The antioxidant effect of imine resveratrol analogues

Article abstract of DOI:10.1016/j.bmcl.2012.06.026

Twenty five Imine resveratrol analogues (IRAs) were synthesized, replacing the CC bond in resveratrol with CN bond, as well as substitution modifications on aromatic rings. Radical scavenging activities against DPPH, along with singlet oxygen quenching capacities were evaluated, and further confirmed using density functional theory calculations (DFT). It was found that IRAs bearing ortho-OH on B ring have better radical scavenging activities against DPPH than resveratrol, these compounds were also found to be effective ¹O ₂ quenchers. Theoretical studies on the reaction mechanism of these compounds with ¹O₂ suggest that the 1,3-addition to a double bond with a -OH group with the formation of allylic hydroperoxide is the most probable reaction route.

Full text of DOI:10.1016/j.bmcl.2012.06.026

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5744–5747
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The antioxidant effect of imine resveratrol analogues
⇑
Jing Lu, Chang Li, Yun-Feng Chai, De-Yu Yang, Cui-Rong Sun
Department of Chemistry, Yuquan Campus, Zhejiang University, No. 38, Zheda Road, Hangzhou 310027, Zhejiang, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty five Imine resveratrol analogues (IRAs) were synthesized, replacing the C@C bond in resveratrol
with C@N bond, as well as substitution modifications on aromatic rings. Radical scavenging activities
against DPPH, along with singlet oxygen quenching capacities were evaluated, and further confirmed
using density functional theory calculations (DFT). It was found that IRAs bearing ortho-OH on B ring have
better radical scavenging activities against DPPH than resveratrol, these compounds were also found to
be effective 1O2 quenchers. Theoretical studies on the reaction mechanism of these compounds with 1O2
suggest that the 1,3-addition to a double bond with a –OH group with the formation of allylic hydroper-
oxide is the most probable reaction route.
Received 24 February 2012
Revised 25 May 2012
Accepted 9 June 2012
Available online 6 July 2012
Keywords:
Imine resveratrol analogue (IRA)
Antioxidant
DPPH
Ó 2012 Elsevier Ltd. All rights reserved.
EPR
Singlet oxygen
Density functional theory calculations (DFT)
Resveratrol, a well-known stilbene phytoalexin present in
grapes, peanuts, mulberries and various medicinal plants, has
attracted a great deal of attention in the past decades mainly be-
cause of its simple structure and significant potential in treating
ageing1 and various human diseases, such as cancer,2,3 neurodegen-
erative diseases,4 cardiovascular diseases5 and stroke.1 Its multiple
activities may be traced back to its antioxidant activity, mainly on
eliminating reactive oxygen species (ROS, including hydroxyl radi-
triolein in micelles.19 Zhou and co-workers studied the scavenging
activity against galvinoxyl radical and antiprolifeactive activity.20
Herein, radical scavenging activities against DPPH, along with sin-
glet oxygen quenching capacities of these IRAs were investigated,
while the mechanism was further deduced using DFT calculations.
The IRAs were prepared as described by Tanaka and Shiraishi
with some modification (Scheme 1)21. Synthetic details, character-
ization (1H NMR, 13C NMR and ESI-MS) and experimental details of
biological evaluation of all IRAs, as well as subsequent DFT calcula-
tion details, are available in the Supplementary data.
DPPH is a relative stable azo radical widely used in antioxidant
activity evaluation. In this study, 25 IRAs’ scavenging activities
against DPPH were performed following our previously reported
cases.22,23 The results presented in Table 1 show that compounds
1–7 have much better scavenging activities compared with
resveratrol, while compounds 8–14 have lower radical scavenging
activities than resveratrol and others have negligible effect on
DPPH-scavenging. Compounds 1–14 which show more or less anti-
oxidant activities all have a OH group on B ring, therefore, it is con-
cluded that OH group on B ring is crucial for these compounds’
radical scavenging activity. Among them, compounds 8–14 have
much lower scavenging activities shows that meta- and para-OH
groups on B ring contribute relatively less to the scavenging activ-
ity. Compounds 1–7 with a same structure moiety scored best,
which indicates that ortho-OH group on B ring is critical in deter-
mination of scavenging activities. The most conceivable reason
for this is that the lone electron pair of the N atom overlap with
the phenolic radical at ortho- position and in this way provide sup-
plementation of an electron to the phenolic radical.19 On the other
hand, only attachment of OH group on A ring cannot make the
cal OH, superoxide anion O2 and singlet oxygen 1O2). Excessive
ROS are considered to be important causative agents to induce the
peroxidation of lipids and oxidative damage of DNA and proteins
in human body6 which result in ageing7 and various disease above
mentioned.8,9 Therefore, the past few years has witnessed many re-
searches focus on the synthesis of new resveratrol analogues and
derivatives aiming to find more potent antioxidants.10–12
Considerable previous researchers found that simple modifica-
tion on resveratrol such as introduction of electron-rich group,13,14
incorporated hybrid moiety15 and elongated the conjugated links16
could significantly enhance its radical scavenging activity. In the
present study, the concept of resveratrol analogue was expanded
by replacing one carbon atom in the C@C bond by one nitrogen
atom, 25 so-called imine resveratrol analogues (IRAs) were synthe-
sized with different substitutions (majorly hydroxyl groups) on
both aromatic rings. Some semblable structures have been studied
in sort of Schiff base. Liu group reported the protective effects of hy-
droxyl-substituted Schiff bases against radical-induced oxidation of
DNA,17 hemolysis of human erythrocytes,18 and peroxidation of
Å
ÅÀ
⇑
Corresponding author. Tel.: +86 571 87953000; fax: +86 571 87951629.
E-mail address: suncuirong@zju.edu.cn (C.-R. Sun).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.026

J. Lu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5744–5747
5745
4
4
2
R
R
water, rt, 2h
3
3
3'
A
B
R
R
CHO
+
H N
R
A
2
3'
N
R
B
2
1
1
2'
1'
R
R
R
R
R
R
1'
2'
R
R
Compounds
R1
R2
R3
H
R4
H
R1’
OH
OH
OH
OH
OH
OH
OH
H
R2’
H
R3’
H
1
H
H
H
2
OH
H
H
H
H
H
3
OH
H
H
H
H
H
4
H
OH
OH
H
H
H
H
5
H
OH
OCH3
H
H
H
H
6
H
H
H
H
7
H
OCH3
H
H
H
H
8
OH
H
H
H
OH
OH
H
H
9
H
OH
H
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
H
H
H
H
OH
OH
OH
OH
OH
H
OH
H
H
H
H
H
H
H
OH
H
H
H
H
H
OH
OCH3
H
OH
OCH3
H
H
H
H
H
H
H
OH
H
H
H
H
OH
OH
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
OH
OH
OH
OH
OCH3
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
CH3
H
H
H
H
H
CH3
OCH3
CH3
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
Scheme 1. Synthetic scheme of imine resveratrol analogues (IRAs) and chemical structures of compounds investigated.
Table 1
DPPH scavenging activities of imine resveratrol analogues (IRAs)
a
a
Compounds
IC50
(lM)
Compounds
IC50 (lM)
1
2
3
4
5
6
7
8
9
23.9 0.5
27.4 0.9
23.1 0.7
24.3 1.0
12.1 0.3
24.9 0.8
28.1 0.9
468.2 5.8
362.1 3.5
10
11
12
13
142.5 1.9
561.3 4.7
94.3 2.0
163.6 2.7
413.4 4.6
>1000
14
Other
Resveratrol
Vitamin C
b
71.2 2.3
19.3 0.6
b
a
IC50 values were expressed as means standard deviation for three
determinations.
b
Resveratrol and vitamin C were used as positive control.
compounds radical scavenger, but it can affect the scavenging abil-
ity of compounds bearing OH group on B ring. IC50 values of com-
pounds 2–4 indicate that meta- or para-OH groups on A ring have
more positive effect than ortho-OH, while methoxy group is nega-
tive on corresponding OH-substituted compounds on scavenging
activity. Notably, compound 5 bearing ortho-OH group on B ring
and the o-diphenolic group on A ring is the most effective one,
which may due to the stabilization effect of the intramolecular
hydrogen bond on resulting o-hydroxyphenoxyl radical.24,25 In
conclusion, these results reveal that the higher radical scavenging
activity of compound 5 is not only related to the the ortho-OH
group on B ring, but also the o-diphenolic group which can stabi-
lize the formed radical.
Figure 1. Quenching effect of compound 4 on TEMP-1O2 adduct formation.
Table 2
Quenching capacities of imine resveratrol ana-
logues on singlet oxygen 1O2
Compounds
IC50(l
M)a
1
2
3
4
5
6
7
3.47 0.13
2.92 0.12
2.18 0.10
0.99 0.06
2.26 0.15
1.42 0.04
1.30 0.06
16.94 0.73
Resveratrolb
Based on these encouraging phenomena, further investigation
on IRAs’ effect on singlet oxygen was also carried out. Singlet oxy-
gen 1O2, a reactive form of molecular oxygen usually generated in
the photosensitized oxidations process, can cause severe damage
a
IC50 values were expressed as means stan-
dard deviation for three determinations.
b
Resveratrol was used as positive control.

5746
J. Lu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5744–5747
O O
1O2
A
B
N
N
HO
HO
O O
1O2
N
N
HO
HO
OOH
1O2
H2O
C
D
N
N
O
O
N
HO
HO
O
1O2
N
N
HO
O
OOH
Scheme 2. Four possible reaction routes between compound 1 and 1O2.
IC value, which is only 0.99
lM, more than 15-fold higher than
50
that of resveratrol, and even better than curcumin (a well-known
1O2 quencher, IC50 = 2.75 M).32
l
Furthermore, compound 1 was representatively studied by DFT
calculations, aiming to give deeper insight into the mechanism of
these compounds’ effect on 1O2. According to some well-accepted
results,31,33–35 four possible oxygen-addition reaction types were
proposed and each type have similar reaction sites, so four corre-
sponding possible reactions are take as examples shown in Scheme
2. (A 1,2-cycloaddition to an isolated double bond; B 1,4-cycload-
dition to two conjugated double bonds; C 1,4-addition to phenols;
D 1,3-addition to a double bond connected to a OH group). All
these possible reaction products were examined to determine the
thermodynamically preferred reaction routes. Ten products are
thermodynamically permitted with energies ranging from À0.014
to À19.45 kcal/mol. On the basis of their degree of
DG, the full path
of three most exergonic reactions (Scheme 2. B, C, D) with energies
ranging from À14.84 to À19.45 kcal/mol were further investigated.
Comparing the three possible competing routes (Fig. 2), route D has
the lowest activation energy, with an energy barrier of 13.10 kcal/
mol relative to the reactants, and the most negative free energy
with
D
G À19.45 kcal/mol, therefore it is predicted to be the most
likely route for these compounds reacting with 1O2.
In conclusion, 25 IRAs were synthesized for radical scavenging
activity research. It was found that seven compounds bearing
ortho-OH on B ring have much better radical scavenging activity
against DPPH than resveratrol. These compounds are also found to
be effective1O2 quenchersbyusingtheEPRspin-trappingtechnique.
Theoretical study of the reaction mechanism suggests that 1,3-addi-
tion to a double bond with OH group to form of an allylic hydroper-
oxide is the most probable reaction route. These results may provide
useful information for designing new drug for treating singlet oxy-
gen mediated diseases such as cataractogenesis,36 pellagra, erythro-
poietic protoporphyria, porphyries,37 ageing and cancer.38
Figure 2. Reaction profile for the three most exergonic reaction routes between
compound 1 and 1O2 using DFT at the B3LYP/6–31 + G(d) level.
26
to a large number of biological targets including lipids,
DNA,27
proteins,26,28 and other important biological molecules.29 Our
group has previously found that resveratrol and its oligomers were
selective 1O2 quenchers but not effective OH and O2 scaveng-
ers.23,30,31 Herein, EPR spin-trapping technique was applied to
determine the quenching activities of selected IRAs as previously
described.31 Compounds 1–7 which show better DPPH scavenging
activities were chosen as the objects of study. The EPR spectra in
Figure 1 reveals that compound 4, as an example, inhibited the for-
mation of TEMP-1O2 adduct in a dose-dependent manner. Only
Å
ÅÀ
Acknowledgment
1.3
l
M compound 4 can obviously inhibit the generation of 1O2,
M caused complete inhibition. The results
Financial support from the NSFC (Nos. 21072174) is gratefully
acknowledged.
and the addition of 37
l
presented in Table 2 demonstrate that all these seven compounds
are effective 1O2 quenchers, which have much better quenching
activities compared with resveratrol. The activity order is like
4 > 7 > 6 > 3 > 5 > 2 > 1 > resveratrol. The introduction of OH group
on A ring and methoxy group on meta- or para-position all in-
creased the quenching activities. Compound 4 have the minimum
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.026.

J. Lu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5744–5747
17. Zhao, F.; Liu, Z.-Q. J. Phys. Org. Chem. 2009, 22, 791.
5747
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