Synthesis, characterization, and antioxidant activity of pyrazolic macrocycle

Article abstract of DOI:10.1007/s00706-014-1298-2

A series of macrocycles containing isoxazoline, dioxazole, and pyrazole were synthesized and characterized by IR, ¹H, and ¹³C NMR, mass spectra, elemental analysis, and X-ray single crystal determination. The antioxidant activity of synthesized compounds was evaluated. One of the compounds exhibits potent DPPH radical scavenging activity, comparable to that of vitamin E.

Full text of DOI:10.1007/s00706-014-1298-2

Monatsh Chem
DOI 10.1007/s00706-014-1298-2
ORIGINAL PAPER
Synthesis, characterization, and antioxidant activity of pyrazolic
macrocycle
•
Hehua Zeng Yongming Zeng Lili Geng
•
•
•
Xueqin Li Xianfeng Meng
Received: 26 June 2014 / Accepted: 5 August 2014
ꢀ Springer-Verlag Wien 2014
Abstract A series of macrocycles containing isoxazoline,
dioxazole, and pyrazole were synthesized and character-
macrocycles containing the pyrazole moiety with the aim
of obtaining new biologically active compounds. In view of
the continued interest in the development of simpler and
more convenient synthetic routes for preparing macrocy-
clic systems, an efficient and useful method is reported
herein to synthesize macrocycles using double different
cycloadditions as cornerstones. In addition, pyrazole
proved to be able to protect various tissues against oxida-
tive stress, predominantly by scavenging deleterious
reactive oxygen species. To the authors’ knowledge, no
investigation of the radical scavenging effects of synthe-
sized pyrazolic macrocycle has yet been undertaken. It
looked promising to support the development of new drugs
and improve the treatment of various diseases.
1
ized by IR, H, and 13C NMR, mass spectra, elemental
analysis, and X-ray single crystal determination. The
antioxidant activity of synthesized compounds was evalu-
ated. One of the compounds exhibits potent DPPH radical
scavenging activity, comparable to that of vitamin E.
Keywords Cycloadditions ꢀ Macrocycles ꢀ Heterocycles ꢀ
Antioxidant activity ꢀ DPPH
Introduction
Heterocyclic compounds, especially nitrogen heterocycles,
are the most important class of compounds in the phar-
maceutical and agrochemical industries, with heterocycles
comprising around 60 % of all drug substances [1–3]. The
pyrazole ring system, in particular, is a very common
structural motif and is found in numerous biologically
active natural products and pharmacologically relevant
therapeutic agents [4–9]. Because of the significance of
these scaffolds in drug discovery and medicinal chemistry,
the development of new methodologies for the construction
of novel heterocycles continues to be a very active field
of research [10–12]. Following reports of these activities,
this study seeks to an efficient synthetic method for
Results and discussion
Synthetic methodology
The synthesis of pyrazolic macrocycle has been accom-
plished by cycloadditive macrocyclization route relies
mainly bifunctional dipole (bis-nitrile oxides) and bifunc-
tional dipolarophile in the presence of a base as
triethylamine as outlined in Scheme 1. The bifunctional
hydroxamic acid chloride 4 was prepared from the corre-
sponding aldehyde 1 in a two-step procedure as given in
Scheme 1. The structures of the cycloadducts were char-
acterized by NMR spectroscopic data. The structures of all
compounds are also confirmed by mass spectral analysis.
The mass spectra show M? peaks at corresponding masses
of respective molecules.
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-014-1298-2) contains supplementary
material, which is available to authorized users.
H. Zeng ꢀ Y. Zeng (&) ꢀ L. Geng ꢀ X. Li ꢀ X. Meng
Department of Chemistry and Applied Chemistry, Changji
University, Changji, People’s Republic of China
e-mail: zym903@126.com
The 1,3-dipolar cycloaddition of nitrile oxide and a,b-
unsaturated ketones led to the formation of macrocycle 6
arising from the nitrile oxide addition to one C=C and C=O
123

H. Zeng et al.
Scheme 1
CHO
Cl
CHO
O
OHC
O
N
N
HO
OH
N
N
C
N
N
b)
a)
2
1
OH
N
Cl
OH
OH
N
OH
N
Cl
N
N
N
N
C
N
C
N
O
O
N
N
H
N
O
O
c)
3
4
R
O
H
O
R
N
O
O
5a-5c
N
N
N
N
N
O
O
d)
6a-6c
=
R
H (a), OCH (b), CH (c)
3
3
Scheme 2
O
N
H
O
H
3C
H
OH
N
O
O
N
H
3C
Cl
Et3N, CH2Cl2 rt
,
4
6
N
Et3N, CH2Cl2 rt
,
N
O
N
7
bonds. All of the cycloadducts 6a–6c were separated
through flash column chromatography using petroleum
ether-ethyl acetate as eluent.
isoxazoline and failed to afford dioxazole (Scheme 2), thus
suggesting that macrocycle 6c might result from further
cycloaddition to the intermediate 7. To confirm this, the
reaction of the intermediate with a nitrile oxide was per-
formed in a separate experiment under the same reaction
conditions as those of the overall reaction. This reaction
furnished macrocycle 6c, suggesting its formation from the
intermediate 7.
With a view to finding the order of occurrence of the
cycloaddition of nitrile oxide to the C=C and C=O bonds
leading to the formation of the cyclophane, the reaction
was intercepted at early stages before completion of the
reaction at 30 min and 1 h and the reaction mixture afford
123

Synthesis, characterization, and antioxidant activity of pyrazolic macrocycle
X-ray analysis
X-ray diffraction study of the compound 6c has shown
that the title compound C44H34N6O5 crystallizes in the
monoclinic system with space grouping C2/c. The title
molecule has a cyclic conformation, which consists of two
pyrazole rings, an isoxazoline ring, and an oxadiazole ring.
The O5, C42, O4, N6, and C44 atoms form a five-mem-
bered ring. X-ray analysis reveals that the five-membered
The structure of macrocycles was further confirmed by
X-ray crystallographic studies of single crystals of 6c. The
structure of the crystal 6c is displayed in Fig. 1, and a
perspective view of the crystal packing in the unit cell is
shown in Fig. 2.
Fig. 1 X-ray crystal structure
of macrocycle 6c
123

H. Zeng et al.
Fig. 2 Crystal packing diagram
of 6c
ring of dioxazole adopts an envelope conformation with the
atom C42 deviating from the plane defined by the atoms
˚
O5, O4, N6, and C44 of 0.375 A. Within the molecule, the
indicates that there are eight independent molecules in the
unit. In the crystal structure, there are no classic hydrogen
bonds. QueryThe crystal structure is stabilized by weak C–
H…N and C–H…O intramolecular hydrogen bonds and
C…p stacking interactions, which are also observed
between the neighboring molecules (Fig. 3; Table 1).
Atoms C40, C24, and C6 act as a hydrogen-bond donor,
respectively, via H…p bond to generate the super-molec-
ular structures. The C atoms are involved in H…p
interactions with the phenyl ring, which increase the sta-
bility of the 3D supramolecular architecture of the
polymer. The perpendicular distance between ring cen-
troids of Cg(2)–Cg(6), Cg(2)–Cg(7), Cg(8)–Cg(3), and
˚
C44–N6 (1.282 A) and the C43–N5 (1.285 A) bonds are
˚
˚
˚
shorter than the C36–N1 (1.347 A) and C39–N3 (1.345 A)
bonds suggesting that the former two bonds have somewhat
higher bond orders than the latter two bonds, because of the
N6–C44 and C43–N5 bonds belong to double bond. The
˚
bond distances of C42–O5 (1.433 A), C28–O1 (1.401 A),
˚
˚
and C41–O3 (1.456 A) are significantly longer than a
˚
normal single C–O bond (1.367(3) A). It is worth to illu-
˚
˚
minate that the C28–O1 (1.401 A), C42–O5 (1.433 A)
˚
bonds are longer than the C36–O1 (1.355 A), C44–O5
˚
(1.372 A) bonds, largely due to intramolecular tension, but
˚
Cg(5)–Cg(6) are 3.820(3) A, 5.875(2) A, 5.728(3) A, and
˚
˚
˚
5.729(2) A, respectively. Furthermore, the bond lengths of
all lie within the range found in aromatic heterocycles [13].
The phenyl rings attached to N1 and N3 are twisted with
respect to the pyrazole ring, the dihedral angles with the
pyrazole ring being 27.3(3)ꢁ and 41.6(3)ꢁ, respectively.
The phenyl groups of Cg(7) is nearly perpendicular to the
oxadiazole ring attached to C42 with angle of 89.48(18)ꢁ.
Similarly, the aryl ring attached to C40 is also twisted from
the plane of the isoxazoline ring, the dihedral angle being
71.07(19)ꢁ. The plane of Cg(5) forms nearly equal dihedral
angles with the other rings, 75.87(17)ꢁ with the pyrazole
ring containing N2, N1 and 69.3(2)ꢁ with the pyrazole ring
containing N3, N4. X-ray crystal structure determination
C40-H…Cg(5), C36-H…Cg(2), and C49-H…Cg(6) corre-
i
spond to 2.95, 2.71, and 2.96 A, respectively [14]. Cg(7) is
˚
the center of gravity of ring (C20, C31, C40, C41, C42, and
C44) and Cg(8)ii is the center of gravity of ring (C34, C48,
C52, C53, C58, and C45); symmetry code: i = X,-Y,-1/
2?Z; ii = -X,Y,1/2-Z.
Evaluation of the antioxidant activity
The model of the scavenging of the stable 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical is extensively used to
123

Synthesis, characterization, and antioxidant activity of pyrazolic macrocycle
Fig. 3 The blue and red dashed lines indicates weak intermolecular interactions (color figure online)
Table 1 Intermolecular and intramolecular interactions in
compounds
100
80
60
40
20
0
D–H…A
D–H
H…A
D…A
D–H…A
Intramolecular interactions
C(9)–H(9A)…O(2)
C(31)–H(31A)…O(1)
C(52)–H(52A)…O(4)
C(53)–H(53A)…N(1)
a
b
c
Vc
0.98
0.93
0.93
0.93
2.50
2.51
2.44
2.50
2.874(4)
2.817(4)
2.944(6)
2.816(5)
102
100
114
100
D–H…A
H…A D…A
D–H…A Symmetry code
Intermolecular interactions
C(9)–H(9A)–Cg(5)
2.95 3.841(3) 152
X,Y,Z
C(36)–H(36A)–Cg(2) 2.71 2.993(5) 99
C(49)–H(49A)–Cg(6) 2.96 3.780(7) 148
X,Y,Z
0.00
0.05
0.10
0.15
0.20
0.25
0.30
1/2-X,1/2-Y,-Z
Concentration/mmol dm-3
Cg2 = O5–N6–C25–C9–C10; Cg5 = C11–C14–C12–C18–C13–
C28; Cg6 = C19–C29–C37–C39–C32–C36
Fig. 4 Scavenging activity of compounds 6a–6c on DPPH radical
evaluate antioxidant activities in less time than other
methods. DPPH is a stable free radical that can accept an
electron or hydrogen radical and thus be converted into a
stable, diamagnetic molecule [15–17]. DPPH has an odd
electron and so has a strong absorption band at 517 nm.
When this electron becomes paired off, the absorption
decreases stoichiometrically with respect to the number of
electrons taken up. Such a change in the absorbance pro-
duced in this reaction has been widely applied to test the
capacity of numerous molecules to act as free radical
scavengers. The scavenging effect of the synthesized
compounds 6a–6c on the DPPH radical was evaluated
[18–22]. All tests and analyses were undertaken on three
replicates and the results averaged. The tests reveal that the
more the concentration of the tested compound, the higher
the radical scavenging activity. Compounds 6a–6c exhib-
ited good radical scavenging activity 80–85 % after
standing 3 h at a final concentration of 0.1 mmol/dm3.
Compounds 6a, 6c exhibited 30–40 % radical scavenging
activity in 30 min incubation at a final concentration of
0.1 mmol/dm3. Compound 6b with methoxy group scav-
enges DPPH radical very fast and the incubated time only
takes 30 min to reach equilibrium. The profiles of the
scavenging effect of compounds 6a–6c on DPPH are
comparable to that of vitamin E, and are presented in
Fig. 4.
123

H. Zeng et al.
mixture was stirred for 50 min. It was then poured into
80 cm3 H2O and extracted with EtOAc. The organic phase
was washed several times with H2O. The solid was
collected by filtration and crystallized from ethanol to
give bis(hydroxamic acid chloride) 4 as a white solid. This
compound was obtained as white crystals in 88 % yield.
M.p.: 180–181 ꢁC; IR (KBr): mꢀ = 3,438 (OH), 3,051 (Ar–
Conclusion
Various biologically active macrocycles that contain two
pyrazole rings, an isoxazoline ring, and an oxadiazole
moiety have been discovered or chemically synthesized.
This work describes an efficient and convenient method for
the synthesis of cyclophane. Macrocycles 6a–6c were
synthesized for the first time by the reaction of 1,3-dipolar
cycloaddition of a,b-unsaturated ketones as key steps in the
presence of triethylamine (Scheme 1). Among these,
compounds 6a–6c exhibit a potent DPPH radical scav-
enging activity, which is comparable to that of vitamin E.
H), 2,974, 1,477 (CH3) cm-1
;
1H NMR (CDCl3,
400 MHz): d = 7.71–6.87 (m, 16H, Ar–H), 2.45 (s, 6H,
–CH3) ppm; 13C NMR (100 MHz, CDCl3): d = 159.2,
152.3, 138.1, 137.6, 137.4, 131.7, 127.9, 126.4, 125.1,
117.5, 29.7, 14.0 ppm; MS (EI): m/z = 580 (M?).
General procedure for the preparation of macrocycles
6a–6c by cycloadditive macrocyclization
Experimental
To
a mixture of bis(hydroxamic acid chloride) 4
All purchased solvents and chemicals were of analytical
grade and were used without further purification. Melting
points were measured on a Mettler FP-5 capillary melting
point apparatus. Elemental analyses were performed on a
Perkin-Elmer 2400 elemental analyzer. The solid-state IR
spectra were recorded from potassium bromide pellet on a
Bruker Tensor 27 Spectrophotometer. The NMR spectra
were recorded on a Varian Inova-400 spectrometer using
CDCl3 as the deuterated solvent and TMS as the internal
standard at room temperature. EI-MS spectra were
obtained with an Agilent 5975 apparatus. The results were
found to be in good agreement with the calculated values.
All reagents were of commercial availability. The com-
pounds 1 [23], 2 [24], and 5 [25] can be synthesized as
described according to the previously reported procedures.
(2 mmol) and the chalcones 5 (2.5 mmol) in 50 cm3
CH2Cl2 was added dropwise a solution of Et3N (5 mmol)
in 10 cm3 CH2Cl2. Then, the solution was stirred at
room temperature for further 12 h. The solid mass sep-
arated out was filtered off, and the solvent was
evaporated in vacuo and the residue was subjected to
flash column chromatography on silica gel (petroleume-
ther:ethyl acetate 10:1) to afford macrocycles 6 as white
crystalline solids.
5,15-Dimethyl-3,8,10,17-tetraphenylcalix[1]arene[2]-
pyrazole[1]oxazole[1]isoxazole (6a, C43H32N6O5)
This compound was obtained as white crystals in 26 %
ꢀ
yield. M.p.: 167–168 ꢁC; IR (KBr): m = 3,051 (Ar–H),
1,567 (C=N), 2,924, 1,490 (CH3) cm-1; H NMR (CDCl3,
1
5,50-[1,4-Phenylenebis(oxy)]bis[3-methyl-1-phenyl-1H-
pyrazole-4-carboxaldehyde] dioxime (3, C28H24N4O4)
To a solution of the dialdehyde 2 (4 mol) and NH2OHꢀHCl
(8 mol) in 60 cm3 EtOH-H2O was added an aqueous solution
of NaOH (8 mol) dropwise. After stirring the suspension for
overnight at 0 ꢁC, the solid was collected by filtration and
washed with water. The solid was crystallized from ethanol to
give dialdoxime 3 as a pale-yellow solid. This compound was
obtained as white crystals in 90 % yield. M.p.:212–213 ꢁC; IR
400 MHz): d = 7.71–6.98 (m, 24H, Ar–H), 4.69–4.67 (d,
1H, H5, Jab = 8.40 Hz), 3.77–3.75 (d, 1H, H4,
Jab = 8.40 Hz), 2.05 (s, 6H, –CH3) ppm; 13C NMR
(100 MHz, CDCl3): d = 158.7, 157.4, 155.4, 152.3,
137.6, 135.0, 130.7, 129.6, 129.0, 128.9, 128.7, 126.8,
123.8, 121.3, 114.2, 109.6, 88.7, 57.5, 29.7, 12.9 ppm; MS
(EI): m/z = 712 (M?).
8-(4-Methoxyphenyl)-5,15-dimethyl-3,10,17-
triphenylcalix[1]arene[2]pyrazole[1]oxazole[1]isoxazole
(6b, C44H34N6O6)
ꢀ
(KBr): m = 3,429 (OH), 3,051 (Ar–H), 2,924, 1,474 (CH3),
2,307 (C=N), 1,086 (C–O–C) cm-1 1H NMR (CDCl3,
;
This compound was obtained as white crystals in 33 %
400 MHz): d = 9.59 (s, 2H, CH=N), 7.58-6.94 (m, 16H,
Ar–H), 2.53 (s, 6H, –CH3)ppm;13CNMR(100 MHz, CDCl3):
d = 160.4, 152.5, 148.8, 137.9, 137.3, 131.5, 128.8, 126.4,
121.2, 119.1, 29.7, 14.0 ppm; MS(EI): m/z = 508 (M?).
ꢀ
yield. M.p.: 194–195 ꢁC; IR (KBr): m = 3,045 (Ar–H),
1,581 (C=N), 2,924, 1,491 (CH3) cm-1; 1H NMR (CDCl3,
400 MHz): d = 7.71–6.87 (m, 23H, Ar–H), 4.66–4.64 (d,
1H, H5, Jab = 8.40 Hz), 3.80 (s, 3H, –OCH3), 3.72–3.70
(d, 1H, H4, Jab = 8.40 Hz), 2.05 (s, 6H, –CH3) ppm; 13C
NMR (100 MHz, CDCl3): d = 160.7, 158.0, 157.3, 152.4,
150.0, 138.0, 135.0, 130.8, 127.4, 127.0, 124.7, 123.1,
122.8, 121.8, 121.0, 119.9, 115.5, 114.0, 109.2, 87.4, 56.6,
55.5, 29.7, 12.9 ppm; MS (EI): m/z = 742 (M?).
5,50-[1,4-Phenylenebis(oxy)]bis[3-methyl-1-phenyl-1H-
pyrazole-4-hydroxamic acid chloride]
(4, C28H22N4O4Cl2)
To a solution of aldoxime 3 (10 mmol) in 12.5 cm3 DMF
was added N-chlorosuccinimide (15 mmol). The resulting
123

Synthesis, characterization, and antioxidant activity of pyrazolic macrocycle
5,15-Dimethyl-8-(4-methylphenyl)-3,10,17-
triphenylcalix[1]arene[2]pyrazole[1]oxazole[1]-
isoxazole (6c, C44H34N6O5)
1H, H5, Jab = 8.40 Hz), 3.83–3.81 (d, 1H, H4,
Jab = 8.40 Hz), 2.49 (s, 3H, –CH3), 2.05 (s, 6H, –CH3)
ppm; 13C NMR (100 MHz, CDCl3): d = 158.6, 157.4,
152.6, 151.6, 138.2, 135.9, 134.3, 134.2, 129.6, 129.0,
126.8, 125.9, 125.7, 121.7, 118.3, 115.4, 111.5, 109.2,
90.6, 59.7, 29.6, 21.5, 12.9 ppm; MS (EI): m/z = 726
(M?).
This compound was obtained as white crystals in 32 %
ꢀ
yield. M.p.: 182–183 ꢁC; IR (KBr): m = 3,038 (Ar–H),
1
1,579 (C=N), 2,922, 1,487 (CH3) cm-1; H NMR (CDCl3,
400 MHz): d = 7.71–6.95 (m, 23H, Ar–H), 4.65–4.63 (d,
Crystal structure determination
Table 2 Crystal data and structure refinement for compound 6c
Empirical formula
C44H34N6O5
The selected crystal with approximate dimensions of
0.57 9 0.41 9 0.7 mm3 was mounted on thin glass fiber
with the aid of an epoxy resin. The XRD data were col-
lected with multi-scan mode at 296(2) K on a Bruker Smart
AXSCCD with a graphite monochromatic Mo-Ka radiation
Formula weight
Temperature/K
726.77
296(2)
˚
Wavelength/A
0.71073
Crystal system
Space group
Monoclinic
˚
(k = 0.71073 A). APEX2 software was used for data
C2/c
reduction and multi-scan absorption correction [26]. A total
of 38,747 reflections (2hmax = 55.04ꢁ) were collected, of
which 9,961 unique reflections (Rint = 0.0349) were used
for structural elucidation. The structures were solved by
direct methods using SHELXS97 [27] and refinement was
˚
a/A
29.396(2)
˚
b/A
20.2506(15)
˚
c/A
16.5737(12)
a/ꢁ
b/ꢁ
c/ꢁ
90
118.345(2)
carried out by the full-matrix least-squares technique on F2
P
90
using SHELXL97 [28]. The function Rx = | x(|F0|2-
3
˚
P
V/A
8,683.1(11)
|F|c2|/ |x(f0)2|1/2 was minimized, where x = [1/r2(F0)2 ?
(aP0)2 ? bP] (P = [F02 ? 2Fc2]/3). The final R1 and wR2
values 0.1097 and 0.3035 are for 5,960 independent
reflections [I [ 2r(I)]. All non-hydrogen atoms were
assigned anisotropic displacement parameters in the
refinement. All hydrogen atoms were added at calculated
positions and refined using a riding model, with C–H dis-
Z
8
Dc/Mg m-3
Crystal size/mm3
1.1119(1)
0.57 9 0.41 9 0.7
1.57–27.52
h range/ꢁ
l/mm-1
0.129
Reflections collected
Data/restraints/parameters
Final R indices [I [ 2r(I)]
R indices (all data)
38,747
9,961/0/497
˚
tances in the range 0.93 A and with their Uiso set at 1.2 (1.4
R1 = 0.1097, wR2 = 0.3035
R1 = 0.1489, wR2 = 0.3515
for methyl groups) times the Ueq values of the appropriate
carrier atoms. Molecular structure was checked using
Table 3 Selected bond lengths/
˚
O(1)–C(7)
1.433(3)
1.439(3)
1.282(4)
1.456(4)
1.504(4)
1.538(5)
1.467(5)
1.352(4)
1.354(5)
1.369(4)
106.0(2)
115.6(3)
117.1(2)
-1.7(5)
67.2(4)
O(1)–C(8)
1.371(3)
1.433(4)
1.420(4)
1.536(5)
1.285(5)
1.457(4)
1.404(4)
1.401(4)
1.383(5)
115.8(3)
103.5(2)
111.1(2)
113.7(3)
-52.7(3)
-165.3(2)
2.1(7)
A, angles/ꢁ, and torsion angles/ꢁ
of compound 6c
O(2)–N(3)
O(2)–C(7)
N(3)–C(8)
O(5)–N(6)
O(5)–C(10)
C(9)–C(10)
C(9)–C(25)
N(6)–C(25)
C(7)–C(10)
C(8)–C(16)
C(25)–C(26)
O(3)–C(14)
O(3)–C(23)
O(4)–C(13)
O(4)–C(24)
N(1)–N(2)
N(4)–N(5)
C(14)–O(3)–C(23)
C(7)–O(1)–C(8)
O(1)–C(7)–C(10)
C(13)–O(4)–C(24)
O(1)–C(7)–C(10)–O(5)
O(2)–C(7)–C(10)–O(5)
O(3)–C(23)–C(26)–C(25)
C(13)–O(4)–C(24)–C(16)
N(3)–O(2)–C(7)
O(1)–C(8)–N(3)
C(7)–C(10)–C(9)
C(8)–C(16)–C(24)–O(4)
O(1)–C(7)–C(10)–C(9)
O(2)–C(7)–C(10)–C(9)
C(14)–O(3)–C(23)–C(26)
-45.5(3)
-65.2(5)
50.3(4)
123

H. Zeng et al.
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Evaluation of the antioxidant activity
The free radical scavenging effect of the synthesized
compounds 6a–6c on the stable DPPH radical was evalu-
ated [15–17]. Various concentrations of the test compound
were dissolved in 12.5 cm3 of methanol in a volumetric
flask, and then 12.5 cm3 of a methanolic solution of DPPH
(0.2 mmol/dm3) were added to obtain a solution of DPPH
(final concentration of DPPH was 0.1 mmol/dm3). The
mixture was shaken vigorously and incubated in a water
bath at 25 ꢁC for 30 min. The absorbance of the remaining
DPPH was determined colorimetrically at 517 nm. The
same reaction conditions were applied for the blank test
compound to evaluate the interference of the title com-
pound on DPPH assay. The scavenging activity of the tested
title compound was measured as the decrease in absorbance
of the DPPH, and the percentage of activity was calculated.
Vitamin E was used as a reference compound. All tests and
analyses were undertaken on three replicates and the results
averaged. Calculated according to the formula
Scavenging activity ð%Þ ¼ ½½ðAb þ AsÞ ꢁ Amꢂ=Abꢂ
ꢃ 100 %
where Ab is the absorbance of 0.1 mmol/dm3 DPPH
methanol solution at 517 nm, As the absorbance of various
concentration solution of test compound at 517 nm, Am the
absorbance of mixture methanol solution at 517 nm.
21. Halliwell B, Gutteridge JMC, Aruoma OI (1987) Anal Biochem
165:215
Acknowledgments We are grateful to Changji University (Grant
No. 2012SSQD020 and 2013 YJZD001) for the financial help.
22. Kontogiorgis C, Hadjipavlou-Litina D (2003) J Enzyme Inhib
Med Chem 18:63
23. Becher J, Olesen P, Knudsen N, Toftlund H (1986) Sulfur Lett
4:175
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