonylmethyl-1,3,4-oxadiazol-5-
yl)pyridine (8).
The compound 3 (2 mmol), 7 (1 mmol) and POCl3 (5 mL) were subjected to
ultrasound irradiation at a frequency of 35 KHz for 35-60 min at laboratory temperature. The
progress of the reaction was checked by TLC. The excess POCl3 was removed in vacuo and the
resultant residue was poured onto crushed ice. The separated solid was filtered, washed with
saturated sodium bicarbonate solution followed by water, dried and recrystallized from 2-propanol.
2,6-Bis(2-(phenylsulfonylmethyl)-1,3,4-oxadiazol-5-yl)pyridine (8a). Yield 92%; White solid,
mp 171-173 oC; IR (KBr cm-1): 1574 (C=N), 1312 & 1152 (SO2); 1H NMR (400 MHz, DMSO-d6):
δ 5.25 (s, 4H, CH2-(C-2)), 7.25-7.81 (m, 13H, Ar-H) ppm; 13C NMR (400 MHz, DMSO-d6): δ 54.6
(CH2), 159.2 (C-2), 157.0 (C-5), 122.4, 125.7, 128.8, 132.4, 136.9, 140.7, 152.2 ppm (aromatic
carbons); HRMS (m/z): 546.0518 [M+ Na]+. Anal. Calcd. for C23H17N5O6S2: C 52.77, H 3.27, N
13.38; Found: C 52.86, H 3.30, N 13.60%.
2,6-Bis(2-(4-methylphenylsulfonylmethyl)-1,3,4-oxadiazol-5-yl)pyridine (8b). Yield 80%; White
o
1
solid, mp 177-179 C; IR (KBr cm-1): 1562 (C=N), 1308 & 1148 (SO2); H NMR (400 MHz,
13
DMSO-d6): δ 2.51 (s, 6H, Ar-CH3), 5.42 (s, 4H, CH2-(C-2)), 7.35-8.07 (m, 11H, Ar-H) ppm; C
NMR (400 MHz, DMSO-d6): δ 22.4 (Ar-CH3), 54.0 (CH2), 158.8 (C-2), 156.9 (C-5), 121.2, 125.0,
127.2, 132.0, 136.0, 140.6, 151.5 ppm (aromatic carbons); HRMS (m/z): 574.0831 [M+ Na]+. Anal.
Calcd. for C25H21N5O6S2: C 54.44, H 3.84, N 12.70; Found: C 54.54, H 3.83, N 12.84%.
2,6-Bis(2-(4-chlorophenylsulfonylmethyl)-1,3,4-oxadiazol-5-yl)pyridine (8c). Yield 85%; White
o
1
solid, mp 192-194 C; IR (KBr cm-1): 1575 (C=N), 1316 & 1157 (SO2); H NMR (400 MHz,
DMSO-d6): δ 5.52 (s, 4H, CH2-(C-2)), 7.40-8.16 (m, 11H, Ar-H) ppm; 13C NMR (400 MHz,
DMSO-d6): δ 54.5 (CH2), 159.6 (C-2), 157.4 (C-5), 122.7, 126.2, 128.9, 133.2, 137.8, 141.5, 153.6
This article is protected by copyright. All rights reserved.

ppm (aromatic carbons); HRMS (m/z): 613.9738 [M+ Na]+. Anal. Calcd. for C23H15Cl2N5O6S2: C
46.63, H 2.55, N 11.82; Found: C 46.75, H 2.57, N 11.98%.
General procedure for the synthesis of 2,6-bis(2-arylsulfonylmethyl-1,3,4-thiadiazol-5-
yl)pyridine (9).
In a sealed tube, a mixture of compound 8 (0.5 mmol), thiourea (2 mmol) and
THF (6 mL) was taken and heated to reflux under ultrasonication at a frequency of 35 KHz for 80-
110 min. The progress of the reaction was checked by TLC. The contents of the flask were
extracted with dichloromethane, washed with water followed by brine solution. The organic layer
was dried over anhydrous sodium sulfate and filtered. Removal of the solvent under reduced
pressure gave a residue which was purified by column chromatography (silica gel, 60-120 mesh)
using hexane-ethyl acetate (3:1) as eluent.
2,6-Bis(2-(phenylsulfonylmethyl)-1,3,4-thiadiazol-5-yl)pyridine (9a). Yield 88%; White solid,
mp 169-171 oC; IR (KBr cm-1): 1568 (C=N), 1321 & 1138 (SO2); 1H NMR (400 MHz, DMSO-d6):
δ 5.45 (s, 4H, CH2-(C-2)), 7.34-7.96 (m, 13H, Ar-H) ppm; 13C NMR (400 MHz, DMSO-d6): δ 54.1
(CH2), 164.4 (C-2), 155.6 (C-5), 122.5, 125.4, 128.2, 134.2, 138.0, 141.7, 152.5 ppm (aromatic
carbons); HRMS (m/z): 578.0061 [M+ Na]+. Anal. Calcd. for C23H17N5O4S4: C 49.72, H 3.08, N
12.60; Found: C 49.81, H 3.09, N 12.78%.
2,6-Bis(2-(4-methylphenylsulfonylmethyl)-1,3,4-thiadiazol-5-yl)pyridine (9b). Yield 86%; White
o
1
solid, mp 166-168 C; IR (KBr cm-1): 1561 (C=N), 1319 & 1131 (SO2); H NMR (400 MHz,
13
DMSO-d6): δ 2.48 (s, 6H, Ar-CH3), 5.39 (s, 4H, CH2-(C-2)), 7.25-7.83 (m, 11H, Ar-H) ppm; C
NMR (400 MHz, DMSO-d6): δ 22.8 (Ar-CH3), 53.5 (CH2), 163.9 (C-2), 154.4 (C-5), 122.0, 125.2,
128.0, 133.7, 137.3, 140.3, 151.3 ppm (aromatic carbons); HRMS (m/z): 606.0374 [M+ Na]+. Anal.
Calcd. for C25H21N5O4S4: C 51.44, H 3.63, N 12.00; Found: C 51.55, H 3.66, N 12.20%.
2,6-Bis(2-(4-chlorophenylsulfonylmethyl)-1,3,4-thiadiazol-5-yl)pyridine (9c). Yield 89%; White
o
1
solid, mp 178-180 C; IR (KBr cm-1): 1572 (C=N), 1327 & 1128 (SO2); H NMR (400 MHz,
DMSO-d6): δ 5.42 (s, 4H, CH2-(C-2)), 7.50-7.98 (m, 11H, Ar-H) ppm; 13C NMR (400 MHz,
DMSO-d6): δ 55.3 (CH2), 164.0 (C-2), 154.8 (C-5), 123.0, 126.6, 128.7, 135.6, 138.5, 142.0, 152.7
ppm (aromatic carbons); HRMS (m/z): 645.9282 [M+ Na]+. Anal. Calcd. for C23H15Cl2N5O4S4: C
44.23, H 2.42, N 11.21; Found: C 44.17, H 2.41, N 11.35%.
General procedure for the synthesis of 2,6-(bis(3-arylsulfonylmethyl-4-amino-1,2,4-
triazol-5-yl)pyridine (10).
The compound 8 (1 mmol), hydrazine hydrate (4 mmol) and n-
butanol (5 mL) were kept under ultrasonication at a frequency of 35 KHz for 55-75 min. Then,
potassium hydroxide (2 mmol) was added to the reaction media. The precipitate obtained was
separated by filtration and treated with concentrated hydrochloric acid to pH ≈ 3. It was washed
This article is protected by copyright. All rights reserved.

with water, dried and purified by column chromatography (silica gel, 160-120 mesh) using hexane-
ethyl acetate (3:1) as eluent.
2,6-(Bis(3-(phenylsulfonylmethyl)-4-amino-1,2,4-triazol-5-yl))pyridine (10a). Yield 89%; White
o
1
solid, mp 174-176 C; IR (KBr cm-1): 3446 & 3338 (NH2), 1559 (C=N), 1324 & 1132 (SO2); H
NMR (400 MHz, DMSO-d6): δ 5.40 (s, 4H, CH2-(C-3)), 5.62 (bs, 4H, NH2), 7.29-7.75 (m, 13H, Ar-
13
H) ppm; C NMR (400 MHz, DMSO-d6): δ 53.2 (CH2), 154.7 (C-3), 150.2 (C-5), 121.5, 126.0,
128.5, 133.4, 137.1, 142.2, 152.3 ppm (aromatic carbons); HRMS (m/z): 574.1056 [M+ Na]+. Anal.
Calcd. for C23H21N9O4S2: C 50.08, H 3.84, N 22.85; Found: C 50.18, H 3.88, N 23.01%.
2,6-(Bis(3-(4-methylphenylsulfonylmethyl)-4-amino-1,2,4-triazol-5-yl))pyridine (10b).
Yield
85%; White solid, mp 188-190 oC; IR (KBr cm-1): 3439 & 3333 (NH2), 1561 (C=N), 1321 & 1126
(SO2); 1H NMR (400 MHz, DMSO-d6): δ 2.44 (s, 6H, Ar-CH3), 5.33 (s, 4H, CH2-(C-3)), 5.63 (bs,
4H, NH2), 7.20-7.69 (m, 11H, Ar-H) ppm; 13C NMR (400 MHz, DMSO-d6): δ 22.5 (Ar-CH3), 54.7
(CH2), 154.6 (C-3), 150.1 (C-5), 121.6, 125.5, 128.3, 133.6, 136.8, 140.8, 152.1 ppm (aromatic
carbons); HRMS (m/z): 602.1369 [M+ Na]+. Anal. Calcd. for C25H25N9O4S2: C 51.80, H 4.35, N
21.75; Found: C 51.92, H 4.37, N 21.97%.
2,6-(Bis(3-(4-chlorophenylsulfonylmethyl)-4-amino-1,2,4-triazol-5-yl))pyridine (10c).
Yield
89%; White solid, mp 198-200 oC; IR (KBr cm-1): 3453 & 3345 (NH2), 1567 (C=N), 1330 & 1137
1
(SO2); H NMR (400 MHz, DMSO-d6): δ 5.37 (s, 4H, CH2-(C-3)), 5.68 (bs, 4H, NH2), 7.31-7.95
(m, 11H, Ar-H) ppm; 13C NMR (400 MHz, DMSO-d6): δ 53.6 (CH2), 155.5 (C-3), 150.7 (C-5),
121.8, 126.4, 129.0, 134.7, 138.6, 142.6, 153.2 ppm (aromatic carbons); HRMS (m/z): 642.0276
[M+ Na]+. Anal. Calcd. for C23H19Cl2N9O4S2: C 44.52, H 3.09, N 20.32; Found: C 44.61, H 3.07, N
20.47%.
General procedure for the synthesis of 2,6-bis(2-arylaminosulfonylmethyl-1,3,4-
oxadiazol-5-yl)pyridine (11).
A mixture of compound 6 (2 mmol), 7 (1 mmol) and POCl3 (5
mL) was subjected to ultrasound irradiation at a frequency of 35 KHz for 30-50 min at laboratory
temperature. The progress of the reaction was monitored by TLC. Removal of the excess POCl3 in
vacuo yielded a residue which was poured onto crushed ice. The separated solid collected by
filtration. It was washed with saturated sodium bicarbonate followed by water, dried and
recrystallized from 2-propanol.
2,6-Bis(2-(phenylaminosulfonylmethyl)-1,3,4-oxadiazol-5-yl)pyridine (11a). Yield 87%; White
solid, mp 167-169 oC; IR (KBr cm-1): 3248 (NH), 1581 (C=N), 1335 & 1141 (SO2); 1H NMR (400
MHz, DMSO-d6): δ 5.45 (s, 4H, CH2-(C-2)), 7.26-7.89 (m, 13H, Ar-H), 10.30 (bs, 2H, NH) ppm;
13C NMR (400 MHz, DMSO-d6): δ 54.4 (CH2), 159.9 (C-2), 156.4 (C-5), 120.5, 124.8, 127.5,
This article is protected by copyright. All rights reserved.

132.7, 136.5, 141.4, 151.8 ppm (aromatic carbons); HRMS (m/z): 576.0736 [M+ Na]+. Anal. Calcd.
for C23H19N7O6S2: C 49.90, H 3.46, N 17.71; Found: C 50.03, H 3.47, N 17.90%.
2,6-Bis(2-(4-methylphenylaminosulfonylmethyl)-1,3,4-oxadiazol-5-yl)pyridine (11b). Yield 89%;
White solid, mp 161-163 oC; IR (KBr cm-1): 3242 (NH), 1577 (C=N), 1327 & 1136 (SO2); 1H NMR
(400 MHz, DMSO-d6): δ 2.26 (s, 6H, Ar-CH3), 5.43 (s, 4H, CH2-(C-2)), 7.21-7.91 (m, 11H, Ar-H),
10.25 (bs, 2H, NH) ppm; 13C NMR (400 MHz, DMSO-d6): δ 22.3 (Ar-CH3), 53.2 (CH2), 158.3 (C-
2), 156.3 (C-5), 120.0, 124.3, 127.3, 132.4, 137.4, 140.4, 151.0 ppm (aromatic carbons); HRMS
(m/z): 604.1049 [M+ Na]+. Anal. Calcd. for C25H23N7O6S2: C 51.63, H 3.99, N 16.86; Found: C
51.71, H 4.01, N 17.03%.
2,6-Bis(2-(4-chlorophenylaminosulfonylmethyl)-1,3,4-oxadiazol-5-yl)pyridine (11c). Yield 91%;
White solid, mp 182-184 oC; IR (KBr cm-1): 3248 (NH), 1581 (C=N), 1335 & 1141 (SO2); 1H NMR
(400 MHz, DMSO-d6): δ 5.51 (s, 4H, CH2-(C-2)), 7.15-7.79 (m, 11H, Ar-H), 10.36 (bs, 2H, NH)
ppm; 13C NMR (400 MHz, DMSO-d6): δ 53.7 (CH2), 159.4 (C-2), 157.8 (C-5), 120.8, 125.8, 127.8,
133.8, 137.6, 141.8, 153.4 ppm (aromatic carbons); HRMS (m/z): 643.9956 [M+ Na]+. Anal. Calcd.
for C23H17Cl2N7O6S2: C 44.38, H 2.75, N 15.75%; Found: C 44.48, H 2.74, N 15.94%.
General procedure for the synthesis of 2,6-bis(2-arylaminosulfonylmethyl-1,3,4-thiadiazol-5-
yl)pyridine (12).
The compound 11 (0.5 mmol), thiourea (2 mmol) and THF (6 mL) were
taken in a sealed tube and heated to reflux under ultrasonication at a frequency of 35 KHz for 60-70
min. The progress of the reaction was monitored by TLC. The reaction mixture was extracted with
dichloromethane, and washed with water followed by brine solution. The organic layer was dried
over anhydrous sodium sulfate. The solvent was removed under vacuum and the resultant residue
was purified by column chromatography (silica gel, 60-120 mesh) using hexane- ethyl acetate (3:1)
as eluent.
2,6-Bis(2-(phenylaminosulfonylmethyl)-1,3,4-thiadiazol-5-yl)pyridine (12a). Yield 91%; White
solid, mp 192-194 oC; IR (KBr cm-1): 3228 (NH), 1591 (C=N), 1324 & 1141 (SO2); 1H NMR (400
MHz, DMSO-d6): δ 5.30 (s, 4H, CH2-(C-2)), 7.33-7.72 (m, 13H, Ar-H), 10.32 (bs, 2H, NH) ppm;
13C NMR (400 MHz, DMSO-d6): δ 54.3 (CH2), 164.1 (C-2), 155.1 (C-5), 120.5, 124.9, 127.0,
134.3, 136.6, 141.6, 152.0 ppm (aromatic carbons); HRMS (m/z): 608.0279 [M+ Na]+. Anal. Calcd.
for C23H19N7O4S4: C 47.17, H 3.27, N 16.74; Found: C 47.24, H 3.30, N 16.90%.
2,6-Bis(2-(4-methylphenylaminosulfonylmethyl)-1,3,4-thiadiazol-5-yl)pyridine (12b). Yield 88%;
White solid, mp 199-201 oC; IR (KBr cm-1): 3211 (NH), 1583 (C=N), 1322 & 1133 (SO2); 1H NMR
(400 MHz, DMSO-d6): δ 2.29 (s, 6H, Ar-CH3), 5.37 (s, 4H, CH2-(C-2)), 7.26-7.90 (m, 11H, Ar-H),
10.41 (bs, 2H, NH) ppm; 13C NMR (400 MHz, DMSO-d6): δ 22.7 (Ar-CH3), 53.6 (CH2), 164.8 (C-
This article is protected by copyright. All rights reserved.

2), 154.5 (C-5), 119.5, 124.5, 127.7, 132.8, 136.2, 140.5, 151.4 ppm (aromatic carbons); HRMS
(m/z): 636.0592 [M+ Na]+. Anal. Calcd. for C25H23N7O4S4: C 48.93, H 3.78, N 15.98; Found: C
49.06, H 3.82, N 16.19%.
2,6-Bis(2-(4-chlorophenylaminosulfonylmethyl)-1,3,4-thiadiazol-5-yl)pyridine (12c). Yield 92%;
White solid, mp 211-213 oC; IR (KBr cm-1): 3236 (NH), 1594 (C=N), 1331 & 1143 (SO2); 1H NMR
(400 MHz, DMSO-d6): δ 5.41 (s, 4H, CH2-(C-2)), 7.33-8.01 (m, 11H, Ar-H), 10.62 (bs, 2H, NH)
ppm; 13C NMR (400 MHz, DMSO-d6): δ 54.5 (CH2), 164.3 (C-2), 156.0 (C-5), 120.6, 125.6, 128.4,
134.6, 137.5, 142.5, 152.8 ppm (aromatic carbons); HRMS (m/z): 675.9500 [M+ Na]+. Anal. Calcd.
for C23H17Cl2N7O4S4: C 42.20, H 2.62, N 14.98; Found: C 42.31, H 2.64, N 15.16%.
General procedure for the synthesis of 2,6-(bis(3-arylaminosulfonylmethyl-4-amino-
1,2,4-triazol-5-yl)pyridine (13).
A solution of compound 11 (1 mmol) and hydrazine hydrate
(4 mmol) in n-butanol (5 mL) was subjected to ultrasound irradiation at a frequency of 35 KHz for
30-45 min. Then, potassium hydroxide (2 mmol) was added to reaction media and the precipitate
obtained was separated by filtration. The resultant solid was treated with concentrated hydrochloric
acid to pH ≈ 3. It was washed with water, dried and purified by column chromatography (silica gel,
160-120 mesh) using hexane- ethyl acetate (3:1) as eluent.
2,6-(Bis(3-(phenylaminosulfonylmethyl)-4-amino-1,2,4-triazol-5-yl))pyridine (13a). Yield 91%;
White solid, mp 189-191 oC; IR (KBr cm-1): 3478 & 3361 (NH2), 3243 (NH), 1596 (C=N), 1334 &
1135 (SO2); 1H NMR (400 MHz, DMSO-d6): δ 5.04 (s, 4H, CH2-(C-3)), 5.60 (bs, 4H, NH2), 7.36-
7.64 (m, 13H, Ar-H), 10.15 (bs, 2H, NH) ppm; 13C NMR (400 MHz, DMSO-d6): δ 55.2 (CH2),
155.6 (C-3), 150.3 (C-5), 120.7, 125.9, 129.2, 134.0, 138.1, 142.3, 153.1 ppm (aromatic carbons);
HRMS (m/z): 604.1274 [M+ Na]+. Anal. Calcd. for C23H23N11O4S2: C 47.50, H 3.99, N 26.49;
Found: C 47.62, H 4.00, N 26.73%.
2,6-(Bis(3-(4-methylphenylaminosulfonylmethyl)-4-amino-1,2,4-triazol-5-yl))pyridine (13b). Yield
o
84%; White solid, mp 180-182 C; IR (KBr cm-1): 3471 & 3365 (NH2), 3237 (NH), 1583 (C=N),
1326 & 1132 (SO2); 1H NMR (400 MHz, DMSO-d6): δ 2.40 (s, 6H, Ar-CH3), 5.30 (s, 4H, CH2-(C-
13
3)), 5.59 (bs, 4H, NH2), 7.38-7.94 (m, 11H, Ar-H), 10.28 (bs, 2H, NH) ppm; C NMR (400 MHz,
DMSO-d6): δ 23.2 (Ar-CH3), 54.7 (CH2), 154.8 (C-3), 149.7 (C-5), 119.8, 124.2, 128.1, 133.5,
137.2, 141.2, 152.3 ppm (aromatic carbons); HRMS (m/z): 632.1587 [M+ Na]+. Anal. Calcd. for
C25H27N11O4S2: C 49.25, H 4.46, N 25.27; Found: C 49.34, H 4.49, N 25.46%.
2,6-(Bis(3-(4-chlorophenylaminosulfonylmethyl)-4-amino-1,2,4-triazol-5-yl))pyridine (13c). Yield
o
89%; White solid, mp 199-201 C; IR (KBr cm-1): 3480 & 3382 (NH2), 3251 (NH), 1598 (C=N),
1
1338 & 1139 (SO2); H NMR (400 MHz, DMSO-d6): δ 5.38 (s, 4H, CH2-(C-3)), 5.70 (bs, 4H,
NH2), 7.57-8.10 (m, 11H, Ar-H), 10.30 (bs, 2H, NH) ppm; 13C NMR (400 MHz, DMSO-d6): δ 55.4
This article is protected by copyright. All rights reserved.

(CH2), 155.2 (C-3), 151.6 (C-5), 121.2, 124.7, 130.0, 134.8, 138.4, 142.8, 153.8 ppm (aromatic
carbons); HRMS (m/z): 672.0494 [M+ Na]+. Anal. Calcd. for C23H21Cl2N11O4S2: C 42.47, H 3.25, N
23.69; Found: C 42.57, H 3.26, N 23.85%.
Antioxidant assays. The compounds 8-13 were evaluated for antioxidant activity by 2,2-
diphenyl-1-picrylhydrazyl,[36, 37] nitric oxide[38, 39] and hydrogen peroxide[40] methods at three 50,75
and 100 µg/mL concentrations.[33]
Acknowledgements. One of the authors M. Madhu Sekhar grateful to University Grants
Commission (UGC), New Delhi, for the sanction of UGC-SRF fellowship. The authors are also
thankful to Prof. Ch. Appa Rao, Department of Biochemistry, S. V. University, Tirupati for
providing necessary facilities to carry out the antioxidant activity.
REFERENCES AND NOTES
[1]
[2]
A. A. Al-Amiery, Med Chem Res 2012, 21, 3204.
G. F. Zha. K. P. Rakesh, H. M. Manukumar, C. S. Shantharam, S. Long, Eur J Med
Chem 2019, 162, 465.
[3]
[4]
[5]
A. C. Sauer, J. G. Leal, S. T. Stefanello, M. T. B. Leite, M. B. Souza, F. A. A.
Soares, O. E. D. Rodrigues, L. Dornelles, Tetrahedron Lett 2017, 58, 87.
G. Yamini, M. Madhusekhar, G. Sravya, V. Padmavathi, A. Padmaja, Monatsh
Chem 2018, 149 (12), 2311.
J. -J. Zhao, X. -F. Wang, B. -L. Li, R. -L. Zhang, B. Li, Y. -M. Liu, C. -W. Li, J. -B.
Liu, B. -Q. Chen, Bioorg Med Chem Lett 2016, 26, 4414.
[6]
[7]
[8]
S. Liras, M. P. Allen, B. E. Segelstein, Synth Commun 2000, 30, 437.
S. L. Gaonkar, K. M. L. Rai, B. Prabhuswamy, Eur J Med Chem 2006, 41, 841.
K. M. Khan, Z. Ullah, M. Rani, S. Perveen, S. M. Haider, M. I. Choudhary, A.
Rahman, W. Voelter, Lett Org Chem 2004, 1, 50.
[9]
T. A. Farghaly, M. A. Abdallah, G. S. Masaret, Z. A. Muhammad, Eur J Med Chem
2015, 97, 320.
[10] M. Zarei, Tetrahedron 2017, 73(14), 1867.
[11] E. Palaska, G. Sahin, P. Kelicen, N. T. Durlu, G. Altinok, Il Farmaco 2002, 57, 101.
[12] L. I. Lutwick, M. W. Rytel, J. P. Yanez, J. N. Galgiani, D. A. Stevens, J Am Med
Assoc 1979, 241, 272.
[13] (a) S. Haider, M. S. Alam, H. Hamid, Eur J Med Chem 2015, 92, 156. (b) F. A. A.
El-Hag, S. M. Gomha, T. A. Farghaley, J Heterocyclic Chem 2017, 54(2), 1172.
This article is protected by copyright. All rights reserved.

[14] T. Plech, B. Kapron, A. Paneth, U. Kosikowska, A. Malm, A. Strzelczyk, P. Staczek,
L. Swiatek, B. Rajtar, M. Polz-Dacewicz, Eur J Med Chem 2015, 97, 94.
[15] Q. Y. Sun, J. M. Xu, Y. B. Cao, W. N. Zhang, Q. Y. Wu, D. Z. Zhang, J. Zhang, H.
Q. Zhao, Y. Y. Jiang, Eur J Med Chem 2007, 42, 1226.
[16] A. M. Abdel-Megeed, H. M. Abdel-Rahman, G. S. Alkaramany, M. A. El-Gendy,
Eur J Med Chem 2009, 44(1), 117.
[17] M. Clemons, R. E. Coleman, S. Verma, Cancer Treat Rev 2004, 30, 325.
[18] M. J. Stocks, D. R. Cheshire, R. Reynolds, Org Lett 2004, 6, 2969.
[19] C. A. Bordner, US Patent June 10, 1952, 2, 600, 689, Chem Abstr 1953, 47, 4373.
[20] E. E. Van Tamelen, J Am Chem Soc 1951, 73, 3444.
[21] C. C. Culvenor, W. Davies, W. E. Savige, J Chem Soc 1952, 4480.
[22] C. C. Price, P. F. Kirk, J Am Chem Soc 1953, 75, 2396.
[23] F. Kiyoshi, T. Senji, Chem Pharm Bull 1960, 8, 908.
[24] N. Linganna, K. M. LokanathaRai, Synth Commun 1998, 28, 4611.
[25] G. D. Henry, Tetrahedron 2004, 60(29), 6043.
[26] C. Ajit, S. N. Pandey, Asian J Pharm Clin Res 2011, 4, 5.
[27] D. H. Vyas, S. D. Tala, J. D. Akbari, M. F. Dhaduk, K. A. Joshi, H. S. Joshi, Indian
J Chem 2009, 48, 833.
[28] T. Murata, M. Shimada, S. Sakakibara, T. Yoshino, H. Kadono, T. Masuda, M.
Shimazaki, T. Shintani, K. Fuchikami, K. Sakai, H. Inbe, K. Takeshita, T. Niki, M.
Umeda, K. B. Bacon, K. B. Ziegelbauer, T. B. Lowingera, Bioorg Med Chem Lett
2003, 13, 913.
[29] A. Rodriguez, W. J. Moran, J Org Chem 2016, 81, 2543.
[30] M. Rouhani, A. Ramazani, S. W. Joo, Ultrason Sonochem 2015, 22, 391.
[31] S. Vahid, M. Ali, Ultrason Sonochem 2012, 19, 300.
[32] G. M. Reddy, A. Muralikrishna, V. Padmavathi, A. Padmaja, T. K. Tilak, C. A. Rao,
Chem Pharm Bull 2013, 61, 1291.
[33] U. Nagarjuna, G. Sravya, V. Padmavathi, A. Padmaja, Med Chem Res 2017, 26,
1574.
[34] M. Madhu Sekhar, U. Nagarjuna, V. Padmavathi, A. Padmaja, N. Vasudeva Reddy,
T. Vijaya, Eur J Med Chem 2018, 145, 1-10.
[35] V. Padmavathi, G. Sudhakar Reddy, A. Padmaja, P. Kondaiah, AliShazia, Eur J Med
Chem 2009, 44, 2106–2112.
[36] M. Burits, F. Bucar, Phytother Res 2000, 14, 323.
This article is protected by copyright. All rights reserved.

[37] M. Cuendet, K. Hostettmann, O. Potterat, W. Dyatmiko, Helv Chim Acta 1997, 80,
1144.
[38] L. C. Green, D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, S. R.
Tannenbaum, Anal Biochem 1982, 126, 131.
[39] L. Marcocci, J. J. Maguire, M. T. Droy-Lefaix, L. Packer, Biochem Biophys Res
Commun 1994, 201, 748.
[40] R. J. Ruch, S. J. Cheng, J. E. Klaunig, Carcinogenesis 1989, 10, 1003.
This article is protected by copyright. All rights reserved.

Fig. 1 In vitro antioxidant activity of 8-13 by DPPH method
Fig. 2 In vitro antioxidant activity of 8-13 by NO method
This article is protected by copyright. All rights reserved.

Fig. 3 In vitro antioxidant activity of 8-13 by H2O2 method
This article is protected by copyright. All rights reserved.

Table 1 In vitro antioxidant activity of 8-13 by DPPH method.
IC50 (μg/mL)
Concentration (μg/mL)
Compound
50
75
100
Mean ± SD
66.26 ± 0.17
Mean ± SD
68.35 ± 0.15
Mean ± SD
71.56 ± 0.21
Mean ± SD
37.73 ± 0.06
8a
75.87 ± 0.11
35.19 ± 0.30
40.23 ± 0.21
45.74 ± 0.16
21.30 ± 0.46
50.96 ± 0.42
59.98 ± 0.20
27.15 ± 0.25
69.50 ± 0.34
78.08 ± 0.21
39.11 ± 0.23
44.43 ± 0.38
49.37 ± 0.26
24.38 ± 0.18
54.13 ± 0.12
61.18 ± 0.25
30.33 ± 0.44
74.19 ± 0.19
-
78.09 ± 0.27
37.22 ± 0.18
43.21 ± 0.12
48.98 ± 0.15
23.19 ± 0.38
53.28 ± 0.36
61.16 ± 0.23
28.11 ± 0.25
73.06 ± 0.28
80.45 ± 0.17
41.06 ± 0.16
47.19 ± 0.35
52.04 ± 0.29
25.36 ± 0.14
58.10 ± 0.23
63.29 ± 0.09
32.10 ± 0.21
75.36 ± 0.16
-
80.46 ± 0.28
41.28 ± 0.42
46.77 ± 0.17
51.27 ± 0.13
26.35 ± 0.33
56.43 ± 0.10
64.04 ± 0.12
31.54 ± 0.40
76.93 ± 0.29
82.65 ± 0.14
44.89 ± 0.13
49.27 ± 0.28
54.50 ± 0.07
28.10 ± 0.05
60.86 ± 0.08
69.74 ± 0.24
36.29 ± 0.16
78.62 ± 0.15
-
32.95 ± 0.08
121.12 ± 0.28
106.90 ± 0.14
97.52 ± 0.16
189.75 ± 0.28
49.05 ± 0.04
41.68 ± 0.06
158.52 ± 0.25
35.97 ± 0.22
32.01 ± 0.10
111.38 ± 0.21
101.48 ± 0.27
50.63 ± 0.05
177.93 ± 0.02
46.18 ± 0.81
40.86 ± 0.11
137.89 ± 0.37
33.69 ± 0.07
-
8b
8c
9a
9b
9c
10a
10b
10c
11a
11b
11c
12a
12b
12c
13a
13b
13c
Ascorbic acid
Blank
(-) No activity, (±) SD
This article is protected by copyright. All rights reserved.

Table 2 In vitro antioxidant activity of 8-13 by NO method.
IC50 (μg/mL)
Concentration (μg/mL)
Compound
50
75
100
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
35.88 ±0.23
69.66 ± 0.14
79.01 ± 0.09
38.29 ± 0.21
47.42 ± 0.18
52.19 ± 0.14
24.89 ± 0.29
57.12 ± 0.31
62.11 ± 0.22
31.24 ± 0.15
75.16 ± 0.26
83.60 ± 0.27
41.72 ± 0.18
50.11 ± 0.22
54.33 ± 0.29
27.71 ± 0.13
60.11 ± 0.09
64.77 ± 0.10
35.10 ± 0.26
78.43 ± 0.16
-
72.25 ± 0.12
81.18 ± 0.04
39.25 ± 0.19
49.15 ± 0.24
54.26 ± 0.10
26.87 ± 0.30
60.80 ± 0.28
65.74 ± 0.17
33.68 ± 0.12
78.07 ± 0.22
86.63 ± 0.31
45.15 ± 0.15
51.79 ± 0.27
57.77 ± 0.34
28.83 ± 0.08
63.33 ± 0.11
68.20 ± 0.09
38.23 ± 0.21
79.57 ± 0.12
-
75.20 ± 0.06
85.29 ± 0.07
42.47 ± 0.22
51.55 ± 0.15
56.49 ± 0.04
28.11 ± 0.29
63.07 ± 0.18
67.13 ± 0.07
36.05 ± 0.11
80.59 ± 0.21
89.36 ± 0.19
48.23 ± 0.08
55.25 ± 0.27
61.41 ± 0.29
34.21 ± 0.05
65.21 ± 0.07
72.46 ± 0.12
40.45 ± 0.15
84.39 ± 0.13
-
8a
8b
31.64 ±0.31
117.73 ±0.51
76.29 ± 0.17
47.90 ± 0.63
177.87 ± 0.61
43.76 ± 0.65
40.25 ± 0.45
138.69 ± 0.24
33.26 ± 0.46
29.90 ± 0.97
103.66 ± 0.80
49.89 ± 0.69
46.01 ± 0.57
146.15 ± 0.40
41.59 ± 0.87
38.59 ± 0.34
123.60 ± 0.43
31.87 ± 0.84
-
8c
9a
9b
9c
10a
10b
10c
11a
11b
11c
12a
12b
12c
13a
13b
13c
Ascorbic acid
Blank
(-) No activity, (±) SD
This article is protected by copyright. All rights reserved.

Table 3 In vitro antioxidant activity of 8-13 by H2O2 method.
IC50 (μg/mL)
Concentration (μg/mL)
Compound
50
75
100
Mean ± SD
67.59 ± 0.21
Mean ± SD
71.92 ± 0.25
Mean ± SD
74.13 ± 0.31
Mean ± SD
36.98 ± 0.30
8a
8b
77.11 ± 0.28
36.25 ± 0.30
43.17 ± 0.23
48.32 ± 0.10
23.13 ± 0.21
54.13 ± 0.34
58.60 ± 0.21
29.63 ± 0.15
73.13 ± 0.08
79.76 ± 0.21
40.03 ± 0.27
46.76 ± 0.22
50.08 ± 0.07
25.84 ± 0.23
56.08 ± 0.31
61.31 ± 0.29
31.27 ± 0.16
76.42 ± 0.08
-
79.37 ± 0.18
39.00 ± 0.05
45.85 ± 0.06
51.25 ± 0.21
25.58 ± 0.10
57.32 ± 0.09
60.03 ± 0.16
32.15 ± 0.13
76.08 ± 0.04
82.15 ± 0.14
42.50 ± 0.15
48.20 ± 0.22
53.75 ± 0.23
29.58 ± 0.08
59.28 ± 0.11
65.36 ± 0.04
33.15 ± 0.21
78.10 ± 0.30
-
82.12 ± 0.22
43.30 ± 0.31
49.22 ± 0.38
55.09 ± 0.07
29.97 ± 0.32
60.15 ± 0.11
66.93 ± 0.07
35.44 ± 0.49
78.26 ± 0.30
85.56 ± 0.23
46.98 ± 0.10
51.32 ± 0.26
57.22 ± 0.08
30.34 ± 0.09
63.77 ± 0.37
68.47 ± 0.40
37.38 ± 0.31
80.29 ± 0.19
-
32.42 ± 0.41
115.47±0.54
101.58 ± 0.45
73.17 ± 0.28
166.83 ± 0.51
46.18 ± 0.69
42.66 ± 0.14
141.08 ± 0.90
34.18 ± 0.84
31.34 ± 0.78
106.42 ± 0.60
97.42 ± 0.96
49.92 ± 0.72
164.79 ± 0.66
44.57 ± 0.62
40.77 ± 0.35
133.76 ± 0.55
32.71 ± 0.21
-
8c
9a
9b
9c
10a
10b
10c
11a
11b
11c
12a
12b
12c
13a
13b
13c
Ascorbic acid
Blank
(-) No activity, (±) SD
This article is protected by copyright. All rights reserved.