Synthesis and evaluation of antioxidant properties of novel 1,2,4-triazole-based schiff base heterocycles

Article abstract of DOI:10.1002/ardp.201300202

A series of 1,2,4-triazole-based Schiff base heterocyclic compounds (5a-f and 8a-i) and phenethylamines (7a-h) were synthesized and evaluated for antioxidant properties by free-radical scavenging, anti-hemolytic activity, lipid peroxidation, and their protective effects against DNA oxidative damage. Compounds 7c, 7d, 7h, 8b, and 8i showed promising DPPH^? radical scavenging activity with the level of inhibition between 86.8% and 94%. Compounds 8a, 8b, 8d, 8g, and 8i were effective against the oxidative hemolysis of human erythrocytes and lipid peroxidation, in a dose-dependent manner, with IC₅₀ values in the range of 55.7-80.7 and 53.2-81.2 μg/mL, respectively. Compounds 8a and 8b were effective against oxidative damage on erythrocyte ghost membrane proteins, and 8g and 8i were able to protect against DNA oxidative damage. 1,2,4-Triazole based Schiff base heterocycles (5a-f, 8a-i) and phenethylamines (7a-h) were synthesized and evaluated for their antioxidant properties by free-radical scavenging, anti-hemolytic activity, lipid peroxidation, and their protective effects against DNA oxidative damage.

Full text of DOI:10.1002/ardp.201300202

922
Arch. Pharm. Chem. Life Sci. 2013, 346, 922–930
Full Paper
Synthesis and Evaluation of Antioxidant Properties of Novel
1,2,4-Triazole-Based Schiff Base Heterocycles
Chandrashekar Aswathanarayanappa1,2, Eswarappa Bheemappa3, Yadav D. Bodke1,
Peethambar S. Krishnegowda4, Srinivas P. Venkata2, and Raghu Ningegowda2
1
Department of Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta, Shimoga,
Karnataka, India
Biocon Ltd., Bangalore, Karnataka, India
Department of PG Studies and Research in Industrial Chemistry, Sir M. V. Government Science College,
2
3
Bommanakatte, Bhadravathi, Karnataka, India
Department of Biochemistry, Kuvempu University, Shankaraghatta, Shimoga, Karnataka, India
4
A series of 1,2,4-triazole-based Schiff base heterocyclic compounds (5a–f and 8a–i) and phenethyl-
amines (7a–h) were synthesized and evaluated for antioxidant properties by free-radical scavenging,
anti-hemolytic activity, lipid peroxidation, and their protective effects against DNA oxidative damage.
Compounds 7c, 7d, 7h, 8b, and 8i showed promising DPPH• radical scavenging activity with the level
of inhibition between 86.8% and 94%. Compounds 8a, 8b, 8d, 8g, and 8i were effective against the
oxidative hemolysis of human erythrocytes and lipid peroxidation, in a dose-dependent manner, with
IC50 values in the range of 55.7–80.7 and 53.2–81.2 mg/mL, respectively. Compounds 8a and 8b were
effective against oxidative damage on erythrocyte ghost membrane proteins, and 8g and 8i were
able to protect against DNA oxidative damage.
Keywords: Anti-hemolytic / Antioxidant / Lipid peroxidation / Schiff base / 1,2,4-Triazole
Received: May 27, 2013; Revised: July 27, 2013; Accepted: August 7, 2013
DOI 10.1002/ardp.201300202
Introduction
The ambient nucleophilic centers present in 3-substituted-4-
amino-5-mercapto-1,2,4-triazoles render them useful syn-
thons for the synthesis of various N-bridged heterocycles.
1,2,4-Triazole, 1,3,4-thiadiazole, and thiadiazine are explored
to the maximum extent owing to their wide spectrum of
pharmacological activities such as antibacterial, anti-fungal
[2, 3], antitubercular [4], anticancer [5], anticonvulsant [6], anti-
inflammatory [7, 8], analgesic [9, 10], antitumor [11], mollus-
cicidal [8, 12], and antiviral [11, 13] activities. Among the
pharmacological profiles of 1,2,4-triazoles, their antimicrobial,
anticonvulsant, and antidepressant properties have been best
documented. Recently, some benzoxazoline, trisubstituted
triazoles, and 4-benzylidenamino derivatives containing tri-
azole and thiadiazole units have been found to be endowed
with excellent free radical scavenging activities [14–16].
Prompted by these observations and in continuation of our
research for biologically active heterocyclic compounds [17], it
was contemplated to synthesize some N-bridged heterocycles
containing 2-methylphenyl and 5-chloro-2-methylphenyl moie-
ties with a view to explore their potency as better chemothera-
peutic agents.
The compounds containing 1,2,4-triazole nucleus represent
an important class of heterocyclic compounds and their
derivatives are characterized with a broad spectrum of
biological activity. Structurally triazole nucleus forms a part
of a wide variety of therapeutically active drug candidates,
including H1/H2 histamine receptor blockers, cholinesterase
active agents, CNS stimulants, antianxiety, and sedative
agents [1]. Many 1,2,4-triazole and 1,3,4-thiadiazole deriva-
tives have been used as “privileged” scaffolds to produce active
pharmaceutical ingredients. Some of the modern-day drugs
like ribavirin (antiviral agent), alprazolam (anxiolytic agent),
fluconazole, itraconazole (anti-fungal agents), and rizatriptan
(antimigrane agent) have triazole nucleus in their structure.
Correspondence: Eswarappa Bheemappa, Department of PG Studies
and Research in Industrial Chemistry, Sir M. V. Government Science
College, Bommanakatte, Bhadravathi 577301, Karnataka, India.
E-mail: eswarappa.bheemappa@gamil.com
Fax: þ91 2808 2303
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Arch. Pharm. Chem. Life Sci. 2013, 346, 922–930
Antioxidant Properties of 1,2,4-Triazole-Based Heterocycles
923
Results and discussion
potassium hydroxide and ethanol under reflux conditions,
resulted in (E)-N-(benzylidene)-3-(phenethylthio)-5-o-tolyl-4H-
1,2,4-triazole-4-amines (7a–h) (Scheme 1). The compounds
5a, 5c, 5d, 5e, 5g, and 5h have been previously reported in the
literature [18].
Chemistry
For the synthesis of title compounds, 4-amino-5-(2-methyl
phenyl)-4H-1,2,4-triazole-3-thiol (4) or 4-amino-5-(5-chloro-2-
methyl phenyl)-4H-1,2,4-triazole-3-thiol (4a) required as start-
ing material was prepared according to a method that
involves the condensation of 2-methylbenzohydrazide (2) or
5-chloro-2-methylbenzohydrazide (2a) with carbon disulfide
and potassium hydroxide to yield potassium dithiocarbazate
3 or 3a, which underwent ring closure with excess of
hydrazine hydrate to produce aminothiol 4 or 4a in good yield
(79–87%). A new series of Schiff base 4-[(E)-benzylideneamino]-
5-(2-methylphenyl)-4H-1,2,4-triazole-3-thiols (5a–h) and 4-[(E)-
benzylideneamino]-5-(5-chloro-2-methylphenyl)-4H-1,2,4-triazole-
3-thiols (8a–i) were prepared by treating compound 4 or 4a
with equimolar amounts of the appropriate benzaldehyde
derivative in presence of catalytic quantity of sulfuric acid and
ethanol under reflux conditions. The Schiff base (5a–h), upon
further reaction with phenethyl bromide, in the presence of
The structure of synthesized compounds was confirmed by
1
analysis of H and 13C NMR spectra; signal for benzylidene-
amino CH proton of 5a–h, 8a–i, and 7a–h was observed at
around 10.17 ppm; triazolethiol SH proton of 5a–h and 8a–i
was observed at around 14.25 ppm; signals for phenethyl
CH2 proton of 7a–h were observed at around 3.0 ppm.
Pharmacology
Antioxidant activity
DPPH free radical scavenging activity: Newly synthesized
compounds were evaluated for their antioxidant activity by
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method as it
is one of the most effective methods for evaluating the
concentration of radical scavenging materials [19]. The DPPH
radical is a stable free radical and its scavenging activity [20]
S
O
OH
O
O
O
NH
NH2
O
NH
NH S-K+
a
b
d
c
R2
R2
N
R2
R2
,
3 3a
,
,
1 1a
2 2a
N
N
N
N
N
SH
SH
S
f
e
N
N
N
N
N
NH2
R2
H
O
R2
R3
R3
,
4 4a
R3
_
_
R2 = H:
7a h
5a h
R2 = H:
, , ,
1 2 3 4
_
R2 = 4-Cl :
,
,
,
R2 = 4-Cl:
1a 2a 3a 4a
8a i
R3
2,4 - F
5a, 7a, 8a
3 - F - 4 - CF3
4 - OCH3
5f, 7f, 8f
5b, 7b, 8b
5c, 7c, 8c
5d, 7d, 8d
5e, 7e, 8e
2,5 - OH
2 - F
3 - F
5g, 7g, 8g
5h, 7h
8h
4 - CH(CH3)2
H -
4 - OH
8i
5 - Br - 2 - OH
Reagents and conditions: (a) MeOH, H2SO4, reflux, 8–10 h; (b) hydrazine hydrate, EtOH, reflux, 8 h; (c) CS ,
2
KOH, EtOH, RT, 5 h; (d) hydrazine hydrate, reflux, 2–6 h; (e) aldehyde, EtOH, H2SO4, reflux, 5–6 h; (f) phenethyl bromide,
EtOH, KOH, reflux, 4–6 h.
Scheme 1. Synthesis of 4-[(E)-benzylideneamino]-5-(2-methylphenyl)-4H-1,2,4-triazole-3-thiol, 4-[(E)-benzylideneamino]-5-(4-chloro-2-
methylphenyl)-4H-1,2,4-triazole-3-thiol, and (E)-N-benzylidene-3-(phenethylthio)-5-o-tolyl-4H-1,2,4-triazol-4-amine derivatives.
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C. Aswathanarayanappa et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 922–930
was determined by the decrease in absorbance at 517 nm, due
to reduction by the antioxidant or reaction with a radical
species, as shown by the equation
DPPH• þ R• ! DPPH ꢀ R
The results of free radical scavenging activity of the newly
synthesized compounds are given in Table 1. Compounds 7c,
7d, 7h, 8b, and 8i were the strongest free radical scavengers
among the compounds tested, with the level of inhibition of
93.9%, 93.6%, 90.0%, 89.2% and 86.8% respectively, followed by
8c, 8d, 8g, and 8h with the level of inhibition of more than
70%. The structure–activity relationship studies reveal that
the free radical scavenging activity of the triazole derivatives
is due to the presence of fluoro group on 2nd or 3rd position
of the benzylideneamino ring (7c, 7d, 8c, 8d); methoxy or
isopropyl group on the 4th position of benzylideneamino
ring (7g, 7h, 8g); and hydroxyl group on 4th position of
benzylideneamino ring (8b, 8i). Compounds 7c, 7d, and 7h
with SH protected with phenethyl group, and fluoro or
isopropyl group on benzylideneamino ring have shown the
level of inhibition equal to or more than BHT.
Figure 1. In vitro protective effects of 1,2,4-triazole-based Schiff
base heterocycles against H2O2-induced hemolysis of human
erythrocytes.
human erythrocytes. These compounds inhibited the hemo-
lysis of human erythrocytes in a dose-dependent manner
with 79.35% as maximum inhibition of erythrocyte hemolysis
In vitro inhibition of human erythrocyte hemolysis
The red blood cells (RBC) are nucleated with intrinsically poor
repair mechanisms; this nature makes them good models to
test the antioxidant capacity of synthetic and natural
compounds in the presence of an oxidative stimulus [21].
Many synthetic and natural compounds have the capacity to
prevent the hemolysis of erythrocyte by hydrogen peroxide
(H2O2) [22]. We examined the ability of compounds 8a, 8b,
8d, 8g, and 8i to inhibit the H2O2-induced hemolysis of
human erythrocytes. The percentage of hemolysis by these
compounds was found to be in the range of 4–5%, which
was comparable with the control (2.3%). Figure 1 shows
the inhibitory effect of different concentrations of these
compounds (25–100 mg/mL) on H2O2-induced hemolysis of
at 100 mg/mL and showed 50% hemolysis inhibition (IC50
)
at concentrations ranging from 55.7 to 80.7 mg/mL (Table 2).
The butylated hydroxytoluene (BHT) exhibited an IC50
value of 51 mg/mL, which is comparable to that of these
compounds. The structure–activity relationship reveals that
anti-hemolysis property of these compounds is due to the
presence of 2,5-dihydroxy group on benzylideneamino ring
(8b). The IC50 values of 8a and 8g are almost the same. The
level of inhibition of 8d and 8g by DPPH method and
hemolysis inhibition is almost equal in either of the cases.
In vitro lipid peroxidation
Lipid peroxides are the mixture of extremely reactive
products of lipid peroxidation, which is a common process
in all biological systems and has deleterious effects on the cell
membrane and deoxyribonucleic acid (DNA) [23]. Malondial-
dehyde (MDA), one of the major products of lipid peroxidation
has been extensively used as an index for lipid peroxidation
and as a marker for oxidative stress. The reaction of MDA with
thiobarbituric acid (TBA) has been widely adopted as a
sensitive assay method for lipid peroxidation [24]. The results
presented in Fig. 2 indicated that lipid peroxidation could be
effectively inhibited by 8a, 8b, 8d, 8g, and 8i. The IC50 value for
lipid peroxidation inhibition on erythrocyte ghost membrane
was found to be in the range of 53.2–81.2 mg/mL. As we can
see from Table 2, compound 8a showed higher inhibition
(77.38%) at the concentration of 100 mg/mL. Compounds 8b,
8d, and 8g showed relatively equal inhibition on lipid
Table 1. DPPH assay in % of 1,2,4-triazole-based Schiff base
heterocycles.
Level of
Level of
Compounds
inhibition (%)
Compounds
inhibition (%)
5a
5b
5f
7a
7b
7c
7d
7e
7f
16.5
19.8
15.3
8a
8b
8c
8d
8e
60.3
89.2
72.5
74.3
55.4
48.7
74.6
75.8
86.8
90.42
30.19
53.53
93.94
93.58
6.46
18.74
65.13
90.00
8f
8g
8h
8i
7g
7h
BHT
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Antioxidant Properties of 1,2,4-Triazole-Based Heterocycles
925
Table 2. In vitro protective effects of 1,2,4-triazole-based Schiff base heterocycles against H2O2-induced haemolysis of human
erythrocyte and lipid peroxidation on erythrocytes ghost membrane.
Level of inhibition of hemolysis
of human erythrocyte (%)
Level of lipid peroxidation
inhibition (%)
Concentration (mg/mL)
25
50
75
100
IC50
25
50
75
100
IC50
BHT
8a
8b
8d
8g
8i
35.43
25.32
27.52
21.48
18.43
11.3
43.74
39.02
48.52
35.34
37.43
29.76
69.06
57.45
63.17
52.79
57.65
45.83
82.37
73.45
79.35
70.53
76.84
63.17
51
43.65
27.18
24.21
26.31
31.28
15.14
57.82
51.32
42.81
39.03
51.32
32.16
69.43
62.17
61.23
57.38
63.62
47.26
79.37
77.38
76.61
72.13
75.31
60.36
36.1
55.5
60.8
64.6
53.2
81.2
64.3
55.7
70
65.6
80.7
peroxidation than 8i. Increased inhibition of 8a is due to the
presence of 2,4-difluoro on benzylideneamino ring.
than the other tested compounds. Increased inhibition of 8b
is due to the presence of hydroxy group on 2nd and 5th
positions of the benzylideneamino ring.
Evaluation of oxidative damage on erythrocyte ghost
membrane proteins by SDS–PAGE
DNA damage protective activity
The change in the protein pattern of membrane proteins
produced by the oxidative stress was observed by the SDS–
PAGE. The ghost membrane was prepared from the hypotonic
lysis of normal human erythrocytes. Membranes were treated
with H2O2 and with and without the test compounds and
analyzed on SDS–PAGE. The membrane protein bands
diminished after 1 h of incubation with H2O2, and very light
new bands of lower molecular weight were observed in the
SDS–PAGE (Fig. 3, lane 1). The protein bands of lanes 2–6,
which were treated with the test compound, were still clearly
evident in the membranes even after 1 h incubation with
H2O2. The compounds 8a and 8b were able to protect better
The efficiency of newly synthesized compounds in preventing
the oxidative damage of DNA induced by H2O2 was evaluated.
The hydroxyl radical generated by the Fenton’s reaction
damages the plasmid DNA, resulting in cleavage of one of the
phosphodiester chains, and produces a relaxed open circular
or relaxed form of DNA. The open circular form of DNA
indicates the formation of linear form of DNA, which in turn
indicates the double-strand breaking into plasmid DNA [25].
The pUC18 plasmid is mainly in supercoiled form (Fig. 4,
lane 7), and when this plasmid is reacted with Fe2þ and H2O2,
the supercoiled form of DNA is converted into an open
circular and a linear form (lane 1). The open circular
Figure 3. SDS–PAGE of RBC membranes showing protective
effects of 1,2,4-triazole-based Schiff base heterocycles against
H2O2-induced oxidative damage on erythrocyte ghost membrane.
Lane 1: membrane proteins treated with H2O2; Lane 2: membrane
proteins, H2O2 and 8a; Lane 3: membrane proteins, H2O2 and 8d;
Lane 4: membrane proteins, H2O2 and 8g; Lane 5: membrane
proteins, H2O2 and 8i; Lane 6: membrane proteins, H2O2 and
8b; Lane 7: membrane proteins (untreated).
Figure 2. In vitro protective effects of 1,2,4-triazole-based Schiff
base heterocycles against H2O2-induced lipid peroxidation on
erythrocyte ghost membrane.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 922–930
Experimental
Chemistry
1H and 13C NMR spectra were recorded with a Bruker Avance
DPX400 spectrometer operating at 400 MHz, with DMSO-d6 as
solvent and Me4Si as internal standard. The chemical shifts are
expressed as d values in parts per million (ppm) and the coupling
constants (J) are given in Hertz (Hz). Purity of the synthesized
compounds was analyzed by HPLC or UPLC method. Mass spectra
were determined with Agilent LC/MS instrument. Flash column
chromatography was performed with silica gel (230–400 mesh;
Merck), and TLC was carried out on precoated silica plates (kiesel
gel 60 F254, BDH). Melting points were determined in open
capillaries on Buchi melting point apparatus and are uncorrect-
ed. All reagents involved in the experiments are commercially
available and used without further purification. The yields are of
purified compounds and are not optimized.
Figure 4. DNA damage protective activity of 1,2,4-triazole-based
Schiff base heterocycles. Lane 1: DNA þ Fenton’s reagent; Lane 2:
DNA þ 8a þ Fenton’s reagent; Lane 3: DNA þ 8d þ Fenton’s re-
agent; Lane 4: DNA þ 8g þ Fenton’s reagent; Lane 5: DNA þ 8i þ
Fenton’s reagent; Lane 6: DNA þ 8b þ Fenton’s reagent, and
Lane 7: DNA.
and linear forms indicate that the OH˙ free radical is
generated from iron-mediated decomposition of H2O2, which
produces both single-strand and double-strand DNA breaks.
No further fragmentation of linear form was observed in the
presence of test compounds (lanes 2–6). From the figure, it is
evident that compound 8g has higher protective effect than 8i
and is comparable to the blank. These compounds prevent
the reaction of Fe2þ ions with H2O2 and directly quench OH˙
free radical by donating hydrogen atom or electron and
therefore protecting the supercoiled plasmid DNA from
OH˙ radical-dependent strand breaks. The increased inhibi-
tion of 8g and 8i is due to the presence of hydroxyl or methoxy
group on the para position of benzylideneamino ring. The
compounds 8a, 8b, and 8d have less ability to scavenge the
OH˙ free radicals, and circular, linear, and supercoiled forms
of DNA are observed (lanes 2, 3, and 6).
Synthesis of methyl-2-methyl benzoate (1) or 5-chloro-2-
methylphenyl benzoate (1a)
To a stirred solution of 2-methyl benzoic acid (73.45 mmol) in dry
methanol (100 mL), three to four drops of con. H2SO4 were added.
The reaction mixture was refluxed for 8–10 h under nitrogen
atmosphere. The reaction was monitored by TLC. After the
reaction completion, methanol was distilled off and the residue
was dissolved in ethyl acetate (200 mL). The organic layer was
washed with 10% sodium bicarbonate solution (50 mL) and water
(50 mL) followed by saturated sodium chloride solution. The
organic layer was treated with anhydrous sodium sulfate, filtered,
and concentrated under vacuum to afford title compound.
Methyl-2-methyl benzoate, pale yellow oil, % yield: 81.74, MF:
C9H10O2, MW: 150.17; 5-chloro-2-methylphenyl benzoate, % yield:
76.2, MF: C9H9ClO2, MW: 183.67.
Synthesis of 2-methylbenzohydrazide (2) or 5-chloro-2-
methylbenzohydrazide (2a)
To a stirred solution of 2-methyl benzoate (1) or 5-chloro-2-
methylphenyl benzoate (1a) (59.93 mmol) in absolute ethanol
(90 mL) hydrazine hydrate (269.68 mmol) was added. The reaction
mixture was refluxed for about 8 h. The reaction was monitored
by TLC. After the reaction completion, the solvent was distilled
under vacuum, ice water (50 mL) was added, and the mixture was
stirred for 15 min. The solid obtained was filtered and dried under
vacuum to yield 2-methylbenzohydrazide (2) as white solid or 5-
chloro-2-methylbenzohydrazide (2a) as off-white solid. 2: % yield:
88.9. m.p.: 152–154.5°C; MF: C8H10N2O; MW: 150.17; [m/z]þ: 151.2.
Conclusion
To summarize, a series of new substituted 4-[(E)-benzylide-
neamino]-5-(2-methylphenyl)-4H-1,2,4-triazole-3-thiol (5a–h),
4-[(E)-benzylideneamino]-5-(5-chloro-2-methylphenyl)-4H-1,2,4-
triazole-3-thiol (8a–i), and (E)-N-(benzylidene)-3-(phenethyl-
thio)-5-o-tolyl-4H-1,2,4-triazole-4-amines (7a–7h) were syn-
thesized and evaluated for antioxidant property by free
radical scavenging method, anti-hemolytic activity, lipid
peroxidation, and protective effect against DNA oxidative
damage. Some of the substituted (E)-N-(benzylidene)-3-(phen-
ethylthio)-5-o-tolyl-4H-1,2,4-triazole-4-amines showed signifi-
cant DPPH free radical scavenging activity, compounds 7c,
7d, and 7h comparable to the standard (BHT). The compound
8b showed the protective effect against H2O2-induced
hemolysis of human erythrocytes, 8g showed an ability to
inhibit lipid peroxidation effectively, and 8g and 8i were able
to protect against DNA oxidative damage. The results exhibit
that some of the synthesized compounds 7c, 7d, 7h, 8b, 8g,
and 8i are potential antioxidant agents.
Synthesis of 2-(2-methylbenzoyl)hydrazinecarbodithiol
acid potassium salt (3) or 2-(5-chloro-2-methylbenzoyl)-
hydrazinecarbodithiol acid potassium salt (3a)
Potassium hydroxide pellets (106.54 mmol) were dissolved in
40 mL of absolute ethanol. To this solution, 2-methylbenzohy-
drazide (2) or 5-chloro-2-methylbenzohydrazide (2a) (53.27 mmol)
was added followed by carbon disulfide (117.19 mmol) and the
contents were stirred at room temperature for 5 h. The reaction
was monitored by TLC. After the reaction completion, di-
ethylether (100 mL) was added and stirred for 10 min. The solid
form was filtered and dried under vacuum to obtain 2-(2-
methylbenzoyl)hydrazinecarbodithiol acid potassium salt (3) as
white solid or 2-(5-chloro-2-methylbenzoyl)hydrazinecarbodithiol
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Arch. Pharm. Chem. Life Sci. 2013, 346, 922–930
Antioxidant Properties of 1,2,4-Triazole-Based Heterocycles
927
acid potassium salt (3a) as off-white solid. 3: % yield: 85.10; m.p.:
167–170°C; MF: C9H9KN2OS2; MW: 264.42; [m/z]þ: 265.3.
5-(4-Chloro-2-methylphenyl)-4-[(E)-(2,4-difluorobenzyli-
dene)amino]-4H-1,2,4-triazole-3-thiol (8a): White solid, 76%;
m.p.: 155–158°C; 1H NMR: 2.37 (s, 3H), 7.28 (t, J1 ¼ 1.6 Hz, 1H), 7.55
(t, J ¼ 1.9Hz, 1H), 7.57 (d, J ¼ 8.4 Hz, 1H), 7.70 (dd, J1 ¼ 1.6 Hz,
J2 ¼ 8.0 Hz, 1H), 7.85 (s, 1H) 8.03 (d, J¼ 6.8 Hz, 1H), 10.07 (s, 1H), 14.3
(s, 1H); 13C NMR: 168.97, 160.45, 151.67, 151.55, 149.44, 137.93,
133.98, 131.42, 129.87, 128.29, 122.39, 120.49, 112.88, 104.54,
Synthesis of 4-amino-5-(2-methyl phenyl)-4H-1,2,4-
triazole-3-thiol (4) or 4-amino-5-(5-chloro-2-methyl
phenyl)-4H-1,2,4-triazole-3-thiol (4a)
103.84, 22.82; IR cmꢀ1, –SH: 2575, –C N–: 1670, Ar–Me: 2925, C–F:
Hydrazine hydrate (45.38 mmol) was added to compound 3 or 3a
(45.38 mmol) and the contents were refluxed for 2 h. The reaction
was monitored by TLC. After the reaction completion, the
reaction mixture was acidified with con. HCl. The precipitate
was filtered and dried under vacuum to obtain compound 4 or 4a.
4-Amino-5-(2-methyl phenyl)-4H-1,2,4-triazole-3-thiol (4), 8.2 g.
White solid, yield: 87.7%, m.p.: 167–170°C; 1H NMR: 2.27
(s, 3H), 5.49 (bs, 2H), 7.18 (t, J ¼ 7.6 Hz, 1H), 7.30 (t, J ¼ 7.2 Hz,
1H), 7.37 (d, J ¼ 7.2 Hz, 2H), 14.25 (s, 1H); MF: C9H10N4S; MW:
206.3, [m/z]þ: 207.4.
–
–
1200. Anal. calcd. for C16H11ClF2N4S: C, 52.50; H, 3.0; N, 15.31%;
found: C, 52.52; H, 3.03; N, 15.34%. MW: 364.80, [m/z]þ: 365.7.
2-((E)-{[3-(4-Chloro-2-methylphenyl)-5-sulfanyl-4H-1,2,4-
triazol-4-yl]imino}methyl)benzene-1,4-diol (8b): Pale brown
solid, 73%; m.p.: 140–144°C; 1H NMR: 2.36 (s, 3H), 7.19–7.22
(m, 3H), 7.39 (s, 1H), 7.49 (d, J ¼ 2.2 Hz, 1H), 7.62–7.69 (m, 1H), 7.91
(s, 1H), 8.86 (s, 1H), 9.85 (s, 1H), 14.17 (s, 1H); 13C NMR: 159.07,
157.45, 155.97, 153.59, 146.48, 142.93, 141.98, 139.40, 136.07,
135.20, 125.37, 119.49, 116.88, 111.49, 107.54, 21.82; IR cmꢀ1
,
General procedure for the synthesis of benzylideneamine
–
–SH: 2950, –C N–: 1690, Ar–Me: 2895, Ar–OH: 3365. Anal. calcd.
–
derivatives (5a, 5b, and 5f; and 8a–i)
for C16H13ClN4O2S: C, 53.21; H, 3.60; N, 15.52%; found: C, 53.24; H,
3.63; N, 15.55%. MW: 360.81, [m/z]þ: 361.7.
To a stirred solution of compound 4 or 4a (4.84 mmol) in ethanol
(10 mL), benzaldehyde (1 eq.) and two to three drops of con. H2SO4
were added and the contents were refluxed for 5 h. The reaction
was monitored by TLC. After the reaction completion, the solvent
was removed under vacuum. To the residue, 5 mL of ice water was
added, stirred for 5 min, and the precipitated solid was filtered
and dried under vacuum. The compounds were purified by
column chromatography using ethyl acetate and petroleum
ether.
5-(4-Chloro-2-methylphenyl)-4-[(E)-(2-fluorobenzylidene)-
amino]-4H-1,2,4-triazole-3-thiol (8c): Off-white solid, 81%;
m.p.: 158–164°C; 1H NMR: 2.37 (s, 3H), 7.36–7.44 (m, 2H), 7.58
(d, J ¼ 8.4 Hz, 1H), 7.67–7.72 (m, 2H), 7.86 (d, J ¼ 1.6 Hz, 1H), 7.95–
7.99 (m, 1H), 10.12 (s, 1H), 14.32 (s, 1H); 13C NMR: 169.97, 168.45,
161.67, 159.59, 149.48, 137.93, 133.98, 130.40, 129.87, 128.29,
126.39, 120.49, 119.88, 104.54, 103.84, 22.52; IR cmꢀ1, –SH: 2572,
–
–C N–: 1665, Ar–Me: 2920; C–F: 1250. Anal. calcd. for
–
C16H12ClFN4S: C, 55.18; H, 3.44; N, 16.09%; found: C, 55.21; H,
4-[(E)-(2,4-Difluorobenzylidene)amino]-5-(2-methyl-
3.41; N, 16.11%. MW: 346.81, [m/z]þ: 347.9.
phenyl)-4H-1,2,4-triazole-3-thiol (5a): Yellow solid, 76%;
m.p.: 119–121.5°C; 1H NMR: 2.27 (s, 3H), 7.18 (t, J ¼ 7.6 Hz, 1H),
7.30 (t, J ¼ 7.2 Hz, 1H), 7.37 (d, J ¼ 7.2 Hz, 1H), 7.45 (q, J ¼ 8 Hz, 3H),
7.78 (q, J ¼ 7.6 Hz, 1H), 10.17 (s, 1H), 14.27 (s, 1H); 13C NMR: 156.87,
138.28, 131.40, 131.15, 130.91, 129.49, 129.39, 126.16, 113.83,
113.60, 105.57, 105.31, 20.14. Anal. calcd. for C16H12F2N4S: C,
58.12; H, 3.63; N, 8.47%; found: C, 58.30; H, 3.65; N, 8.49%. MW:
330.35, [m/z]þ: 331.6.
5-(4-Chloro-2-methylphenyl)-4-[(E)-(3-fluorobenzylidene)-
amino]-4H-1,2,4-triazole-3-thiol (8d): Yellow solid, 75%; m.p.:
147–152°C; 1H NMR: 2.36 (s, 3H), 7.46–7.50 (m, 1H), 7.57–7.64
(m, 2H), 7.68–7.73 (m, 3H), 7.85 (s, 1H), 9.80 (s, 1H), 14.31 (s, 1H);
13C NMR: 163.67, 162.45, 160.67, 159.59, 146.48, 137.93, 135.98,
130.40, 129.87, 127.29, 126.39, 120.49, 116.88, 104.54, 103.84,
22.12; IR cmꢀ1, –SH: 2542, –C N–: 1675, Ar–Me: 2930, C–F: 1300.
–
–
Anal. calcd. for C16H12ClFN4S: C, 55.18; H, 3.44; N, 16.09%; found:
2-[(E)-{[3-(2-Methylphenyl)-5-sulfanyl-4H-1,2,4-triazol-4-
C, 55.20; H, 3.42; N, 16.10%. MW: 346.81, [m/z]þ: 347.8.
yl]imino}methyl]benzene-1,4-diol (5b): Pale yellow solid,
72%; m.p.: 145–147°C; 1H NMR: 2.24 (s, 3H), 6.91–6.97 (m, 1H),
7.34 (t, J ¼ 7.2 Hz, 1H), 7.38 (d, J ¼ 7.6 Hz, 1H), 7.41–7.46 (m, 2H),
7.51 (dd, J1 ¼ 2.4 Hz, J2 ¼ 6.2 Hz, 1H), 7.61 (d, J ¼ 2.8 Hz, 1H), 10.78
(s, 1H), 10.83 (s, 1H), 11.2 (s, 1H), 14.25 (s, 1H). Anal. calcd. for
4-Bromo-2-[(E)-{[3-(4-chloro-2-methylphenyl)-5-sulfanyl-
4H-1,2,4-triazol-4-yl]imino}methyl]phenol (8e): Pale yellow
solid, 80%; m.p.: 139–143°C; 1H NMR: 2.37 (s, 3H), 6.93 (t,
J ¼ 5.2 Hz, 1H), 7.51–7.61 (m, 3H), 7.87–7.90 (m, 2H), 10.05 (s, 1H),
10.90 (s, 1H), 14.22 (s, 1H); 13C NMR: 167.07, 165.45, 164.97,
157.59, 156.48, 142.93, 141.98, 139.40, 136.07, 127.29, 125.37,
C
16H14N4O2S: C, 58.82; H, 4.28; N, 17.15%; found: C, 58.84; H, 4.30;
N, 17.18%. MW: 326.37, [m/z]þ: 327.5.
119.49, 113.88, 111.44, 107.54, 22.42; IR cmꢀ1, –SH: 2740, –C N–:
–
–
4-{(E)-[3-Fluoro-4-(trifluoromethyl)benzylidene]amino}-5-
1595, Ar–Me: 2893, Ar–OH:3350; C–Br: 580. Anal. calcd. for
(2-methylphenyl)-4H-1,2,4-triazole-3-thiol (5f): Pale yellow
solid, 71%; m.p.: 131.5–134°C; 1H NMR: 2.21 (s, 3H), 7.31
(t, J ¼ 7.6 Hz, 1H), 7.37 (d, J ¼ 7.6 Hz, 1H), 7.43–7.48 (m, 2H),
7.73–7.80 (m, 2H), 7.91 (t, J ¼ 8 Hz, 1H), 10.13 (s, 1H), 14.34 (s, 1H);
13C NMR: 162.19, 161.47, 149.80, 138.29, 131.34, 131.25, 130.98,
128.98, 128.93, 126.23, 125.29, 116.79, 116.57, 20.14. Anal. calcd.
for C17H12F4N4S: C, 53.63; H, 3.15; N, 14.72%; found: C, 53.65;
H, 3.18; N, 14.73%. MW: 380.36, [m/z]þ H2O: 399.5.
C
16H12BrClN4OS: C, 45.32; H, 2.83; N, 13.21%; found: C, 45.35; H,
2.86; N, 13.25%. MW: 423.71, [m/z]þ: 424.65.
5-(4-Chloro-2-methylphenyl)-4-{(E)-[2-fluoro-4-(trifluoro-
methyl)benzylidene]amino}-4H-1,2,4-triazole-3-thiol (8f):
Pale yellow solid, 77%; m.p.: 120–123°C; 1H NMR: 2.37 (s, 3H),
7.56–7.60 (m, 1H), 7.70 (dd, J1 ¼ 1.7 Hz, J2 ¼ 8.2 Hz, 1H), 7.78
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928
C. Aswathanarayanappa et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 922–930
(d, J ¼ 1.6 Hz, 1H), 7.83–8.06 (m, 3H), 10.06 (s, 1H), 14.37 (s, 1H);
13C NMR: 160.67, 155.45, 149.67, 137.59, 135.48, 132.93, 131.98,
128.40, 128.07, 127.29, 126.39, 120.49, 112.88, 104.54, 103.84,
–
C–F: 1270. Anal. calcd. for C17H11ClF4N4S: C, 49.07; H, 2.64; N,
13.47%; found: C, 49.10; H, 2.67; N, 13.50%. MW: 414.80, [m/z]þ:
415.7.
(t, J ¼ 7.3 Hz, 2H), 6.90–7.81 (m, 11H), 8.02 (s, 1H), 8.49 (s, 1H). Anal.
calcd. for C24H20F2N4S: C, 66.28; H, 4.60; N, 12.88%; found: C,
66.32; H, 4.65; N, 12.92%. MW: 434.50, [m/z]þ: 435.0.
102.11, 21.42; IR cmꢀ1, –SH: 2540; –C N–: 1575; Ar–Me: 2830;
–
(E)-2-((3-(Phenethylthio)-5-o-tolyl-4H-1,2,4-triazol-4-yl-
imino)methyl)benzene-1,4-diol (7b): Pale yellow solid, 64%;
m.p.: 215–217°C; 1H NMR: 2.40 (s, 3H), 3.1 (t, J ¼ 7.4 Hz, 2H), 3.28
(t, J ¼ 7.2 Hz, 2H), 7.10–7.93 (m, 11H), 8.12 (s, 1H), 8.5 (s, 1H), 10.26
(s, 1H), 10.29 (s, 1H). Anal. calcd. for C24H22N4O2S: C, 66.89; H,
5.11; N, 13.00%; found: C, 66.93; H, 5.15; N, 13.04%. MW: 430.52,
[m/z]þ: 431.8.
5-(4-Chloro-2-methylphenyl)-4-[(E)-(4-methoxybenzyli-
dene)amino]-4H-1,2,4-triazole-3-thiol (8g): Pale brown solid,
76%; m.p.: 159–163°C; 1H NMR: 2.37 (s, 3H), 3.81 (s, 3H), 7.03–7.12
(m, 3H), 7.56 (d, J ¼ 8.4Hz, 1H), 7.68 (d, J ¼ 0.8 Hz, 1H), 7.78–7.86
(m, 2H), 9.5 (s, 1H), 14.22 (s, 1H); 13C NMR: 168.57, 165.45, 164.67,
157.59, 156.48, 142.93, 141.98, 137.40, 129.07, 127.29, 125.39,
119.49, 112.88, 110.44, 107.54, 55.84, 22.42; IR cmꢀ1, –SH: 2640,
–
(E)-N-(2-Fluorobenzylidene)-3-(phenethylthio)-5-o-tolyl-
4H-1,2,4-triazole-4-amine (7c): Off-white solid, 61%; m.p.:
198–201°C; 1H NMR: 2.38 (s, 3H), 3.08 (t, J ¼ 7.3 Hz, 2H), 3.31
(t, J ¼ 7.3 Hz, 2H), 7.06–7.84 (m, 12H), 8.12 (s, 1H), 8.42 (s, 1H). Anal.
calcd. for C24H21FN4S: C, 69.14; H, 5.04; N, 13.44%; found: C, 69.18;
H, 5.08; N, 13.47%. MW: 416.51, [m/z]þ: 417.3.
–C N–: 1565, Ar–Me: 2813, Ar–MeO: 1150. Anal. calcd. for
–
C17H15ClN4OS: C, 56.72; H, 4.17; N, 15.57%; found: C, 56.75; H,
4.20; N, 15.60%. MW: 358.84, [m/z]þ: 359.6.
4-[(E)-Benzylideneamino]-5-(4-chloro-2-methylphenyl)-
(E)-N-(3-Fluorobenzylidene)-3-(phenethylthio)-5-o-tolyl-
4H-1,2,4-triazole-3-thiol (8h): Off-white solid, 70%; m.p.: 125–
129°C; 1H NMR: 2.36 (s, 3H), 7.57 (dd, J1 ¼ 1.6 Hz, J2 ¼ 8.2 Hz, 2H),
7.61–7.63 (m, 1H), 7.70 (dd, J1 ¼ 2 Hz, J2 ¼ 8.4 Hz, 2H), 7.87
(d, J ¼ 0.8 Hz, 2H), 7.89 (s, 1H), 9.71 (s, 1H), 14.28 (s, 1H);
13C NMR: 167.57, 155.45, 149.67, 137.59, 135.48, 133.93, 131.98,
128.40, 129.07, 127.29, 126.39, 119.49, 112.88, 107.54, 102.84,
4H-1,2,4-triazole-4-amine (7d): Off-white solid, 67%; m.p.:
171–173.5°C; 1H NMR: 2.41 (s, 3H), 3.12 (t, J ¼ 7.2 Hz, 2H), 3.34
(t, J ¼ 7.4 Hz, 2H), 7.07–7.92 (m, 12H), 8.09 (s, 1H), 8.39 (s, 1H);
13C NMR: 164.00, 163.65, 161.22, 161.15, 131.92, 131.83, 131.67,
131.57, 131.49, 130.87, 130.89, 130.79, 130.73, 129.29, 129.21,
129.02, 128.84, 128.54, 127.69, 118.46, 114.57, 36.21, 28.34, 19.8.
Anal. calcd. for C24H21FN4S: C, 69.14; H, 5.04; N, 13.44%; found: C,
69.19; H, 5.08; N, 13.48%. MW: 416.51, [m/z]þ: 417.1.
22.42; IR cmꢀ1, –SH: 2640, –C N–: 1565, Ar–Me: 2813. Anal. calcd.
–
–
for C16H13ClN4S: C, 58.23; H, 3.94; N, 6.98%; found: C, 58.25; H,
3.97; N, 6.70%. MW: 328.82, [m/z]þ: 329.7.
(E)-2-((3-(Benzylthio)-5-o-tolyl-4H-1,2,4-triazol-4-ylimino)
4-[(E)-{[3-(4-Chloro-2-methylphenyl)-5-sulfanyl-4H-1,2,4-
methyl)-4-bromo-phenol (7e): Pale yellow solid, 58%; m.p.:
129–133°C; 1H NMR: 2.27 (s, 3H), 3.01 (t, J ¼ 7.3 Hz, 2H), 4.84
(t, J ¼ 7.3 Hz, 2H), 6.70–7.94 (m, 11H), 7.98 (s, 1H), 8.32 (s, 1H),
10.26 (s, 1H); 13C NMR: 162.55, 156.01, 137.71, 137.25, 130.91,
130.24, 129.29, 129.18, 128.78, 128.72, 128.67, 128.53, 127.99,
127.75, 127.51, 126.42, 126.34, 126.11, 122.34, 119.68, 114.45,
37.01, 19.88. Anal. calcd. for C23H19BrN4OS: C, 57.57; H, 3.96; N,
11.68%; found: C, 57.60; H, 3.99; N, 11.70%. MW: 479.39, [m/z]þ:
480.9.
triazol-4-yl]imino}methyl]phenol (8i): Yellow solid, 81%; m.p.:
135–139°C; 1H NMR: 2.35 (s, 3H), 6.83–6.93 (m, 3H), 7.54
(d, J ¼ 4.8 Hz, 1H), 7.66–7.75 (m, 3H), 7.86 (s, 1H), 9.38 (s, 1H),
14.19 (s, 1H); 13C NMR: 157.07, 155.45, 154.97, 147.59, 146.48,
142.93, 140.98, 139.40, 136.07, 127.20, 125.37, 119.49, 116.88,
–
2890; Ar–OH: 3370. Anal. calcd. for C16H13ClN4OS: C, 55.68; H,
3.77; N, 16.24%; found: C, 55.70; H, 3.80; N, 16.25%. MW: 344.81,
[m/z]þ: 345.75.
111.49, 107.54, 21.72; IR cmꢀ1, –SH: 2940; –C N–: 1695; Ar–Me:
–
General procedure for the synthesis of (E)-N-(2-
benzylidene)-3-(phenethylthio)-5-o-tolyl-4H-1,2,4-
triazole-4-amine derivatives (7a–7h)
(E)-N-(3-Fluoro-4-(trifluoromethyl)benzylidene)-3-(phen-
ethylthio)-5-o-tolyl-4H-1,2,4triazole-4-amine (7f): Off-white
solid, 65%; m.p.: 204–206°C; 1H NMR: 2.41 (s, 3H), 3.02
(t, J ¼ 7.4 Hz, 2H), 3.33 (t, J ¼ 7.4 Hz, 2H), 6.94–7.84 (m, 11H),
7.96 (s, 1H), 8.39 (s, 1H). Anal. calcd. for C25H20F4N4S: C, 61.91; H,
4.12; N, 11.55%; found: C, 61.95; H, 4.15; N, 11.59%. MW: 484.51,
[m/z]þ: 485.8.
To a stirred solution of 5 (1 eq.) in absolute ethanol (10 mL),
potassium hydroxide (2.5 eq.) was added followed by phenethyl-
bromide (1 eq.). The reaction mixture was refluxed for 4 h and
the reaction was monitored by TLC. After the completion of
reaction, solvent was distilled off and the product from the
residue was extracted using ethyl acetate (25 mL). The organic
layer was washed with water and saturated sodium chloride
solution, dried with anhydrous sodium sulfate, and concentrated
under vacuum to afford title compounds. These compounds were
purified by column chromatography using ethyl acetate and
petroleum ether.
(E)-N-(4-Methoxybenzylidene)-3-(phenethylthio)-5-o-tolyl-
4H-1,2,4-triazol-4-amine (7g): Pale yellow solid, 61%; m.p.:
199–201°C; 1H NMR: 2.39 (s, 3H), 3.09 (t, J ¼ 7.0 Hz, 2H), 3.41
(t, J ¼ 7.1 Hz, 2H), 3.96 (s, 3H), 7.01–7.67 (m, 12H), 8.02 (s, 1H), 8.46
(s, 1H); 13C NMR: 160.79, 155.34, 145.45, 140.53, 139.86, 132.23,
132.09, 132.01, 131.12, 131.02, 129.54, 128.43, 127.78, 126.34,
125.65, 124.54, 123.87, 122.65, 120.98, 115.98, 114.87, 55.26,
36.21, 28.34, 19.89. Anal. calcd. for C25H24N4OS: C, 70.0; H, 5.60; N,
13.06%; found: C, 70.05; H, 5.64; N, 13.10%. MW: 428.55, [m/z]þ:
429.6.
(E)-N-(2,4-Difluorobenzylidene)-3-(phenethylthio)-5-o-
tolyl-4H-1,2,4-triazole-4-amine (7a): Pale yellow solid, 57%;
m.p.: 209–211°C; 1H NMR: 2.39 (s, 3H), 3.07 (t, J ¼ 7.2 Hz, 2H), 3.44
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Arch. Pharm. Chem. Life Sci. 2013, 346, 922–930
Antioxidant Properties of 1,2,4-Triazole-Based Heterocycles
929
(E)-N-(4-Isopropoxybenzylidene)-3-(phenethylthio)-5-o-
540 nm by spectrophotometer to determine the hemolysis.
Likewise, the erythrocytes were treated with 100 mM H2O2
without inhibitors (test compounds) to obtain a complete
hemolysis. The absorbance of the supernatant was measured
under the same condition. The inhibitory effect of these
compounds was compared with that of standard antioxidant
BHT. To evaluate the hemolysis induced by the test compounds,
erythrocytes were pre-incubated with 50 mL of test compound for
1 h and the hemolysis was determined. The percentage of
hemolysis was calculated by taking hemolysis caused by 100 mM
H2O2 as 100%. The IC50 values were calculated from the plots as
the antioxidant concentration required for the inhibition of 50%
hemolysis.
tolyl-4H-1,2,4-triazol-4-amine (7h): Off-white solid, 67%;
m.p.: 162–166°C; 1H NMR: 1.1 (d, J ¼ 2.4 Hz, 6H), 2.38 (s, 3H),
3.07 (t, J ¼ 4.6 Hz, 2H), 3.36 (q, J ¼ 2 Hz, 2H), 4.1 (m, 1H), 7.21–7.91
(m, 12H), 8.09 (s, 1H), 8.41 (s, 1H). Anal. calcd. for C27H28N4S: C,
73.53; H, 6.35; N, 12.70%; found: C, 73.57; H, 6.39; N, 12.75%. MW:
440.60, [m/z]þ: 441.6.
Pharmacology
Antioxidant activity
DPPH free radical scavenging activity: Free radical-
scavenging capacities of test compounds were determined
according to the previously reported procedure [26], using the
stable DPPH radical. This is the most commonly used method for
screening of antioxidant activity of newly synthesized organic
compounds. This method is based on the reduction of free radical
DPPH by free radical scavengers. The procedure involves the
measurement of decrease in absorbance of DPPH [27] at 517 nm,
which is proportional to the activity of free radical scavenger
added to DPPH reagent solution. A stock solution of test
compounds (1 mg/mL) and DPPH (0.004%) was prepared in 95:5
methanol/water. To 3 mL of freshly prepared DPPH solution in a
test tube, stock solution of test compound (100 mL) was added and
reacted for 15 min, and the absorbance was measured at 517 nm
using UV–Visible spectrophotometer (Shimadzu UV-1800, Japan).
BHT was used as a reference standard and dissolved in distilled
water to make the stock solution with the same concentration
(1 mg/mL). Ascorbic acid was used as control sample, and 95%
methanol served as blank. Free radical inhibition in % (I%) was
calculated as
In vitro assay for the inhibition of lipid peroxidation on
erythrocyte ghost membrane
The erythrocyte ghost membranes were prepared following
the procedure of Fairbanks et al. [28] by hypotonic lysis of
erythrocytes in 5 mM phosphate buffer (pH 8.0) with the addition
of 1 mM EDTA to the lysis buffer. Protein content in the
membrane was determined according to the method described
by Lowry et al. [29] using bovine serum albumin as standard.
The erythrocyte ghost membrane lipid peroxidation was
performed according to the method of Stocks and
Dormandy [30] with slight modifications. The test compound
(25–100 mg concentration) was added to 1 mL of erythrocyte ghost
membrane suspension (200 mg protein) followed by 100 mL of
100 mM H2O2. After incubation for 1 h at 37°C, the reaction was
stopped by adding 2 mL of 0.25 M HCl containing 15% trichloro-
acetic acid and 0.375% TBA. The reaction mixture was boiled for
15 min, cooled, and centrifuged, and the absorbance of the
supernatant was measured at 532 nm. The appropriate blanks
and controls were run along with the test samples. The
percentage of inhibition was calculated and plotted against
the concentration of the samples. The IC50 values were calculated
from the plots as the antioxidant concentration required for the
inhibition of 50% lipid peroxidation.
Ablank ꢀ Asample
I% ¼
ꢁ 100
Ablank
where Ablank is the absorbance of the control reaction (containing
all reagents except the test compound) and Asample is the
absorbance of the test sample.
Hemolytic activity
Evaluation of oxidative damage on erythrocyte ghost
RBC suspension preparation: Blood was collected through
vein puncture from fully informed and consenting healthy
volunteers, into tubes containing ethylenediaminetetraacetic
acid (EDTA) as anticoagulant. Samples were centrifuged at
2000 rpm, for 5 min at 4°C. Plasma and buffy coat was then
carefully removed and discarded; RBCs were washed three times
with saline phosphate buffer (PBS: 125 mM NaCl and 10 mM
sodium phosphate, pH 7.4), and finally re-suspended in PBS to
produce an RBC suspension at 2% v/v hematocrit.
membrane proteins by SDS–PAGE
The oxidative modification on erythrocyte ghost membrane
proteins was determined by SDS–PAGE according to the method
described by Carini et al. [31] and Ajila et al. [32] with slight
modifications. The oxidation of membrane protein was induced
by addition of 100 mL of 200 mM H2O2 to 1 mL of solution
containing 200 mg of protein and with or without test compound
(100 mg of AAE) and incubated for 1 h. Appropriate controls were
run along with the test samples, SDS–PAGE was performed on
10% discontinuous gel according to the method of Laemmli [33].
The protein bands were visualized by staining with Coomassie
brilliant blue.
In vitro inhibition of human erythrocyte hemolysis: The
inhibition of human erythrocyte hemolysis by newly synthesized
compounds was evaluated according to the procedure described
by Tedesco et al. [22] with due modifications. The human
erythrocyte hemolysis was performed with H2O2 as a free radical
initiator. To 100 mL of 2% v/v suspension of erythrocytes in PBS,
100 mL of test compound with different concentrations (25, 50,
75, and 100 mg/mL in PBS, pH 7.4) was added followed by 100 mM
H2O2 (in PBS, pH 7.4). The reaction mixture was shaken gently
while being incubated at 37°C for 3 h. Then, it was diluted with
8 mL of PBS and centrifuged at 3000 rpm for 10 min. The
absorbance of the resulting supernatant liquid was measured at
DNA damage protective activity
Induction of DNA scission by Fenton’s reagent was measured on
pUC18 DNA according to the procedure described by Lee et al. [34]
with slight modifications. A mixture of 10 mL of synthesized
compound (concentration 1 mg/mL) and 0.5 mg of DNA was
incubated for 10 min at 37°C followed by addition of 10 mL of
Fenton’s reagent (30 mM of H2O2, 50 mM of ascorbic acid, and
80 mM of FeCl3). The final volume of mixture was made up to
20 mL and the mixture was incubated for 30 min at 37°C. The DNA
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

930
C. Aswathanarayanappa et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 922–930
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The authors thank M/s. Biocon Ltd., Bangalore, India for providing
laboratory facility for necessary laboratory works and analytical
support. We also gratefully acknowledge Dr. Kiran Kumar Kothakonda
and Mr. Arun Sindhe for their support for data analysis and writing
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