Diaryl-1,2,4-oxadiazole antioxidants: Synthesis and properties of inhibiting the oxidation of DNA and scavenging radicals

Article abstract of DOI:10.1016/j.biochi.2012.12.005

Six 1,2,4-oxadiazole derivatives were prepared in order to compare their abilities to protect DNA against radical-mediated oxidation and to scavenge radicals. These derivatives had a structure based on disubstituted 1,2,4-oxadiazole, in which a vanillin group (A ring) and a substituted benzene group (B ring) were the substituents. The functional group at B ring was assigned as ortho- or meta-hydroxylbenzene group, ortho-chlorobenzene group, no group contained, and pyridine group or vanillin group at B ring. It was found that the compound with two vanillin groups attaching to oxadiazole can trap 2.05 radicals in protecting DNA against 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation, and the compound with an ortho-hydroxylbenzene group at B ring can trap 1.78 radicals. The compound with an ortho-chlorobenzene group at B ring exhibited the highest ability to inhibit ^·OH-induced oxidation of DNA, while the compound with a meta-hydroxylbenzene group at B ring inhibited Cu²⁺/glutathione (GSH)-induced oxidation of DNA efficiently. The ortho- and para-hydroxylbenzene groups at B ring made the compounds possess the highest rate constant (k) in scavenging 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+.) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH). Therefore, only a few hydroxyl groups can markedly enhance the activity of the core-branched antioxidant, which may be a novel structural feature in designing antioxidant.

Full text of DOI:10.1016/j.biochi.2012.12.005

Biochimie 95 (2013) 842e849
Contents lists available at SciVerse ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Diaryl-1,2,4-oxadiazole antioxidants: Synthesis and properties
of inhibiting the oxidation of DNA and scavenging radicals
*
Chao Zhao, Zai-Qun Liu
Department of Organic Chemistry, College of Chemistry, Jilin University, No. 2519 Jiefang Road, Changchun 130021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Six 1,2,4-oxadiazole derivatives were prepared in order to compare their abilities to protect DNA against
radical-mediated oxidation and to scavenge radicals. These derivatives had a structure based on disub-
stituted 1,2,4-oxadiazole, in which a vanillin group (A ring) and a substituted benzene group (B ring)
were the substituents. The functional group at B ring was assigned as ortho- or meta-hydroxylbenzene
group, ortho-chlorobenzene group, no group contained, and pyridine group or vanillin group at B ring. It
was found that the compound with two vanillin groups attaching to oxadiazole can trap 2.05 radicals in
protecting DNA against 2,20-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation, and
the compound with an ortho-hydroxylbenzene group at B ring can trap 1.78 radicals. The compound with
an ortho-chlorobenzene group at B ring exhibited the highest ability to inhibit $OH-induced oxidation of
DNA, while the compound with a meta-hydroxylbenzene group at B ring inhibited Cu2þ/glutathione
(GSH)-induced oxidation of DNA efficiently. The ortho- and para-hydroxylbenzene groups at B ring made
the compounds possess the highest rate constant (k) in scavenging 2,20-azinobis(3-ethylbenzothiazoline-
6-sulfonate) cationic radical (ABTSþ.) and 2,20-diphenyl-1-picrylhydrazyl radical (DPPH). Therefore, only
a few hydroxyl groups can markedly enhance the activity of the core-branched antioxidant, which may
be a novel structural feature in designing antioxidant.
Received 13 October 2012
Accepted 1 December 2012
Available online 12 December 2012
Keywords:
1,2,4-Oxadiazole derivatives
Antioxidant
Oxidation of DNA
Free radical
Kinetics
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
[12,13]. We also reported a result on the dendritic antioxidant with
pyrazole being the core and three benzene rings being the branches.
1,2,4-Oxadiazole, an important heterocycle with five members, is
usually found in marine organisms, Phidiana militaris [1], or can be
synthesized via many methods [2]. The derivativesof this oxadiazole
possess various biological activities such as antidiabetic, anti-
inflammatory [3], immunosuppressive [4], antimicrobial [5,6], and
antitumor [7e9]. Especially, 1,2,4-oxadiazole derivatives are bene-
ficial for interacting with DNA [10] and inhibiting bacterial [11]. This
phenomenon gives us an important information that 1,2,4-
oxadiazole may be a powerful core to construct functional groups
in the drug design. Hence, some phenol moieties are connected with
1,2,4-oxadiazole in order to obtain higher bioactivities than phenol
moieties employed alone [9], and phenol-substituted 1,2,4-
oxadiazole implies an attractive direction for the development of
novel antioxidants. In addition, 4-aminomethylbenzylamine and
tris(2-aminoethyl)amine have been used as the core for construct-
ing dendritic antioxidants with polyphenols attaching. The obtained
dendritic antioxidants exhibited relative high abilities to scavenge
radical and to inhibit the oxidation of low density lipoprotein
In the pyrazole-related antioxidant one phenolic hydroxyl group can
play the antioxidative role in protecting DNA against radical-
induced oxidation [14]. This motivated us to test whether few
phenolic hydroxyl groups can also function as active antioxidant
when 1,2,4-oxadiazole is used as the core. As shown in Scheme 1, we
herein designed diaryl-1,2,4-oxadiazole antioxidants, in which
a vanillin group acted as the A ring, and the groups at B ring changed
in order to screen the influence of the various substituents on the
antioxidant effectiveness of the whole molecule of diaryl-
substituted 1,2,4-oxadiazoles. Comparing with the structures of
pyrazole-related antioxidants as we have reported previously [14],
the benefit in the structure of diaryl-1,2,4-oxadiazole antioxidants
was only two benzene rings contained, and the antioxidant activity
will be derived from the interaction between diaryl groups and
1,2,4-oxadiazole moiety.
The radical-mediated oxidation of DNA is related to many
diseases [15], the inhibitive effect on the oxidation of DNA and the
ability to trap radicals are major index to evaluate the capacity of an
antioxidant [16]. Some chemical experimental systems are usually
applied to mimic DNA undergoing radical-induced oxidation,
in which 2,20-azobis(2-amidinopropane hydrochloride) (AAPH,
* Corresponding author. Tel.: þ86 138 4306 7908.
E-mail address: zaiqun-liu@jlu.edu.cn (Z.-Q. Liu).
0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.biochi.2012.12.005

C. Zhao, Z.-Q. Liu / Biochimie 95 (2013) 842e849
843
Scheme 1. Synthetic routine of oxadiazole derivatives.
ReN]NeR, R ¼ eCMe2C(]NH)NH2), Cu2þ/glutathione (GSH),
hydroxyl radical (.OH) act as the radical resources [17e19]. In
addition, the radical-scavenging capacities of antioxidants can be
estimated by reacting with 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) cationic radical (ABTSþ.) [20] and 2,20-diphenyl-1-
picrylhydrazyl radical (DPPH) [21]. Presented here is a study on
the effects of six oxadiazole derivatives on inhibiting Cu2þ/GSH-,
.OH-, and AAPH-induced oxidation of DNA and on trapping ABTSþ.
and DPPH.
a well-stirred ethanolic solution of K2CO3 and refluxed for 4 h. The
mixture was then concentrated in vacuum. The residue was dis-
solved in CH3COOC2H5, filtered, and washed with 10% aqueous
NaOH and saturated NaCl solution for three times. The organic layer
was dried over MgSO4 and concentrated in vacuum. The solids were
recrystallized with ethanol to yield compound 1 as a colorless
crystal (2.23 g, 92%). m.p. 62e63 ꢀC.
The compound 2 was derived from compound 1 treated by I2 in
NH3 aqueous solution. A tetrahydrofuran (15 mL) solution of
compound 1 (2.42 g, 10 mmol) was mixed with I2 (2.79 g, 11 mmol)
in 28% aqueous solution of NH3 (90 mL). The mixture was stirred for
12 h at room temperature and extracted with CHCl3 (3 ꢁ15 mL). The
organic solution was washed with water, 10% aqueous solution of
Na2S2O3, and brine, then dried over MgSO4. The organic solutionwas
removed in vacuum. The residue was recrystallized with ethanol to
yield compound 2 as a colorless crystal (2.27 g, 95%). m.p. 89e90 ꢀC.
The amidoxime was produced from compound 2. The compound
2 (2.39 g, 10 mmol) and hydroxylamine hydrochloride (1.39 g,
20 mmol) were mixed in ethanol (30 mL) under stirring, and 10 mL
of 10% aqueous solution of KOH was added. The mixture was
refluxed for 18 h under stirring and then cooled to room tempera-
ture. The ethanol was removed in vacuum, the residue was dissolved
in CH3COOC2H5, dried over MgSO4, and filtered, and concentrated in
vacuum. The residue was recrystallized with ethanol to afford pure
amidoxime as an off-white powder (2.45 g, 90%). m.p. 162e163 ꢀC.
The hydroxyl group in the reagent for preparing compound 3
should be etherized by reacting with benzyl chloride. The operation
was similar to the treatment of compound 1. Then, the aldehyde
2. Materials and methods
2.1. Materials and instrumentation
Diammonium salt of 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) (ABTS salt), and DPPH were purchased from Fluka
Chemie GmbH, Buchs, Switzerland. AAPH, GSH, and the naked DNA
sodium salt were purchased from Acros Organics, Geel, Belgium.
Other agents were of analytical grade and used directly. The
structures of the obtained compounds were identified by 1H NMR
(Varian Mercury 300 NMR spectrometer).
2.2. Synthesis and structural characterization of oxadiazole
derivatives
The compound 1 can be prepared by the reaction between
vanillin and benzyl chloride. Briefly, a mixture of vanillin (1.52 g,
10 mmol) and benzyl chloride (1.39 g, 11 mmol) was added to

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C. Zhao, Z.-Q. Liu / Biochimie 95 (2013) 842e849
group should be oxidized by the following operation. Briefly, the
aldehyde (2.42 g, 10 mmol), NaH2PO4 (0.36 g, 3 mmol), H2O2 (35%
aqueous solution, 1.07 mL, 11 mmol) were dissolved in 60 mL of
CH3CN and H2O (5:1, v:v). A 15 mL of aqueous solution of NaClO2
(1.27 g, 14 mmol) was added dropwisely at 10 ꢀC. The mixture was
stirred at room temperature for 90 min, then Na2S2O3 (0.22 g,
1.40 mmol) was added. The mixture was stirred for 5 min to
decompose the excess H2O2, diluted by aqueous solution of NaCl
and extracted by CH3COOC2H5 (3 ꢁ 30 mL). The organic layer was
washed by aqueous solutions of NaCl and NaHCO3 in turn. The
alkaline extract was acidified by concentrated HCl and extracted by
CH3COOC2H5. The organic layer was dried over Na2SO4 and evap-
orated to afford compound 3, which was recrystallized with hexane
to give a white solid (2.58 g), yield w100%. m.p. 171e172 ꢀC.
Furthermore, the acyl chloride was prepared by the reaction of
compound 3 with SOCl2. Briefly, the compound 3 (2.58 g, 10 mmol)
was suspended in 50 mL of dichloromethane. After a drop of
(CH3)2NCHO was added, SOCl2 (1.16 mL, 16 mmol) was dropwisely
added. The mixture was stirred at room temperature for 1 h then
heated at 40 ꢀC until the formed gas was removed completely. The
solvent and excess of reagents were removed in vacuum, and the
acyl chloride was used without further purification.
(PBS1: 6.1 mM Na2HPO4, 3.9 mM NaH2PO4), and oxadiazole deriv-
atives were dissolved in dimethyl sulfoxide (DMSO). Then, 2.0 mg/
mL DNA, 5.0 mM Cu2þ, 3.0 mM GSH, and 0.4 mM oxadiazole
derivatives were mixed to form a solution. The solution was poured
into test tubes, and each test tube contained 2.0 mL. The test tubes
were incubated at 37 ꢀC to initiate the oxidation of DNA, and three
of them were taken out at every 30 min and cooled immediately.
PBS1 solution of EDTA (1.0 mL, 30.0 mM) was added to chelate Cu2þ
,
followed by adding 1.0 mL of thiobarbituric acid (TBA) solution
(1.00 g of TBA and 0.40 g of NaOH dissolved in 100 mL of PBS1) and
1.0 mL of 3.0% trichloroacetic acid aqueous solution. The test tubes
were heated in boiling water for 30 min and cooled to room
temperature, 1.5 mL of n-butanol was added and shaken vigorously
to extract thiobarbituric acid reactive substance (TBARS) whose
absorbance was measured at 535 nm.
2.4. .OH-induced oxidation of DNA test
.OH was generated by mixing H2O2 with tetra-
chlorohydroquinone (TCHQ, dissolved in DMSO as the stock solu-
tion) as the description in a literature [19]. DNA and H2O2 were
dissolved in phosphate buffered solution (PBS2: 8.1 mM Na2HPO4,
The synthesis of oxadiazole derivative was carried out by the
reaction of amidoxime (1 mmol) and acyl chloride (1.1 mmol) in dry
pyridine (5 mL) under reflux for 5 h. Then, the mixture was poured
into water (100 mL). The crude product was used without further
purification. TiCl4 (0.24 mL, 1.1 mmol, dissolved in 10 mL of CH2Cl2)
was dropwisely added to 15 mL of CH2Cl2 solution of 3,5-bis(4-
benzyloxy-3-methoxyphenyl)-1,2,4-oxadiazole (0.49 g, 1 mmol)
under nitrogen atmosphere and stirred for 5 h at room tempera-
ture. The reaction was quenched by adding methanol, and the
solvent was evaporated in vacuum. The residue was purified on
silica gel column chromatography with CH3COOC2H5epetroleum
ether (1:4, v:v) being eluent to afford oxadiazole derivative.
4-(5-(4-Hydroxy-3-methoxyphenyl)-1,2,4-oxadiazol-3-yl)-2-
1.9 mM NaH2PO4, 10.0 mM EDTA). DNA (2.0 mg/mL), 4.0 mM TCHQ,
2.0 mM H2O2, and 0.4 mM oxadiazole derivatives (dissolved in
DMSO as the stock solution) were mixed to form a solution. The
solution was poured into test tubes, and each test tube contained
2.0 mL. The test tubes were incubated at 37 ꢀC for 30 min and
cooled immediately. The following operation was the same as in
Cu2þ/GSH-induced oxidation of DNA except EDTA was not added.
The absorbance in the control experiment and in the presence of
oxadiazole derivatives was assigned as A0 and Adetect, respectively.
.
The effects of oxadiazole derivatives on OH-induced oxidation of
DNA were expressed by Adetect/A0 ꢁ 100.
methoxyphenol (HMOP). Yield 71%. m.p. 211e212 ꢀC. 1H NMR
2.5. AAPH-induced oxidation of DNA test
(DMSO-d6, 300 MHz) d: 10.18 (s, 1H), 9.76 (s, 1H), 7.66 (dd, 1H,
The experiment of AAPH-induced oxidation of DNA was per-
formed as the description in a literature [17]. Briefly, 2.0 mg/mL
DNA, 40 mM AAPH, and a certain concentration of oxadiazole
derivatives (dissolved in DMSO as the stock solution) were mixed to
form a solution. The solution was poured into test tubes, and each
test tube contained 2.0 mL. The test tubes were incubated at 37 ꢀC
to initiate the oxidation of DNA, and three of them were taken out
at every 2 h and cooled immediately. The following operation was
J ¼ 2.1 Hz, 8.1 Hz), 7.61 (d, 1H, J ¼ 2.1 Hz), 7.54e7.57 (m, 2H), 7.00 (d,
1H, J ¼ 8.1 Hz), 6.95 (d, 1H, J ¼ 8.1 Hz), 3.91 (s, 3H), 3.87 (s, 3H).
2-Methoxy-4-(5-phenyl-1,2,4-oxadiazol-3-yl)phenol
(PHOP).
: 9.80
Yield: 82%, m.p. 134e135 ꢀC. 1H NMR (DMSO-d6, 300 MHz)
d
(s, 1H), 8.19 (d, 2H, J ¼ 7.8 Hz), 7.57e7.74 (m, 5H), 6.97 (d, 1H,
J ¼ 7.8 Hz), 3.88 (s, 3H).
4-(5-(2-Hydroxyphenyl)-1,2,4-oxadiazol-3-yl)-2-methoxyphenol
(2HOP). Yield: 77%, m.p. 145e146 ꢀC. 1H NMR (DMSO-d6, 300 MHz)
.
the same as in OH-induced oxidation of DNA except the heating
period was 15 min after TBA and trichloroacetic acid were added.
The absorbance of TBARS was plotted vs the incubation period.
d
: 10.57 (s, 1H), 9.79 (s, 1H), 7.99 (dd, 1H, J ¼ 1.8 Hz, 8.4 Hz), 7.02e
7.60 (m, 5H), 6.96 (d, 1H, J ¼ 8.4 Hz), 3.88 (s, 3H).
4-(5-(2-Chlorophenyl)-1,2,4-oxadiazol-3-yl)-2-methoxyphenol
(CHOP). Yield: 68%, m.p. 168e169 ꢀC. 1H NMR (DMSO-d6, 300
MHz)
d
: 9.82 (s, 1H), 8.16 (dd, 1H, J ¼ 1.5 Hz, 7.8 Hz), 7.56e7.79
2.6. Scavenging DPPH and ABTSþ.
(m, 5H), 6.98 (d, 1H, J ¼ 7.8 Hz), 3.88 (s, 3H).
4-(5-(3-Hydroxyphenyl)-1,2,4-oxadiazol-3-yl)-2-methoxyphenol
DPPH was dissolved in 50 mL of ethanol to make the absorbance
around 1.00 at 517 nm (εDPPH ¼ 4.09 ꢁ 103 Mꢂ1 cmꢂ1). ABTSþ. was
produced from 2.0 mL of a mixture containing 4.0 mM ABTS
aqueous solution and 1.41 mM K2S2O8 after kept for 16 h and
diluted by 100 mL of ethanol. The absorbance of ABTSþ. solution
(3HOP). Yield: 70%, m.p.197e198 ꢀC.1H NMR (DMSO-d6, 300 MHz)
d:
10.07 (s, 1H), 9.79 (s, 1H), 7.08e7.62 (m, 6H), 6.96 (d, 1H, J ¼ 7.5 Hz),
3.87 (s, 3H).
2-Methoxy-4-(5-(pyridin-3-yl)-1,2,4-oxadiazol-3-yl)phenol (PYOP).
Yield: 64%, m.p. 130e131 ꢀC. 1H NMR (DMSO-d6, 300 MHz)
d: 9.83 (s,
was around 1.00 at 734 nm ðεABTS ¼ 1:6 ꢁ 104 Mꢂ1 cmꢂ1Þ. The
þ,
1H), 9.33 (s,1H), 8.90 (s,1H), 8.54(d,1H, J ¼ 7.8Hz), 7.57e7.72 (m, 3H),
DMSO solutions of oxadiazole derivatives (0.1 mL) were added to
6.98 (d, 1H, J ¼ 7.8 Hz), 3.89 (s, 3H).
1.9 mL of DPPH with the final concentrations of oxadiazole deriv-
atives being 500
(0.1 mL) were added to 1.9 mL of ABTSþ. with final concentrations of
HMOP, PHOP, CHOP, 3HOP, and PYOP being 250 M and the final
M. The decreases of the absor-
mM. The DMSO solutions of oxadiazole derivatives
2.3. Cu2þ/GSH-induced oxidation of DNA test
m
Cu2þ/GSH-induced oxidation of DNA was carried out following
a previous report [18] with a slight modification. Briefly, DNA,
CuSO4, and GSH were dissolved in phosphate buffered solution
concentration of 2HOP being 125 m
bance of these radicals were recorded at 25 ꢀC with a certain time
interval.

C. Zhao, Z.-Q. Liu / Biochimie 95 (2013) 842e849
845
2.7. Statistical analysis
possesses the highest activity among these oxadiazole derivatives.
It can be concluded that meta-hydroxylphenyl group at B ring
shows the highest antioxidant effectiveness in this case.
All the data were the average value from at least three indepen-
dent measurements with the experimental error within 10%. The
equationswere analyzedbyone-way ANOVA in Origin 8 professional
software, and p < 0.001 indicated a significant difference.
.
3.2. Effects of oxadiazole derivatives on OH-induced oxidation of
DNA
.OH can be produced from H2O2 catalyzed by TCHQ [19]. The
ribosyl moiety in DNA can be oxidized by OH to form carbonyl
3. Results and discussion
.
3.1. Effects of oxadiazole derivatives on Cu2þ/GSH-induced
oxidation of DNA
species [26]. Fig. 2 outlines that the absorbance of TBARS generated
.
from OH-induced oxidation of DNA in the blank experiment is
assigned as 100%. The absorbances of TBARS in the presence of
0.40 mM oxadiazole derivatives are compared with that of the
blank experiment. The percentage of TBARS in the presence of PYOP
(71.5%) is lower than that of PHOP (87.6%), implying that the pyri-
dine group in PYOP exhibits stronger antioxidant effect than
benzene group in PHOP. The percentages of TBARS in the presence
of 2HOP, 3HOP, and HMOP are 67.0%, 61.5%, and 69.7%, respectively,
lower than that of PHOP (87.6%), demonstrating that the phenolic
hydroxyl group enhances the antioxidant effect at either ortho-,
meta-, or para-position. It is worthy to note that CHOP reduces the
percentage of TBARS to the lowest value (57.3%), indicating that
A large body of clinic and biochemical evidences elucidates that
the pathogenesis of many fatal diseases is related to the free
radical-initiated oxidation of organic tissues [22,23]. Many efforts
are contributed to designing antioxidants and characterizing the
antioxidant effectiveness [24]. As shown in Scheme 1, the synthetic
strategy for 1,2,4-oxadiazoles is based on the reaction between
amidoxime and acyl chloride, followed by the cyclization and the
dehydration. The variation in the substituents takes place in ring B,
while a hydroxyl group with an ortho-methoxyl group at ring A is
kept in order to estimate the interaction between the groups at ring
B and ring A in protecting DNA and scavenging radicals.
.
CHOP possesses the highest ability to protect DNA against OH-
The intracellular GSH and Cu(II) may destroy DNA because the
produced GSH radical (GS.) can drive DNA to form carbonyl species,
which can be detected after reacting with TBA to form colorful TBA
reactive substance (TBARS) [25]. As shown in Fig. 1, the absorbance
line of the blank experiment locates above all the other lines and
increases with the reaction period, indicating that the amount of
TBARS increases in the mixture of DNA, Cu2þ and GSH. The addi-
tions of 0.4 mM oxadiazole derivatives do not inhibit the increase of
TBARS, but the absorbance lines are below that of the blank
experiment. Hence, oxadiazole derivatives are able to protect DNA
against GS.-induced degradation. The addition of CHOP decreases
the absorbance line below that of the blank experiment, demon-
strating that the vanillin group at A ring with an ortho-chloroben-
zene group at B ring (CHOP) exhibits weak ability to inhibit GS.-
induced degradation of DNA. On the other hand, the vanillin group
at A ring and ortho- or para-hydroxylphenyl at B ring (2HOP and
HMOP) and even without any group at B ring (PHOP and PYOP) can
also inhibit GS.-induced degradation of DNA, and their antioxidant
capacities are better than that of CHOP. In particular, the lowest
position of the absorbance line of 3HOP indicates that 3HOP
induced oxidation.
3.3. Effects of oxadiazole derivatives on AAPH-induced oxidation of
DNA
The guanine bases in DNA can be oxidized by radicals generated
from AAPH [27], and the oxidized products can be followed by
measuring TBARS. As shown in Fig. 3, the absorbance of TBARS
increases in the blank experiment, indicating that the amount of
the oxidized products generated from AAPH-induced oxidation of
DNA increases with the incubation period.
The absorbance of TBARS does not increase at the beginning of
the reaction when oxadiazole derivatives are added. Thus, oxadia-
zole derivatives can retard the oxidation of DNA for a period, which
can be measured by the cross-point from the tangent lines for the
inhibition and oxidation period. As shown in Fig. 4, the inhibition
period (tinh) increases linearly with the concentrations of oxadia-
zole derivatives, and the quantitative equations of these lines are
listed in Table 1.
It is proved that the inhibition period (tinh) is proportionally
related to the concentration of the antioxidant as shown as equa-
tion (1) [28].
0.75
0.50
tinh ¼ ðn=RiÞ½antioxidantꢃ
(1)
0.25
0.00
Blank
PYOP
3HOP
HMOP
180
PHOP
CHOP
2HOP
120
0
60
Reaction period (min)
Fig. 1. The increase of the absorbance at 535 nm in the absence and presence of
0.40 mM oxadiazole derivatives in the mixture of 2.0 mg/mL DNA, 5.0 mM Cu2þ, and
3.0 mM GSH.
Fig. 2. The percentages of TBARS in the mixture of 2.0 mg/mL DNA, 4.0 mM H2O2,
2.0 mM TCHQ, and 0.40 mM oxadiazole derivatives at 37 ꢀC for 30 min.

846
C. Zhao, Z.-Q. Liu / Biochimie 95 (2013) 842e849
Fig. 3. The variation of the absorbance of TBARS in the mixture of 2.0 mg/mL DNA, 40 mM AAPH, and various concentrations of oxadiazole derivatives at 37 ꢀC.
The n is the stoichiometric factor, which means the number of the
radical-propagation terminated by one molecule of the antioxidant.
The Ri is the initiation rate of the radical-induced reaction. When
equation (1) is employed in AAPH-induced oxidation of DNA, Ri is
assumed to be equal to the generation rate (Rg) of radicals
(Rg ¼ (1.4 ꢄ 0.2) ꢁ 10ꢂ6 [AAPH] sꢂ1 [28]). This is because both
sodium salt of DNA and AAPH are dissolved in water, and radicals
generated from AAPH attack DNA at the same phase [29]. Thus,
values of n of oxadiazole derivatives are the product of the coeffi-
cients of the equation (see Table 1) multiplying Ri ¼ Rg
¼
1.4 ꢁ 10ꢂ6 ꢁ 40 mM sꢂ1 ¼ 3.36
m
M minꢂ1 and listed in Table 1 as
well. For example, the coefficient in tinh w [HMOP], 0.61, multi-
plying 3.36 m
M minꢂ1 results in the n of HMOP, 2.05, indicating that
HMOP can trap 2.05 radicals in protecting DNA against AAPH-
induced oxidation. Accordingly, the n value provides an order for
antioxidant effectiveness as HMOP
> 2HOP > CHOP >
PYOP > 3HOP w PHOP. The n value of 3HOP (0.87) is similar to that
Table 1
The equations of tinh w [oxadiazole derivatives], and n of oxadiazole derivatives in
protecting DNA against AAPH-induced oxidation.a
Antioxidant
tinh (min) ¼ (n/Ri) [antioxidant (
m
M)] þ constantb
n
PHOP
CHOP
2HOP
PYOP
3HOP
HMOP
tinh (min) ¼ 0.24 [PHOP] þ 92.54
tinh (min) ¼ 0.44 [CHOP] þ 17.28
tinh (min) ¼ 0.53 [2HOP] þ 27.24
tinh (min) ¼ 0.31 [PYOP] þ 42.19
tinh (min) ¼ 0.26 [3HOP] þ 68.43
tinh (min) ¼ 0.61 [HMOP] þ 87.90
0.81
1.48
1.78
1.04
0.87
2.05
a
Ri ¼ Rg ¼ 1.4 ꢁ 10ꢂ6 [AAPH] sꢂ1 ¼ 3.36
m
M minꢂ1 when 40 mM AAPH was
Fig. 4. The linear relationship between the concentrations of oxadiazole derivatives
and inhibition period (tinh) in protecting DNA against AAPH-induced oxidation.
employed, thus, n ¼ coefficient ꢁ 3.36
m .
M minꢂ1
b
The constant was generated from the linear regression analysis.

C. Zhao, Z.-Q. Liu / Biochimie 95 (2013) 842e849
847
of PHOP (0.81) without hydroxyl group attaching to B ring,
implying that meta-hydroxyl as in 3HOP almost does not provide
additional antioxidant capacity for 3HOP. But when the benzene at
B ring (as PHOP) is replaced by pyridine ring to form PYOP, the n
value increases from 0.81 (PHOP) to 1.04 (PYOP), indicating that
pyridine ring ameliorates the antioxidant effectiveness. Further-
more, a Cl atom at ortho-position of B ring enhances the n value to
1.48 (CHOP), and an ortho-hydroxyl group increases the n value to
1.78 (2HOP). This result reveals that the contribution of substituent
at B ring to the antioxidant capacity follows vanillin group > ortho-
hydroxylbenzene group > ortho-chlorobenzene group > pyridine
group > meta-hydroxylbenzene and benzene group itself. The
antioxidant effectiveness of oxadiazole derivatives is even higher
than that of chroman-4-one. We have measured n value of 3-(40-
hydroxybenzylidene)-7-methoxychroman-4-one (p-HBMC, struc-
ture in Scheme 2) at the same experimental system, and the n of p-
HBMC is only 0.50 [29], lower than those of oxadiazole derivatives.
The n values of diaryl-1,2,4-oxadiazoles used herein are close to
those of triaryl-pyrazoles (ranging from 1.58 to 1.8) [14].
Fig. 5. Decay of 60.75
m
M ABTSþ. in the presence of 125
mM 2HOP and 250
m
M other
compounds.
3.4. Rate constant of oxadiazole derivatives to scavenge ABTSþ. and
DPPH
dihydropyrimidine in trapping ABTSþ. and DPPH [33], and herein,
the k values of oxadiazole derivatives are also calculated and listed
in Tables 2 and 3. Since there is no substituent attaching to B ring in
Free radical-scavenging property is the basic feature for
a compound to be an antioxidant [30]. ABTSþ. and DPPH are usually
used to test the ability of an antioxidant to trap radicals [31,32]. The
measurement of the rate constants (k) of an antioxidant to trap
ABTSþ. and DPPH reveals the radical-scavenging kinetics. As shown
in Figs. 5 and 6, in the presence of oxadiazole derivatives the
concentrations of ABTSþ. and DPPH decrease with the reaction
period increasing, demonstrating that oxadiazole derivatives can
quench ABTSþ. and DPPH efficiently.
After the data in Figs. 5 and 6 are input into statistical software,
the relationship between the concentrations of ABTSþ. and DPPH
([radical]) and reaction period (t) fits for equation (2), an expo-
nential function, and the results are listed in Tables 2 and 3.
PHOP, the k values of PHOP (9.45 mMꢂ1 sꢂ1 and 4.08 Mꢂ1 sꢂ1
)
reveal the ability of vanillin group at A ring to trap ABTSþ. and
DPPH, respectively. When the benzene group at B ring is replaced
by pyridine group to form PYOP, the k values of PYOP decrease to
6.62 mMꢂ1 sꢂ1 and 0.82 Mꢂ1 sꢂ1, respectively, demonstrating that
the electron-withdrawing group like pyridine decreases the
radical-scavenging ability of the vanillin group at A ring. In addi-
tion, the k values of CHOP are similar to those of PHOP, indicating
that an ortho-chloro group cannot markedly influence the radical-
scavenging property. But the remarkable enhancement of k value
of 3HOP in trapping DPPH implies that meta-hydroxyl group at B
ring can increase the ability of 3HOP to donate its hydrogen atom to
DPPH radical. Moreover, it can be found that the k values of HMOP
and 2HOP in trapping either ABTSþ. or DPPH are much higher than
those of other oxadiazole derivatives. This result demonstrates that
ortho- and para-hydroxyl group at B ring plays the major role in
trapping radicals.
½radicalꢃ ¼ Aeꢂðt=aÞ þ Beꢂðt=bÞ þ C
(2)
Moreover, as shown as equation (3), the differential style of
equation (2) indicates the relationship between the reaction rate
(r ¼ ꢂd[radical]/dt) and the reaction period (t). The results are listed
in Tables 2 and 3 as well.
ꢂd½radicalꢃ=dt ¼ r ¼ ðA=aÞeꢂðt=aÞ þ ðB=bÞeꢂðt=bÞ
(3)
The reaction rate at t ¼ 0 (r0) can be calculated by equation (3)
when t is assigned to be 0, and the results are involved in
Tables 2 and 3. According to the concept of reaction rate, the
reaction rate at t ¼ 0 (r0) correlates with the concentrations of
radical and antioxidant at the same time point as shown in equa-
tion (4), in which k is the rate constant.
r0 ¼ k½radicalꢃ0½antioxidantꢃ0
(4)
The concentrations of radical and antioxidant at t ¼ 0 together
with r0 are known, and k can be calculated by equation (4). This
method has been applied to calculate the rate constant (k) of
Scheme 2. Structure of p-HBMC.
Fig. 6. Decay of 293.89 mM DPPH in the presence of 500 mM oxadiazole derivatives.

848
C. Zhao, Z.-Q. Liu / Biochimie 95 (2013) 842e849
Table 2
Equation of [ABTSþ.] w t and its differential style (ꢂd[ABTSþ.]/dt w t), reaction rate at t ¼ 0 (r0), and rate constant (k).a
Compound
Equation of [ABTSþ.] w t
Equation of ꢂd[ABTSþ.]/dt w t
r0
(
m
M sꢂ1
)
k (mMꢂ1 sꢂ1
)
d½ABTSþ,
ꢃ
t
t
t
t
½ABTSþ,ꢃ ¼ 22:59eꢂ þ 22:54e
ꢂ
ꢂ
ꢂ9:97
ꢂ9:97
0:16
0:16
PHOP
þ 15:57
þ 15:12
¼ 141:19eꢂ þ 2:26e
143.45
144.35
9.45
dt
d½ABTSþ,
dt
ꢃ
t
t
t
t
CHOP
½ABTSþ,ꢃ ¼ 22:80eꢂ þ 22:77e
¼ 142:50eꢂ þ 1:85e
9.50
ꢂ12:32
ꢂ12:32
0:16
0:16
d½ABTSþ,
dt
ꢃ
ꢃ
ꢃ
t
t
t
t
½ABTSþ,ꢃ ¼ 22:23eꢂ þ 22:20e
ꢂ11:87
2HOP
PYOP
3HOP
¼ 444:60eꢂ þ 1:87e
446.47
100.55
58.53
58.79
6.62
3.85
ꢂ11:87
0:05
0:05
þ 16:28
þ 15:38
þ 14:17
ꢂ
ꢂ
ꢂ
d½ABTSþ,
dt
t
t
t
t
½ABTSþ,ꢃ ¼ 22:68eꢂ þ 22:64e
¼ 98:61eꢂ þ 1:94e
ꢂ11:66
ꢂ11:66
0:23
0:23
d½ABTSþ,
dt
t
t
t
t
½ABTSþ,ꢃ ¼ 23:23eꢂ
þ 23:26e
¼ 1:80eꢂ
þ 56:73e
ꢂ0:41
ꢂ0:41
12:90
12:90
d½ABTSþ,
dt
ꢃ
t
t
t
t
½ABTSþ,ꢃ ¼ 22:63eꢂ þ 22:57e
ꢂ14:57
HMOP
¼ 377:17eꢂ þ 1:55e
378.72
24.94
ꢂ14:57
0:06
0:06
þ 15:42
ꢂ
a
The concentration of 2HOP was 125 mM, and the concentrations of other compounds were 250 m mM.
M. The concentration of ABTSþ. was 60.75
Table 3
Equation of [DPPH] w t and its differential style (ꢂd[DPPH]/dt w t), reaction rate at t ¼ 0 (r0), and rate constant (k).a
Compound
Equation of [DPPH] w t
Equation of ꢂd[DPPH]/dt w t
r0
(
m
M sꢂ1
)
k (Mꢂ1sꢂ1
)
d½DPPHꢃ
dt
d½DPPHꢃ
dt
d½DPPHꢃ
dt
d½DPPHꢃ
dt
d½DPPHꢃ
dt
d½DPPHꢃ
dt
t
t
t
t
ꢂ23:14
¼ 0:12eꢂ
¼ 0:15eꢂ
¼ 0:26eꢂ
¼ 0:06eꢂ
¼ 0:07eꢂ
¼ 0:20eꢂ
þ 0:48e
þ 0:72e
þ 4:51e
þ 0:06e
þ 3:03e
0.60
0.87
4.77
0.12
3.10
3.08
4.08
ꢂ23:14
½DPPHꢃ ¼ 43:23eꢂ
½DPPHꢃ ¼ 36:20eꢂ
½DPPHꢃ ¼ 41:26eꢂ
½DPPHꢃ ¼ 20:29eꢂ
½DPPHꢃ ¼ 39:96eꢂ
½DPPHꢃ ¼ 42:24eꢂ
þ 11:12e
þ 212:69
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
366:81
366:81
PHOP
t
t
t
t
ꢂ12:20
ꢂ12:20
248:47
248:47
CHOP
þ 8:77e
þ 237:36
5.92
t
t
t
t
ꢂ14:42
ꢂ14:42
159:94
159:94
2HOP
þ 65:04e
þ 20:29e
þ 23:53e
þ 16:05e
þ 187:35
þ 227:50
32.46
0.82
t
t
t
t
ꢂ318:82
ꢂ318:82
318:82
318:82
PYOP
t
t
t
t
ꢂ7:77
ꢂ7:77
608:09
608:09
3HOP
þ 230:34
þ 235:60
21.10
20.96
t
t
t
t
ꢂ5:57
ꢂ5:57
214:64
214:64
HMOP
þ 2:88e
a
The concentrations of oxadiazole derivatives were 500
m
M, and the concentration of DPPH was 293.89
mM.
[4] N.A. Bokach, A.V. Khripoun, V.Y. Kukushkin, M. Haukka, A.J.L. Pombeiro,
A route to 1,2,4-oxadiazoles and their complexes via platinum-mediated 1,3-
dipolar cycloaddition of nitrile oxides to organonitriles, Inorg. Chem. 42
(2003) 896e903.
[5] J.M. dos Santos Filho, A.C.L. Leite, B.G. de Oliveira, D.R.M. Moreira, M.S. Lima,
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1,2,4-Oxadiazole is a useful group to construct diaryl-1,2,4-
oxadiazole antioxidants. In this core-branched antioxidant, only
a few hydroxyl groups can exhibit high antioxidant properties. In
the presence of a vanillin group at A ring, only one para- or ortho-
hydroxyl at B ring can trap radicals and protect DNA against AAPH-
induced oxidation. An ortho-chloro group even possesses high
.
ability to inhibit OH-induced oxidation of DNA, while a meta-
hydroxyl group at B ring acts as an efficient antioxidant factor to
protect DNA against Cu2þ/GSH-induced oxidation. The benefit of
core-branched antioxidant is due to that few hydroxyl groups play
efficient role in protecting DNA and in trapping radicals. Therefore,
core-branched type may be a novel structural feature in designing
antioxidant.
oxadiazole moiety at C-5 through
a straight-chain carbon and oxygen
atoms, Eur. J. Med. Chem. 44 (2009) 3571e3576.
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D.A. Learmonth, Discovery of a long-acting, peripherally selective inhibitor of
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G. Calvaruso, S. Buscemi, N. Vivona, A. Pace, Synthesis, characterization,
Acknowledgment
cellular uptake and interaction with native DNA of
a bis(pyridyl)-1,2,4-
Financial support from Jilin Provincial Foundation for Natural
Science, China, (201115017) is acknowledged gratefully.
oxadiazole copper(II) complex, Dalton Trans. 39 (2010) 9140e9145.
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