Antioxidant activities and transition metal ion chelating studies of some hydroxyl Schiff base derivatives

Article abstract of DOI:10.1007/s00044-011-9648-7

Several hydroxyl Schiff base (HSB) compounds (1-10) with good radical scavenging activity (RSA) were designed. Compounds 6, 7, and 10 showed better RSAs than the common synthetic antioxidant 2,6-diterbutyl- 4-methylphenol (BHT) in DPPH and ABTS assays. To probe whether these HSB compounds may exert their antioxidant effect through transition metal ion chelation, the copper and ferrous chelating abilities of them were investigated. It was found by fluorescence quenching spectra that the binding constants K_a were in the range of 0.85×10³-7.30×10⁴ M^-1. Further study was carried out by the complexation of a representative compound 5 with ferrous ion in mass spectrum. A 2:1 5-ferrous complex was readily formed in a methanol-water solution (v:v, 8:2), which confirmed that the chelation happened when the HSB compounds were treated with transition metal ions. The above results indicated that the transition metal ion chelation play an important role in their antioxidant abilities. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9648-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1341–1346
DOI 10.1007/s00044-011-9648-7
ORIGINAL RESEARCH
Antioxidant activities and transition metal ion chelating studies
of some hydroxyl Schiff base derivatives
•
Ye Zhang Biqun Zou Kai Wang Yingming Pan
•
•
•
•
Hong Liang Xianghui Yi Hengshan Wang
•
Received: 9 December 2010 / Accepted: 16 April 2011 / Published online: 2 May 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract Several hydroxyl Schiff base (HSB) com-
pounds (1–10) with good radical scavenging activity (RSA)
were designed. Compounds 6, 7, and 10 showed better
RSAs than the common synthetic antioxidant 2,6-diter-
butyl-4-methylphenol (BHT) in DPPH• and ABTS• assays.
To probe whether these HSB compounds may exert their
antioxidant effect through transition metal ion chelation,
the copper and ferrous chelating abilities of them were
investigated. It was found by fluorescence quenching
spectra that the binding constants Ka were in the range of
0.859103–7.309104 M-1. Further study was carried out
by the complexation of a representative compound 5 with
ferrous ion in mass spectrum. A 2:1 5-ferrous complex was
readily formed in a methanol–water solution (v:v, 8:2),
which confirmed that the chelation happened when the
HSB compounds were treated with transition metal ions.
The above results indicated that the transition metal ion
chelation play an important role in their antioxidant
abilities.
Keywords Hydroxyl Schiff base derivatives ꢀ
Antioxidants activity ꢀ Transition metal ion chelation ꢀ
Radical scavenging activity
Introduction
It is well known that the homeostasis of free radical and
transition metal ion and Fenton reaction play an important
part in the course of aging (Hipkiss, 2005; Halliwell and
Gutteridge, 1999; Perez et al., 2009). Some age-related
diseases such as atherosclerosis, Alzheimer’s, and Parkin-
son diseases, are characteristically associated with free
radical and transition metal ion catalyzed oxidative damage
to carbohydrates, lipids, proteins, and nucleic acids (Hip-
kiss, 2005; Halliwell and Gutteridge, 1999). The homeo-
stasis modulation of free radicals and transition metal ion,
as well as Fenton reaction, is generally maintained by the
balanced system of antioxidant defences (Hipkiss, 2005;
Halliwell and Gutteridge, 1999; Perez et al., 2009). How-
ever, only endogenous antioxidant defenses are not entirely
efficient, exogenous antioxidants are also required. The
exogenous antioxidants, which have good RSAs and tran-
sition metal ion chelation ability, could not only diminish
the cumulative effects of free radicals and oxidative dam-
ages, but also maintain the transition metal ion homeostasis
modulation and Fenton reaction (Hipkiss, 2005; Halliwell
and Gutteridge, 1999; Perez et al., 2009).
Y. Zhang
School of Chemistry and Chemical Engineering, South Central
University, Hunan 410083, People’s Republic of China
Y. Zhang ꢀ B. Zou ꢀ Y. Pan ꢀ H. Liang (&) ꢀ H. Wang (&)
Key Laboratory for the Chemistry and Molecular Engineering of
Medicinal Resources, College of Chemistry & Chemical
Engineering, Guangxi Normal University, Guilin 541004,
People’s Republic of China
Some HSB derivatives, such as compound 1 and 4
(Fig. 1), have been showed to have good RSAs (Tang and
Liu, 2007), it would be very interesting to explore if, tran-
sition metal ion chelation play an important role in their
antioxidant abilities. In addition, a look at the structures of
these HSB derivatives shows that they contain ‘‘metal ion-
binding motifs’’ and thus are expected to bind transition
e-mail: hliang@gxnu.edu.cn
H. Wang
e-mail: wang_hengshan@yahoo.com.cn
Y. Zhang ꢀ K. Wang ꢀ X. Yi (&)
Department of Chemistry and Engineering Technology, Guilin
Normal College, Guilin 541001, People’s Republic of China
e-mail: yixianghui2008@yahoo.com.cn
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Med Chem Res (2012) 21:1341–1346
HO
HO
HO
OH
N
HO
N
HO
N
N
N
HO
HO
4
1
2
3
5
N
N
O
OH
N
N
HO
N
N
N
HO
HO
HO
9
10
6
7
8
Fig. 1 Structure of the HSB compounds
metal ion. Therefore, other HSB derivatives were rationally
designed (Fig. 1) and the antioxidant activities of all the HSB
derivatives were evaluated, especially with regard to their
transition metal ion chelation abilities.
concentrations of HSB compounds can be calculated by
Eq. 1. Then, the values of IC50 that the effective concen-
tration at which 50% of DPPH• or ABTS• were scavenged,
were calculated to evaluate the radical activity from Eq. 1:
Scavenging DPPHꢁðor ABTSꢁÞð%Þ
Absref ꢂ Absdetect
¼
ꢃ 100%:
ð1Þ
Materials and methods
Absref
The procedure of fluorescence spectrum studies
on transition metal ion chelation
Reagents and apparatus
2,6-Diter-butyl-4-methylphenol (BHT), 2,20-Azino-di(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS•) and 1,1-
diphenyl-2-picrylhydrazyl (DPPH•) were purchased from
Sigma Chemicals Co. (St. Louis, MO, USA). Other
chemicals were purchased from China National Medicine
Group Shanghai Corporation (Shanghai, China). All
chemicals and solvents used were of analytical grade. The
UV-1100 spectrophotometer (Beijing Rayleigh Analytical
Instrument Corporation, Beijing, China) were used to
evaluate the radical activity. The fluorescence spectroscopy
was scanned by the RF-5301 spectrophotometer. The NMR
study was carried out by the instrument NMR (BRUKER
AVANCE 500, BRUKER company, Switzerland), and the
mass spectral studies were done using BRUKER
ESQUIRE HCT instrument (BRUKER DALTON com-
pany, USA).
At room temperature, the ten compounds, ferrous nitrate
and copper nitrate were dissolved in DMF-water (v:v =
7:3) to the proper concentration, respectively. After addi-
tion of Fe2? or Cu2? solution (in serial concentration) to
the Schiff bases solution, respectively, the mixture was
allowed to stand for 5 min (until the stable) with inter-
mittent shaking, and the fluorescence spectroscopy was
then scanned.
The procedure of mass spectrum studies on transition
metal ion chelation
A mixture of 5 (30 lM) and ferrous sulfate (10 lM) in
methanol–water solution (v:v, 8:2) was allowed stand for
0.5 h with intermittent shaking, and the mass spectrum was
then scanned.
Free radical scavenging activity
Results and discussion
The experiments of HSB compounds to scavenge DPPH
and ABTS free radical were performed following our pre-
vious reports (Pan et al., 2007, 2010). In brief, the stock
solution of DPPH• was prepared by dissolving DPPH• in
ethanol directly to make the absorbance *1.00 (Absref) at
517 nm. ABTS• aqueous (4.0 mM, 2.00 ml) was oxidized
by 1.41 mM K2S2O8 for 16 h, and then diluted with 100 ml
of ethanol to make the absorbance *0.70 (Absref) at
734 nm. The addition of various concentrations of HSB
derivatives solution in DMF decreased Absref to a stable
value (Absdetect). Thus, the scavenging percentage of
DPPH• or ABTS• in the presence of different
DPPH and ABTS RSAs evaluations were classical assays
in antioxidant activity studies and offer rapid techniques
for screening the RSAs of the compounds. The RSAs of the
HSB compounds were estimated using these two methods
(Pan et al., 2007, 2010). The values of IC50 for compounds
1–10 were tested to evaluate their radical activities
(Table 1). The IC50 of BHT was also determined for
comparison.
As showed in Table 1, all the compounds exhibited good
potent inhibition of DPPH and ABTS radical, since that the
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Med Chem Res (2012) 21:1341–1346
1343
Table 1 IC50 of the HSB compounds in scavenging of ABTS• and DPPH•
Compounds
DPPH• IC50 (lM)
ABTS• IC50 (lM)
Compounds
DPPH• IC50 (lM)
ABTS• IC50 (lM)
1
2
3
4
5
6
a
76.05 (71.50a)
466.46 (0.092bmg/ml)
406.93
117.44 (107.00a)
98.49 (25.00a)
10.83 (20.00a)
6.03 (10.30a)
33.46
7
20.82 (20.77a)
398.62
4.16 (8.21a)
4.93
8
3.68
9
143.32
59.41 (50.24a)
3.99
6.14 (9.97a)
4.68 (8.76a)
5.16 (10.20a)
7.07 (10.19a)
10
BHT
65.80
36.98
The values in parenthesis were from reference (Tang and Liu 2007; Wang et al., 2007)
The IC50 of the lowest RSA compound 2 was also equal to 0.092 mg/ml
b
IC50 of the lowest RSA compound 2 (0.092 mg/ml) was
ions (Cu2? and Fe2?) into the HSB compounds solution,
the fluorescence intensity decreased gradually as in Fig. 2.
Fluorescence quenching can be dynamic, confirmed by
Stern–Volmer equation (1) (Eftink, 1991; Lakowica and
Weber, 1973; Lakowicz, 1999), or static, confirmed by the
modified Stern–Volmer equation (2) (Eftink, 1991; La-
kowica and Weber, 1973; Lakowicz, 1999), and that would
give the information about the binding to transition metal
ions.
further lower than the standard value 10 mg/ml (Lee et al.
2007). Compounds 6, 7, and 10 were shown to be more
potent inhibition of DPPH radical than the common syn-
thetic antioxidant BHT (IC50 65.80 lM), with IC50 of
10.83, 20.82, and 59.41 lM, respectively, while compound
6 displayed the best potent activity. The order of their RSAs
was: 6 [ 7 [ 10 [ BHT [ 1 [ 5 [ 4 [ 9 [ 8 [ 3 [ 2.
On the basis of this observation, it can be suggested that the
presence of electron-donating groups in the para-position of
benzene moiety have an important influence on their DPPH
RSAs.
For dynamic quenching, the fluorescence data can be
described by the Stern–Volmer equation (2) to confirm the
mechanism.
In addition, all the compounds displayed stronger ABTS
RSAs than that of BHT (IC50 36.98 lM), while compound
3 showed the best activity (IC50 3.68 lM) and compound 2
displayed the lowest (IC50 33.46 lM). The order was listed
as follow: 3 [ 9 [ 7 [ 4 [ 8 [ 5 [ 1 [ 10 [ 6 [ 2 [
BHT. From this observation, it could be concluded that the
position and number of hydroxyl groups, as well as elec-
tron-donating groups was very important to their ABTS
RSAs.
F0
¼ 1 þ Kqs0cðQÞ ¼ 1 þ KSVcðQÞ
ð2Þ
F
where F0 and F are the steady-state fluorescence intensities
in the absence and presence of quencher (transition metal
ions), respectively, c(Q) the concentration of quencher
(Cu2? or Fe2?), s0 the average lifetime of the molecule
without any quencher and the fluorescence lifetime of the
biomolecule is 10-8 s (Eftink, 1991; Lakowica and Weber,
1973). From the Stern–Volmer plot (Fig. 3a), the values of
Stern–Volmer quenching constant (KSV) and bimolecular
quenching constant (kq) for the quenchers can be
determined.
To probe whether they may exert their antioxidant effect
through transition metal ion chelation, the chelating abili-
ties with Fe2? and Cu2? were investigated by fluorescence
quenching spectroscopy. Upon addition of transition metal
a
b
25000
20000
15000
10000
5000
0
25000
1
1
20000
12
10
15000
10000
5000
0
450
500
550
600
450
500
550
600
Wavelength (nm)
Wavelength (nm)
Fig. 2 Emission spectra of the representative compound 5 in the
presence of various concentrations of transition metal ion Cu2?
(a) and Fe2? (b). The concentrations of Cu2?(Fe2?) 1 ? 10
(1 ? 12) were: 0, 4.0, 6.0, 7.0, 10.0, 11.0, 12.0, 13.0, 14.0,
15.0 9 10-5 (0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0,
15.0 9 10-5) mol l-1, respectively
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Med Chem Res (2012) 21:1341–1346
Fig. 3 Stern–Volmer plots
7
6
5
4
3
2
1
0
a
b
5
4
3
2
1
0
(a) and Modified Stern–Volmer
plots (b) for the quenching of 5
by transition metal ion Fe2?
0
5
10
15
20
25
30
35
0
3
6
9
C-1(Fe2+)/103L.mol-1
C(Fe2+)/(10-5mol.L-1)
Table 2 Binding constants of the HSB compounds
Products
Ksv/Cu2? (M-1
)
kq/Cu2? (M-1 s-1
)
Ksv/Fe2? (M-1
)
kq/Fe2? (M-1 s-1
)
Ka/Cu2? (M-1
)
Ka/Fe2? (M-1
)
1
2
3.49 9 103
5.20 9 103
1.10 9 105
7.21 9 104
6.07 9 104
1.53 9 104
2.69 9 104
2.49 9 103
2.80 9 103
1.17 9 103
3.49 9 1011
5.20 9 1011
1.10 9 1013
7.21 9 1012
6.07 9 1012
1.53 9 1012
2.69 9 1012
2.49 9 1011
2.80 9 1011
1.17 9 1011
4.09 9 103
7.03 9 103
2.61 9 104
1.43 9 105
6.70 9 104
5.19 9 103
7.40 9 103
6.60 9 103
8.91 9 103
0.90 9 103
4.09 9 1011
7.03 9 1011
2.61 9 1012
1.43 9 1013
6.70 9 1012
5.19 9 1011
7.40 9 1011
6.60 9 1011
8.91 9 1011
0.90 9 1011
3.17 9 103
1.39 9 104
3.02 9 103
4.43 9 103
1.26 9 104
2.36 9 104
3.05 9 103
1.45 9 104
3.51 9 103
0.85 9 103
1.62 9 104
1.47 9 104
3.21 9 103
9.67 9 103
6.48 9 103
7.30 9 104
5.03 9 103
2.47 9 104
6.25 9 103
1.81 9 103
3
4
5
6
7
8
9
10
In Table 2, kq was much greater than the value of the
maximum scatter collision quenching constant 2.0 9
1010 M-1 s-1 (Eftink, 1991; Lakowica and Weber, 1973),
which indicated that the fluorescence quenching was
caused by a specific interaction, and the quenching was
mainly arisen by complex formation (Maurice and Camillo,
1981), while dynamic collision could be negligible in the
concentration studied (Papadopoulou et al., 2005). In
addition, the UV–Vis absorption spectra of the compounds
with transition metal ions system (Fig. 4) were also found
to be obviously different, which confirmed that the
quenching was mainly a static quenching process. There-
fore, the mechanism of fluorescence quenching was a static
quenching procedure and the quenching data must be
analyzed by the modified Stern–Volmer equation (3)
(Lakowica and Weber, 1973):
As can be seen in Table 2, the binding constants Ka were
in the range of 0.859103–7.309104 M-1. Their orders for
the chelation with copper and ferrous ions were: 6 [ 8 [ 2
[ 5 [ 4 [ 9 [ 1 [ 7 [ 3 [ 10 and 6 [ 8 [ 1 [ 2 [ 4
[ 5 [ 9 [ 7 [ 3 [ 10, respectively. Compound 6 showed
the best transition metal ions chelating abilities with Fe2?
and Cu2?, with binding constants of 2.36 9 104 and
7.30 9 104 M-1, respectively. Based on the above obser-
vation, it could be summarized that the position of
hydroxyl group and electron-donating atmosphere had
important influence on their transition metal ion chelation
ability. The above results suggested that the HSB com-
pounds have good chelation abilities with transition metal
ions and the transition metal ion chelation play an impor-
tant role in their antioxidant abilities.
Further studies on the chelation of transition metal ions
by HSB compounds were carried out by the complexation
of the representative compound 5 with ferrous ion in a
mass spectrometer, since that its salen structure may have
good complexation ability. A 8:2 (v:v) mixture of methanol
and water was used as the solvent.
F0
F0
1
1
¼
¼
þ :
ð3Þ
F0 ꢂ F DF faKacðQÞ fa
In this case, DF was the difference of fluorescence
intensity in absence and presence of the quencher at the
concentration c(Q), fa was the fraction of accessible fluo-
rescence. From the modified Stern–Volmer plots (Fig. 3b),
the corresponding binding constants (Ka) could be obtained
(Table 2).
Figure 5 showed the mass spectra for a mixture of
30 lM of 5 with 10 lM of freshly prepared ferrous solu-
tion. Species corresponding to the formation of a 2:1
complex between 5 and ferrous ion was observed in this
case: a 5-Fe2? complex (m/z = 480, [5-Fe(II)-(5-H)]?,
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Med Chem Res (2012) 21:1341–1346
1345
Fig. 4 The UV–Vis absorption
spectra of compound 5 in the
absence and presence of Cu2?
(a) and Fe2? (b). The
1
8
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1
7
b
3.0
2.5
2.0
1.5
1.0
0.5
0.0
a
concentrations of Cu2?(Fe2?
)
1 ? 7 (1 ? 8) were: 0, 2.0,
3.0, 4.0, 5.0, 9.0, 14.0 9 10-5
(0, 3.0, 4.0, 5.0, 7.0, 9.0, 11.0,
12.0 9 10-5) M, respectively
300
400
500
300
400
500
Wavelength (nm)
Wavelength (nm)
Fig. 5 Mass spectra of 30 lM 5 with 10 lM Fe2? in a methanol–water solution (v:v, 8:2)
Fig. 5). A close look at both the ?MS and –MS patterns of
the 5-Fe complex (Fig. 5) suggested that they fit well the
above result. This observation confirmed that the chelation
of transition metal ions happened when the HSB com-
pounds were treated with transition metal ions.
transition metal ions. The results indicated that the HSB
derivatives may exert their antioxidant effect through
transition metal ion chelation and the transition metal ion
chelation play an important role in their antioxidant
abilities.
Acknowledgments This study was supported by the National Nat-
ural Science Foundation of China (No. 20762001), the Project of the
Key Laboratory of Medicinal Chemical Resources and Molecular
Engineering, Guangxi Normal University, China (No.0630006-
5D09), and the Guangxi Department of Education research project
(No. 200807MS075, 200807MS076, 200911MS281, 200911MS282).
Conclusion
In conclusion, we have shown that the rationally designed
HSB derivatives have good RSAs and chelation with
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Med Chem Res (2012) 21:1341–1346
Appendix: synthesis of the derivatives
Ar-H), 7.10 (d, J = 8.56 Hz, 2H, Ar-H), 6.77 (m, 4H, Ar-
H), 3.00 (s, 6H, CH3). EMS: m/z: 241 [M ? H]?.
1
Synthesis
Compound 8: H NMR (DMSO,500HZ) d: 9.48 (s, 1H,
OH), 8.38 (d, J = 8.78 Hz, 1H, N=CH), 7.63 (d, J = 7.39
Hz, 2H, Ar-H), 7.33-7.42 (3H, m, Ar-H), 7.26 (d,
J = 15.96 Hz, 1H, Ar-H), 7.07-7.12 (m, 3H, Ar-H), 6.76
(d, 2H, J = 7.76 Hz, =CH). EMS: m/z: 224 [M ? H]?.
General procedure for the preparation of 1–10: the mixture
of aminophenol (5 mmol), aromatic aldehydes (6 mmol),
ethanol (10 ml), and triethylamine (0.1 mmol) was refluxed
at 90ꢁC for 2 h and then filtered to give crystal or powder
of HSB derivatives.
1
Compound 9: H NMR (DMSO,500HZ) d: 9.77 (s, 1H,
OH), 8.39 (d, J = 8.78 Hz, 1H, N=CH), 7.77 (d, J = 7.39
Hz, 2H, Ar-H), 7.35–7.44 (3H, m, Ar-H), 7.28 (d,
J = 15.96 Hz, 1H, Ar-H), 7.09–7.14 (m, 3H, Ar-H), 6.73
(d, 2H, J = 7.76 Hz, =CH). EMS: m/z: 224 [M ? H]?.
Spectra data for compounds 1–10
1
Compound 10: H NMR (DMSO, 500 Hz) d: 9.90 (s,
1
Compound 1: H NMR (DMSO, 500HZ) d: 9.50 (s, 1H,
1H, OH), 8.41 (s, 1H, N=CH), 7.83 (d, J = 7.37 Hz, 1H),
7.20 (d, J = 8.73 Hz, 2H), 7.07 (t, J = 11.62 Hz, 1H), 6.80
(d, J = 8.32, 2H), 6.67 (d, J = 7.47 Hz, 1H), 6.53-6.46 (m,
1H). EMS: m/z:188 [M ? H]?.
OH), 8.62 (s, 1H, N=CH), 7.90 (d, J = 7.67 Hz, 2H, Ar-H),
7.49–7.51 (m, 3H, Ar-H), 7.21 (d, J = 8.57 Hz, 2H, Ar-H),
6.82 (d, J = 8.57 Hz, 2H, Ar-H). EMS: m/z: 198
[M ? H]?.
1
Compound 2: H NMR (DMSO, 500HZ) d: 8.29 (d,
J = 7.20 Hz, 1H, N=CH), 7.90 (d, J = 8.50 Hz, 1H, Ar-
H), 7.68 (dd, J = 8.79 Hz, 5.5 Hz, 2H, Ar-H), 7.62 (t,
J = 7.26 Hz, 1H, OH), 7.51 (d, J = 7.38 Hz, 1H, Ar-H),
7.37 (d, J = 7.27 Hz, 1H, Ar-H), 7.29 (s, 2H, Ar-H), 6.83
(t, J = 7.30 Hz, 1H, Ar-H), 6.65 (t, J = 7.57 Hz, 1H, Ar-
H), 6.18 (d, J = 7.5 Hz, 1H, Ar-H). EMS: m/z: 198
[M ? H]?
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Compound 3: 1H NMR (DMSO,500HZ) d: 13.12 (s, 1H,
OH), 9.64 (s, 1H, OH), 8.91 (s, 1H, N=CH), 7.67 (d,
J = 7.53 Hz, 1H, Ar-H), 7.42 (d, J = 7.75 Hz, 1H, Ar-H),
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Compound 6: 1H NMR (DMSO,500HZ) d: 9.42 (1s, 1H,
OH), 8.58 (s, 1H, N=CH), 7.82 (d, 2H, J = 8.84Hz, Ar-H),
7.28 (d, J = 7.87Hz, 1H, Ar-H), 7.14 (d, J = 7.94Hz, 1H,
Ar-H), 7.01 (d, J = 8.16Hz, 1H, Ar-H), 6.91 (d,
J = 7.5Hz, 1H, Ar-H), 6.78 (d, J = 8.9Hz, 2H, Ar-H),
3.01 (s, 6H, CH3). EMS: m/z: 241 [M ? H]?
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1
Compound 7: H NMR (DMSO, 500HZ) d: 9.31 (1s,
1H, OH), 8.39 (s, 1H, N=CH), 7.70 (d, J = 8.67 Hz, 2H,
123