Radical-scavenging activity and mechanism of resveratrol-oriented analogues: Influence of the solvent, radical, and substitution

Article abstract of DOI:10.1021/jo9007095

(Chemical Equation Presented). Resveratrol (3,5,4′-trihydroxy-trans- stilbene, 3,5,4′-THS) is a well-known natural antioxidant and cancer chemopreventive agent that has attracted much interest in the past decade. To find a more active antioxidant and investigate the antioxidative mechanism with resveratrol as the lead compound, we synthesized 3,5-dihydroxy-trans-stilbene (3,5-DHS), 4-hydroxy-trans-stilbene (4-HS) 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4′-dihydroxy-trans-stilbene (4,4′-DHS), 4-hydroxy-3-methoxy-trans-stilbene (3-MeO-4-HS), 4-hydroxy-4′-methoxy- trans-stilbene (4′-MeO-4-HS), 4-hydroxy-4′-methyl-trans-stilbene (4′-Me-4-HS), 4-hydroxy-4′-nitro-trans-stilbene (4′-NO ₂-4-HS), and 4-hydroxy-4′-trifluoromethyl-trans-stilbene (4′-CF₃-4-HS). The radical-scavenging activity and detailed mechanism of resveratrol and its analogues (ArOHs) were investigated by the reaction kinetics with galvinoxyl (GO^?) and 2,2-diphenyl-1- picrylhydrazyl (DPPH^?) radicals in ethanol and ethyl acetate at 25°C, using UV-vis spectroscopy. It was found that the reaction rates increase with increasing the electron-rich environment in the molecules, and the compound bearing o-dihydroxyl groups (3,4-DHS) is the most reactive one among the examined resveratrol analogues. The effect of added acetic acid on the measured rate constant for GO^?-scavenging reaction reveals that in ethanol that supports ionization solvent besides hydrogen atom transfer (HAT), the kinetics of the process is partially governed by sequential proton loss electron transfer (SPLET). In contrast to GO^?, DPPH ^? has a relatively high reduction potential and therefore enhances the proportion of SPLET in ethanol. The relatively low rate constants for the reactions of ArOHs with GO^? or DPPH^? in ethyl acetate compared with the rate constants in ethanol prove that in ethyl acetate these reactions occur primarily by the HAT mechanism. The contribution of SPLET and HAT mechanism depends on the ability of the solvent to ionize ArOH and the reduction potential of the free radical involved. Furthermore, the fate of the ArOH-derived radicals, i.e., the phenoxyl radicals, was investigated by the oxidative product analysis of ArOHs and GO^? in ethanol. The major products were dihydrofuran dimers in the case of resveratrol, 4,4′-DHS, and 4-HS and a dioxane-like dimer in the case of 3,4-DHS. It is suggested from the oxidative products of these ArOHs that the hydroxyl group at the 4-position is much easier to subject to oxidation than other hydroxyl groups, and the dioxane-like dimer is formed via an o-quinone intermediate.

Full text of DOI:10.1021/jo9007095

Radical-Scavenging Activity and Mechanism of Resveratrol-Oriented
Analogues: Influence of the Solvent, Radical, and Substitution
Ya-Jing Shang, Yi-Ping Qian, Xiao-Da Liu, Fang Dai, Xian-Ling Shang, Wen-Qiang Jia,
Qiang Liu, Jian-Guo Fang,* and Bo Zhou*
State Key Laboratory of Applied Organic Chemistry, Lanzhou UniVersity, Lanzhou, Gansu 730000, China
bozhou@lzu.edu.cn; fangjg@lzu.edu.cn
ReceiVed April 8, 2009
Resveratrol (3,5,4′-trihydroxy-trans-stilbene, 3,5,4′-THS) is a well-known natural antioxidant and cancer
chemopreventive agent that has attracted much interest in the past decade. To find a more active antioxidant
and investigate the antioxidative mechanism with resveratrol as the lead compound, we synthesized 3,5-
dihydroxy-trans-stilbene (3,5-DHS), 4-hydroxy-trans-stilbene (4-HS) 3,4-dihydroxy-trans-stilbene (3,4-DHS),
4,4′-dihydroxy-trans-stilbene (4,4′-DHS), 4-hydroxy-3-methoxy-trans-stilbene (3-MeO-4-HS), 4-hydroxy-4′-
methoxy-trans-stilbene (4′-MeO-4-HS), 4-hydroxy-4′-methyl-trans-stilbene (4′-Me-4-HS), 4-hydroxy-4′-nitro-
trans-stilbene (4′-NO2-4-HS), and 4-hydroxy-4′-trifluoromethyl-trans-stilbene (4′-CF3-4-HS). The radical-
scavenging activity and detailed mechanism of resveratrol and its analogues (ArOHs) were investigated by
the reaction kinetics with galvinoxyl (GO•) and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radicals in ethanol
and ethyl acetate at 25 °C, using UV-vis spectroscopy. It was found that the reaction rates increase with
increasing the electron-rich environment in the molecules, and the compound bearing o-dihydroxyl groups
(3,4-DHS) is the most reactive one among the examined resveratrol analogues. The effect of added acetic
acid on the measured rate constant for GO•-scavenging reaction reveals that in ethanol that supports ionization
solvent besides hydrogen atom transfer (HAT), the kinetics of the process is partially governed by sequential
proton loss electron transfer (SPLET). In contrast to GO•, DPPH• has a relatively high reduction potential and
therefore enhances the proportion of SPLET in ethanol. The relatively low rate constants for the reactions of
ArOHs with GO• or DPPH• in ethyl acetate compared with the rate constants in ethanol prove that in ethyl
acetate these reactions occur primarily by the HAT mechanism. The contribution of SPLET and HAT
mechanism depends on the ability of the solvent to ionize ArOH and the reduction potential of the free radical
involved. Furthermore, the fate of the ArOH-derived radicals, i.e., the phenoxyl radicals, was investigated by
the oxidative product analysis of ArOHs and GO• in ethanol. The major products were dihydrofuran dimers
in the case of resveratrol, 4,4′-DHS, and 4-HS and a dioxane-like dimer in the case of 3,4-DHS. It is suggested
from the oxidative products of these ArOHs that the hydroxyl group at the 4-position is much easier to subject
to oxidation than other hydroxyl groups, and the dioxane-like dimer is formed via an o-quinone intermediate.
Introduction
in at least 70 plant species, a number of which are dietary
components including grapes, mulberries, and peanuts.1 Its
relatively high concentration in red wine (0.1-14.3 mg/L)1 has
led to its being proposed as the main protagonist for the so-
called “French paradox”; that is, despite fat-rich diets, mortality
Resveratrol (3,5,4′-trihydroxy-trans-stilbene) (Figure 1) is a
naturally occurring phytoalexin with a stilbene structure found
* To whom correspondence should be addressed. Fax: (+86) 931-8915557.
10.1021/jo9007095 CCC: $40.75  2009 American Chemical Society
Published on Web 05/27/2009
J. Org. Chem. 2009, 74, 5025–5031 5025

Shang et al.
and apoptosis-inducing activity on human promeolocytic leu-
kemia cells.8 In the antioxidant reaction of resveratrol, the
hydrogen abstraction from 4′-OH is more favorable than that
from 3-OH or 5-OH as evidenced by stationary γ-radiolysis
and pulse radiolysis experiments,4i,j oxidation product analysis,9
experimental X-ray structure,10 and theoretical calculations.11
It was reported previously that the 4′-OH in resveratrol is
responsible for its biological activities.12 Therefore, it is of
interest to extend the research and study the structure-activity
relationship of resveratrol-oriented analogues by the introduction
of electron-donating (ED) and electron-withdrawing groups
(EW) in the ortho- or para-position of 4-OH. On the other hand,
phenol antioxidants (ArOH) react with free oxygen-centered and
nitrogen-center radicals (X•) via three different mechanisms
(Scheme 1): (1) a one-step hydrogen atom transfer from phenol
to X• (HAT mechanism), (2) a sequential proton loss electron
transfer process from phenoxide anion (ArO-) to X• (SPLET
mechanism), and (3) an electron-transfer process from ArOH
to X• follwed by proton transfer (ET-PT mechanism).13 To
investigate the detailed antioxidant mechanism and the structure
basis for the antioxidant activity of resveratrol, we report herein
a quantitative kinetic study of the scavenging reaction of
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FIGURE 1. Molecular structures of resveratrol and its analogues.
from coronary heart disease is lower in France than in other
countries due to the moderate consumption of red wine.2 This
compound has attracted much attention during the last years
due to its simplicity in structure, its low toxicity in in vivo
system, and its activities against a collection of diseases
including heart disease, aging, and cancer.1 The cancer chemo-
prevention effects, one of the most striking biological activities,
elicited by resveratrol can be traced back to its antioxidant
activity, because free-radical-mediated peroxidation of mem-
brane lipids and oxidative damage of DNA might play a
causative role in cancer.3 Therefore, the past decade has
witnessed tremendous interest in the antioxidant activity of
resveratrol,4 and an effective effort in the synthesis of new
resveratrolanalogues,aimingtofindmoreeffectiveantioxidants4e,h
and cancer chemopreventive agents.5
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Chem. 2004, 52, 7279–7285.
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G.; Forti, L.; Pagnoni, U. M.; Albini, A.; Prosperi, E.; Vannini, V. J. Biol. Chem.
2001, 276, 22586–22594.
In our ongoing research project on bioantioxidants, we
previously found that simple structural modification of resvera-
trol could significantly enhance its antioxidant activity against
free radical-induced lipid peroxidation,6 prooxidant activity on
DNA damage in the presence of Cu(II) ions,7 and cytotoxicity
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(b) Foti, M. C.; Daquino, C.; Ceraci, C. J. Org. Chem. 2004, 69, 2309–2314.
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Kawashima, T.; Ohkubo, K.; Kanazawa, H.; Inami, K.; Mochizuki, M.; Fukuhara,
K.; Okuda, H.; Ozawa, T.; Itoh, S.; Fukuzumi, S.; Ikota, N. Org. Biomol. Chem.
2005, 3, 626–629. (f) Litwinienko, G.; Ingold, K. U. Acc. Chem. Res. 2007, 40,
222–230.
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658, 68–94.
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J. 2003, 17, 1195–1214.
5026 J. Org. Chem. Vol. 74, No. 14, 2009

Mechanism of ResVeratrol-Oriented Analogues
SCHEME 1. Antioxidative Mechanisms of Phenol
Antioxidants (ArOH): (1) HAT Mechanism; (2) SPLET
Mechanism; and (3) ET-PT Mechanism
resveratrol and its analogues (ArOHs) toward galvinoxyl (GO•)
or 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical in ethanol and
ethyl acetate at 25 °C. The resveratrol-oriented analogues studied
were 3,5-dihydroxy-trans-stilbene (3,5-DHS), 4-hydroxy-trans-
stilbene (4-HS), 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4′-
dihydroxy-trans-stilbene (4,4′-DHS), 4-hydroxy-3′-methoxy-
trans-stilbene (3-MeO-4-HS), 4-hydroxy-4′-methoxy-trans-
stilbene (4′-MeO-4-HS), 4-hydroxy-4′-methyl-trans-stilbene (4′-
Me-4-HS), 4-hydroxy-4′-nitro-trans-stilbene (4′-NO2-4-HS), and
4-hydroxy-4′-trifluoromethyl-trans-stilbene (4′-CF3-4-HS). The
oxidation products of ArOHs obtained in the presence of GO•
in ethanol were also studied to help elucidate the structure-
activity relationship and the fate of the ArOH-derived radicals.
The structure-activity relationship and reaction mechanism are
discussed based on the kinetic results and oxidation products
analysis obtained in this study, providing insight into the
mechanistic details and the development of resveratrol-oriented
antioxidants.
FIGURE 2. Spectral changes observed upon addition of resveratrol
(50 µM) to an ethanol solution of GO• (5 µM) at 298 K (interval: 3
min). Inset: Plot of the pseudo-first-order rate constant (kobs) vs. the
concentration of resveratrol.
TABLE 1. Rate Constants for Radical-Scavenging Reactions of
ArOHs at 25 °Ca
k (M-1 s-1) (GO•)
k (M-1 s-1) (DPPH•)
ArOHs
ethanol
ethyl acetate
ethanol
ethyl acetate
Resveratrol
3,5-DHS
4-HS
3,4-DHS
4,4′-DHS
3-MeO-4-HS
4′-MeO-4-HS
4′-Me-4-HS
4′-NO2-4-HS
4′-CF3-4-HS
29.8
2.1
29.4
15.7
1.1
9.7
95.1
1.2
1.6
0.05
0.6
83.1
6.4
9.1
1.9
1.1
0.3
106.9
b
2147.6
414.0
1095.6
202.9
92.3
1468.0
108.6
101.0
33.0
15.7
-
2982.2
2298.1
358.0
151.0
6.1
c
c
-
-
9.9
3.2
23.5
0.7
a Data are the averages of three reproducible determinations with a
deviation of less than (10%. b Reaction rate is too fast to be
determined. c Reaction rate could not be measured because of spectral
overlapping.
Results
Radical-Scavenging Reactions of Resveratrol and Its
Analogues in Ethanol and Ethyl Acetate. GO• is a relatively
stable oxygen radical and has been widely used for evaluating
antioxidant activities. Direct measurements of the rate of the
reaction between ArOHs and GO• were performed in ethanol
at 25 °C by UV-vis spectroscopy. Upon addition of resveratrol
to an ethanol solution of GO•, the absorption band at 428 nm
due to GO• disappeared immediately as shown in Figure 2. The
decay of GO• obeyed pseudo-first-order kinetics when the
concentration of resveratrol was maintained at more than 10-
fold excess of the concentration of GO•. Plotting this pseudo-
first-order rate constant (kobs) versus the concentration of
resveratrol gave a straight line (the inset of Figure 2), from
which the second-order rate constant (k) for the GO•-scavenging
reaction by resveratrol could be obtained. Other ArOHs gave
the same second-order kinetics and their second-order rate
constants were listed in Table 1. It can be seen from Table 1
that GO•-scavenging activity of ArOHs follows the sequence
of 3,4-DHS > 3-MeO-4-HS > 4,4′-DHS > 4′-MeO-4-HS > 4′-
Me-4-HS > resveratrol ≈ 4-HS > 4′-CF3-4-HS > 3,5-DHS. The
GO•-scavenging activity of 4-HS is higher than that of 3,5-DHS
and is similar to that of resveratrol, indicating that hydrogen
transfer from 4′-OH of resveratrol to GO• takes place. By
comparing the k values for 4-HS, 3,4-DHS, 4,4′-DHS, 3-MeO-
4-HS, 4′-MeO-4-HS, and 4′-Me-4-HS, it is clear that the
introduction of ED groups, such as methyl, methoxy, and
hydroxyl, in the ortho- or para-position of 4-OH, remarkably
increases the GO•-scavenging activity. The compound bearing
o-dihydroxyl groups (3,4-DHS) is the most reactive one among
the ArOHs and its k value is 70 times larger than that of 4-HS
or resveratrol. On the other hand, the reaction rate of 4′-CF3-
4-HS is 3 times smaller than that of 4-HS, and this may be due
to the presence of an EW group, trifluoromethyl.
It has been reported that ethyl acetate and ethanol have
identical hydrogen bond-accepting activity (ꢀ2 ) 0.45).13c
H
However, ethyl acetate has much lower dielectric constants
(ε ) 6.02)13c than ethanol (ε ) 24.30)13c and hence has a lower
ability to support ionization of the substrate. To investigate the
GO•-scavenging reaction mechanism by ArOHs, the reaction
kinetics was also performed in ethyl acetate at the same
temperature. The results are summarized in Table 1. The
structure-activity relationship obtained in ethyl acetate is similar
to that obtained in ethanol. However, the rate constants for the
reactions of GO• with ArOHs in ethyl acetate are 2-10 times
smaller than that in ethanol.
The DPPH• has been widely used to measure the radical-
trapping abilities of natural antioxidants and served the mecha-
nistic studies in the past.13 In contrast to GO• (E0red vs. SCE )
0.05 V),14 DPPH• has a relatively high reduction potential (E0
red
vs. SCE ) 0.18 V),14 thereby facilitating the electron transfer
process. The reaction rates of DPPH• with ArOHs were
determined by monitoring the decay of DPPH• at 517 nm in
ethanol and ethyl acetate, and the results are summarized in
(14) Nakanishi, I.; Kawashima, T.; Ohkubo, K.; Iizuka, Y.; Inami, K.;
Mochizuki, M.; Urano, S.; Itoh, S.; Miyata, N.; Fukuzumi, S. J. Chem. Soc.,
Perkin Trans. 2 2002, 1520–1524.
J. Org. Chem. Vol. 74, No. 14, 2009 5027

Shang et al.
TABLE 2. Effect of Acetic Acid on Rate Constants of ArOHs in Ethanol at 25 °Ca
k (M-1 s-1) (GO•)
k (M-1 s-1) (DPPH•)
[CH3COOH] (µM)
[CH3COOH] (µM)
ArOHs
0
10
50
1000
5000
10000
0
10
50
1000
5000
10000
Resveratrol
3,5-DHS
4-HS
3,4-DHS
4,4′-DHS
3-MeO-4-HS
4′-MeO-4-HS
4′-Me-4-HS
4′-NO2-4-HS
4′-CF3-4-HS
29.8
2.1
29.4
22.6
1.4
20.2
14.5
1.0
9.0
11.6
0.8
4.7
11.3
0.8
4.6
10.9
0.8
4.6
95.1
1.2
54.1
0.9
26.0
0.7
3.9
0.5
3.9
2.5
0.5
2.1
2.0
0.5
1.9
106.9
61.4
33.5
b
c
c
c
c
c
2147.6
414.0
1095.6
202.9
92.3
1516.6
231.0
515.2
109.8
37.8
686.6
93.4
294.6
36.8
15.5
508.0
68.0
135.2
21.4
11.7
462.6
57.2
117.6
19.2
10.8
432.6
64.4
111.8
18.3
9.1
-
-
-
-
-
-
2982.2
2298.1
358.0
151.0
6.1
1331.5
919.2
260.5
91.9
562.7
448.3
130.0
57.8
2.6
41.9
81.9
9.4
27.2
27.7
6.2
27.0
28.0
5.9
6.5
0.6
3.9
2.9
0.5
0.5
2.9
0.4
0.43
d
d
d
d
d
d
-
-
-
-
-
-
4.5
21.3
9.9
5.9
3.0
1.6
1.5
1.3
23.5
12.3
a The k values were given with errors usually less than 10%. b Reaction rate is too fast to be determined. c No determination. d Reaction rate could not
be measured because of spectral overlapping.
TABLE 3. Oxidative Dimerization of ArOHs with Galvinoxyl
Table 1. A similar structure-activity relationship was also
obtained in the case of DPPH•, that is, the k value increases
with the introduction of ED groups (methyl, methoxy, and
hydroxyl) and decreases with the introduction of EW groups
(nitro and trifluoromethyl), but the reaction rate for the two
radicals (DPPH• and GO•) was appreciably different. In ethanol
that supports ionization solvent the reaction rate is quicker in
the case of DPPH• than in the case of GO•. The differences and
details will be discussed in the following sections.
Effect of Acetic Acid on the Rates of Radical-Scaveng-
ing Reactions. In solvents that support ionization, such as
alcohols and water, ArOH may be in equilibrium with the
corresponding phenolate anion (ArO-) [eq 1], which is a much
stronger electron donor as compared to the parent ArOH. If
ArO- acts as the electron donor and the SPLET mechanism is
operative in solvents that support ionization, the addition of acid
will suppress the ionization of ArOH and the measured rate
constant might therefore be expected to decrease. When the
electron donor is the parent molecule (ArOH) and the ET-PT
mechanism occurs, the addition of acid will increase or remain
the measured rate constant.13d Recently, Litwinienko and Ingold
have clearly demonstrated the occurrence of SPLET in the
reaction of DPPH• with some phenolic compounds in methanol
and ethanol by studying the effect of added acetic acid on the
measured rate constant.13a,c,d
Radical in Ethanol at Room Temperature
ArOHs
time (h)
conversion (%)
isolated yield (%)
resveratrol
4-HS
4,4′-DHS
3,4-DHS
8
8
4
3
78
85
80
41
42
56
87
100
Oxidative Products of Resveratrol and Its Analogues in
the Presence of GO• in Ethanol. Whether the electron-transfer
or the hydrogen atom-transfer process from ArOH to free radical
will result in the formation of the same phenoxyl radical, ArO•.
To investigate the fate of ArO•, we also isolated and identified
the reaction products of resveratrol, 4-HS, 4,4′-DHS, and 3,4-
DHS with GO• in ethanol at room temperature (Table 3). The
major products (Figure 3) were dihydrofuran dimers in the case
of resveratrol, 4-HS, and 4,4′-DHS and a dioxane-like dimer in
the case of 3,4-DHS by characterizing with HRMS (ESI) and
1D and 2D NMR (see the Supporting Information). It is
suggested from the oxidative products that the hydroxyl group
at the 4-position is much easier to subject to oxidation than
other hydroxyl groups, and the dioxane-like dimer is formed
via an o-quinone intermediate (vide infra). The oxidative product
analysis also suggests the importance of o-dihydroxyl groups
and 4-hydroxyl groups. It is worth noting that the oxidative
coupling products of ArOHs in the presence of GO• were
obtained in good yields and conversions (Table 3). This provides
an efficient approach for the synthesis of resveratrol-based
dimers.
ArOH h ArO- + H+
(1)
As an example, the oxidation of 3,4-DHS in the presence of
GO• and the structural characterization of its dimer will be
discussed. The HRMS (ESI) analysis of the major product
showed an [M+H]+ of 423.1597 (calculation: 423.1591),
To rationalize the mechanism and actual electron donor, the
effect of acetic acid on the radical-scavenging rates of ArOHs
in ethanol was examined. The results are summarized in Table
2. Addition of acetic acid to the ethanol-containing resveratrol
from 10 µM to 10 mM reduced the rate of the GO•-scavenging
reaction by a factor of 3 and to the limiting value (Table 2),
suggesting that the actual electron donor is ArO- and SPLET
does occur in nonacidified ethanol and acid reduces the rate by
eliminating SPLET to leave only HAT (vide infra). The limiting
value and 5- to 10-fold decline in the rate of the GO•-scavenging
reaction were also found in the case of 3,4-DHS and other
analogues (Table 2). Similar results were obtained in the case
of DPPH•, but the effect of acetic acid on the reaction rate
became much more pronounced. For example, the rate was
decreased 50- and 100-fold for resveratrol and 4,4′-DHS (Table
2), respectively. The mechanistic details in the radical-scaveng-
ing reaction will be discussed in the following sections.
FIGURE 3. Oxidation products of resveratrol and its analogues
obtained in the presence of GO• in ethanol.
5028 J. Org. Chem. Vol. 74, No. 14, 2009

Mechanism of ResVeratrol-Oriented Analogues
SCHEME 2. GO•-Scavenging Reaction Mechanism by Resveratrol in Ethanol
demonstrating the structure of a dehydrodimer, and its 1H NMR
spectrum gave additional information that allowed its identifica-
tion with the structure. Despite the fact that the product was a
dimer, only two phenolic OH (a singlet at δ 8.01), two aliphatic
protons (doublets at δ 4.92 and 5.05, J ) 8.0 Hz, H-7 and H-8),
two trans-olefinic protons (two doublets at δ 7.13 and 7.19,
with a large coupling constant of 16.0 Hz, H-7′ and H-8′), and
thirteen aromatic protons were present. The correct structural
assignments could be made on the basis of homonuclear
bidimensional correlation (1H,1H-COSY), heteronuclear 1H,13C-
HMQC, and long-range 1H,13C-HMBC correlation experiments.
The long-range correlation between the doublet at δ 6.51
(H-6) and the aliphatic carbon at δ 81.1 (related to the doublet
at δ 4.92, C-7), as well as between the doublet at δ 6.77 (H-2)
and the carbon at δ 81.1 (related to the doublet at δ 4.92, C-7),
allowed us to establish that the 3,4-dihydroxybenzoyl moiety
was bonded to C-7. The long-range correlation between the
multiplet at δ 7.26 (H-10 and H-14) and the aliphatic carbon at
δ 81.3 (related to the doublet at δ 5.05, C-8) allowed us to
establish that the phenyl moiety was bonded to C-8. All these
data were consistent with the proposed “closed” and dioxane-
like dimer formed via an o-quinone intermediate (vide infra).
activity of nine resveratrol-oriented analogues with the different
structural features that enables us to deduce a clear picture on
the structural determinants for the activity and a structure-activity
relationship. Furthermore, the study on the reaction kinetics
gives us important information for understanding the reactive
mechanism of these antioxidants.
Structure-Activity Relationship. The oxidative product of
resveratrol demonstrates that the hydroxyl group at the 4′-
position is much easier to subject to oxidation than other
hydroxyl groups. This is in accordance with that obtained from
the UV-visible spectra change mentioned above and the
previous observation by stationary γ-radiolysis and pulse
radiolysis experiments,4i,j experimental X-ray structure,10 and
theoretical calculations.11 Theoretical calculations10 showed that
the most stable phenolate in resveratrol is obtained after
extraction of the proton in the 4′-position, which is ∼7 kcal/
mol lower than those having extracted the protons in the 3-
and 5-position. Therefore, the 4′-OH in resveratrol provides the
most acidic hydrogen and its removal from the 4′-OH is highly
preferred. Pulse radiolysis experiments also indicated that
spectral and kinetic properties of the phenoxyl radicals show
great similarity between resveratrol and 4-HS; thus, 4′-OH of
resveratrol scavenges free radicals more effectively than its
3-OH and 5-OH.4i,j The oxidation reaction of 4′-OH resulted
in the formation of phenoxyl radicals or semiquinone (“A”, “B”,
and “C”), whose existence was also elucidated by the electron
paramagnetic resonance4h and pulse radiolysis experiments.4i,j
Successively, the coupling of one radical “B” and one radical
“C”, followed by tautomeric rearrangement and intramolecular
nucleophilic attack to the intermediate quinone, gave the
dihydrofuran dimer as shown in Scheme 2.
3
Moreover, more than 8 values for the J coupling constant
between H-7 and H-8 in the dimer suggested a predominant
pseudo-trans-axial arrangement for these two aliphatic protons.
Furthermore, enantiomeric composition of the dimer was
evaluated by HPLC analysis on a chiral column and it was found
that the dimer was a racemic mixture (see the Supporting
Information).
Discussion
Resveratrol’s significant antioxidant and cancer chemopre-
vention activities, along with its low molecular weight and lack
of toxicity, make this molecule an ideal lead compound for
development of new antioxidants and cancer chemoprevention
agents. Also, the detailed antioxidant mechanism of resveratrol
and its analogues is important for understanding their biological
activities. The present work studied the radical-scavenging
It can be seen from Table 1 that the radical-scavenging
activity of ArOHs increases with the introduction of ED groups
(methyl, methoxy, and hydroxyl) in the ortho- or para-position
of 4-OH. On the contrary, the activity decreases in the presence
of EW groups (trifluoromethyl and nitro). It has been proven
that the bond dissociation energy (BDE) of O-H regulates the
J. Org. Chem. Vol. 74, No. 14, 2009 5029

Shang et al.
SCHEME 3. GO•-Scavenging Reaction Mechanism by
3,4-DHS in Ethanol
of the rate constant for the HAT process and the rate constant
for reaction of radical with the phenoxide anion (SPLET)
[eq 2]. Schemes 2 and 3 show cooperation between hydrogen-
abstraction and electron-transfer processes in the GO•-scaveng-
ing reaction by resveratrol and 3,4-DHS in ethanol. The
antioxidant mechanism of the resveratrol, 3,5-DHS, and 4-HS
was well investigated previously by pulse radiolysis experiments
and their anionic phenoxyl radicals (semiquinone anion) were
also identified from the transient absorption spectra in alkaline
solution.4j It should be pointed out that the relative contribution
of SPLET and HAT processes in the radical-scavenging reaction
depends on the experimental conditions such as the ability of
the solvent to ionize ArOHs and the character of the attacking
radical. The relatively low rate constants for the reactions of
ArOHs with GO• in ethyl acetate compared with the rate
constants in ethanol (Table 1) clearly indicates that in ethyl
acetate having low ability to ionize ArOHs the reactions occur
primarily by the HAT mechanism. In contrast to GO•, DPPH•
has a relatively high reduction potential and therefore more
easily undergoes the electron-transfer reaction. The extremely
high rate constants for the reactions of ArOHs with DPPH• in
ethanol compared with the rate constants in ethyl acetate (Table
1), taken together with the dramatic decreases in the rate
constants in ethanol which are produced by added acetic acid
(Table 2), prove that in ethanol these reactions proceed primarily
via the SPLET mechanism.
antioxidative potency in phenolic compounds.15 Therefore,
enhancement in the radical-scavenging activity of ArOHs can
be explained by the fact that ED groups reduce the BDE, and
EW groups have the reverse effect. The compound bearing
o-dihydroxyl groups (3,4-DHS) is the most reactive one among
the examined ArOHs. It can also be understood because the
oxidative intermediate, o-hydroxyphenoxyl radicals, is more
stable due to the intramolecular hydrogen bonding interaction,
as evidenced recently from experiments by spectrophotometric
measurement16 and theoretical calculations.15a The theoretical
calculation showed that the hydrogen bond in the o-OH
phenoxyl radical is approximately 4 kcal/mol stronger than that
in the parent catechol, and that the BDE of catechol is 9.1 kcal/
mol lower than that of phenol and 8.8 kcal/mol lower than that
of resorcinol.15a In addition, it should be easier to further oxidize
the o-OH phenoxyl radical and/or o-semiquinone anion to form
the final o-quinone (Scheme 3) as evidenced from the formation
of the dioxane-like dimer of 3,4-DHS (Figure 3) in the presence
of GO• in ethanol at room temperature. Obviously, this dimer
is the [4+2] Diels-Alder adduct of the o-quinone intermediate
and another molecule of 3,4-DHS.
V ) kHAT[ArOH][GO•] + kSPLET[ArO-][GO•]
(2)
Conclusion
Resveratrol and its analogues (ArOHs), that is, 3,5-DHS,
4-HS, 3,4-DHS, 4,4′-DHS, 3-MeO-4-HS, 4′-MeO-4-HS, 4′-Me-
4-HS, 4′-NO2-4-HS, and 4′-CF3-4-HS, are effective scavengers
against GO• and DPPH• in ethanol and ethyl acetate. The
observation that the activity of ArOHs increases with increasing
the electron-rich environment in the molecules, and the com-
pound bearing o-dihydroxyl groups (3,4-DHS) is the most
reactive one among the examined ArOHs, gives us useful
information for antioxidant drug design. The SPLET and HAT
mechanisms are responsible for the radical-scavenging reaction
and the relative contribution depends on the ability of the solvent
to ionize ArOH and the reduction potential of the free radical
involved.
Mechanism. Table 2 shows that the GO•-scavenging reaction
rates of ArOHs in ethanol were decreased approximately 3- to
10-fold by the addition of acetic acid. This result is fully
consistent with the partial dissociation of these ArOHs and the
occurrences of SPLET in nonacidified ethanol. It is also
noticeable that the limiting rate constants of ArOHs in ethanol
at high concentration of acetic acid are similar to rate constants
for the same GO•-scavenging reaction in ethyl acetate having
similar hydrogen bond-accepting activity and low ability to
ionize ArOHs (Tables 1 and 2). This indicates that in acidified
ethanol, these limiting rate constants correspond to the HAT
reaction. Therefore, the experimental rate constant is the sum
Experimental Section
Materials. Resveratrol and its analogues, that is, 3,5-DHS, 4-HS,
3,4-DHS, 4,4′-DHS, 3-MeO-4-HS, 4′-MeO-4-HS, 4′-Me-4-HS, and
4′-NO2-4-HS, were synthesized by the Wittig-Horner reaction.17
4′-CF3-4-HS was synthesized by the Perkin reaction.18 Their
structures were fully identified by using 1H and 13C NMR and EI-
MS (see the Supporting Information) and the data were consistent
with those reported in the literature.18 The purity (>98%) of each
compound was all checked by using high-performance liquid
chromatography (HPLC).
(17) (a) Thakkar, K.; Geahlen, R. L.; Cushman, M. J. Med. Chem. 1993, 36,
2950–2955. (b) Bachelor, F. W.; Loman, A. A.; Snowdon, I. R. Can. J. Chem.
1970, 48, 1554–1557. (c) Heynekamp, J. J.; Weber, W. M.; Hunsaker, L. A.;
Gonzales, A. M.; Orlando, R. A.; Deck, L. M.; Vander Jagt, D. L. J. Med. Chem.
2006, 49, 7182–7189.
(18) Solladie, G.; Pasturel-Jacope, Y.; Maignan, J. Tetrahedron 2003, 59,
3315–3321.
(15) (a) Wright, J. S.; Johnson, E. R.; Dilabio, G. A. J. Am. Chem. Soc.
2001, 123, 1173–1183. (b) Zhang, H. Y.; Sun, Y. M.; Wang, X. L. Chem.sEur.
J. 2003, 9, 502–508. (c) Sushma, M.; Nakanishi, I.; Ohkubo, K.; Uto, Y.;
Kawashima, T.; Hori, H.; Fukuhara, K.; Okuda, H.; Ozawa, T.; Ikota, N.;
Fukuzumi, S.; Anzai, K. Chem. Commun. 2008, 626, 628.
(16) Foti, M.; Ruberto, G. J. Agric. Food Chem. 2001, 49, 342–348.
5030 J. Org. Chem. Vol. 74, No. 14, 2009

Mechanism of ResVeratrol-Oriented Analogues
The galvinoxyl radical (GO•) (from Acros) and the 2,2-diphenyl-
1-picrylhydrazyl (DPPH•) (from Aldrich-Sigma) radical were
purchased with the highest purity available and used as received.
Spectral and Kinetic Measurement. Typically, an aliquot of
ArOHs at more than 10-fold excess of the concentration of GO•
was added to a quartz (10 mm i.d.) that contained GO• (5 × 10-6
M) in ethanol solution. This led to a hydrogen-transfer reaction
from ArOH to GO•. UV-visible spectra changes associated with
this reaction were measured at room temperature with a Hitachi
557 spectrophotometer. The rates of hydrogen transfer were
determined by monitoring the absorbance change at 428 nm due
to GO•. In the case of DPPH•, the rates followed second-order
kinetics and were measured by monitoring the decay of DPPH• at
517 nm ([ArOH]/[DPPH•] ) 1/1). The rates in the presence of acid
were determined in the same manner.
131.7, 132.4, 138.5, 142.5, 160.4; HRMS (ESI) for C28H22O2 m/z
calcd for [M + H]+ 391.1693, found 391.1689, error ) 1.0 ppm.
4,4′-DHS (55 mg, 0.26 mmol) and 110 mg of GO• (0.26 mmol)
were mixed in 30 mL of ethanol and the solution was stirred for
4 h at room temperature. The products were purified by silica gel
chromatography and eluted with chloroform-methanol (15:1/v:v)
to afford dimer (32 mg) and recover unreacted 4.4′-DHS (16 mg)
with the conversion and yield of 80% and 56%, respectively.
Dimer of 4,4′-DHS: 1H NMR (400 MHz, (CD3)2CO) δ 4.53 (d,
J ) 8.8 Hz, 1H), 5.43 (d, J ) 8.8 Hz, 1H), 6.80 (d, J ) 8.4 Hz,
2H), 6.83 (d, J ) 8.8 Hz, 2H), 6.85 (d, J ) 8.4 Hz, 2H), 6.87 (d,
J ) 8.4 Hz, 1H), 6.96, 6.97 (AB system, d, J ) 18.4 Hz, 2H), 7.05
(d, J ) 8.8 Hz, 2H); 7.16 (br s, 1H), 7.24 (d, J ) 8.4 Hz, 2H),
7.37 (d, J ) 8.4 Hz, 1H), 7.39 (d, J ) 8.4 Hz, 2H); 13C NMR (100
MHz, (CD3)2CO) δ 57.5, 94.5, 110.2, 116.2 (2C), 116.4 (2C), 116.6
(2C), 123.4, 126.6 (2C), 126.9, 128.1 (2C), 128.4 (2C), 128.7, 130.3
(2C), 130.5, 132.4, 132.9, 133.4, 157.6, 157.9, 158.5, 160.3; HRMS
(ESI) for C28H22O4 m/z calcd for [M + H]+ 423.1591, found
423.1595, error ) 0.9 ppm.
Oxidation Products Analysis of Resveratrol and Its Ana-
logues in the Presence of Galvinoxyl Radical in Ethanol.
Resveratrol (60 mg, 0.26 mmol) and 220 mg of GO• (0.52 mmol)
were mixed in 30 mL of ethanol and the solution was stirred for
8 h at room temperature. The products were purified by silica gel
chromatography and eluted with chloroform-methanol (5:1/v:v)
to afford dimer (24 mg) and recover unreacted resveratrol (29 mg)
with the conversion and yield of 78% and 41%, respectively.
3,4-DHS (54 mg, 0.25 mmol) and 107 mg of GO• (0.25 mmol)
were mixed in 30 mL of ethanol and the solution was stirred for
3 h at room temperature. The products were purified by silica gel
chromatography and eluted with chloroform-methanol (10:1/v:v)
to afford dimer (47 mg) with the yield of 87%. The mixture first
eluted down was repurified by silica gel chromatography and eluted
with petroleum ether and gave 99 mg of galvinoxy-H.
Dimer of 3,4-DHS: 1H NMR (400 MHz, (CD3)2CO) δ 4.92 (d,
J ) 8.0 Hz, 1H), 5.05 (d, J ) 8.0 Hz, 1H), 6.51 (d, J ) 8.0 Hz,
1H), 6.68 (d, J ) 8.0 Hz, 1H), 6.77 (d, J ) 2.0 Hz, 1H), 6.97 (d,
J ) 8.0 Hz, 1H), 7.13, 7.19 (AB system, d, J ) 16.0 Hz, 2H), 7.17
(d, J ) 8.0 Hz, 1H), 7.23 (m, 2H), 7.24 (d, J ) 7.8 Hz, 1H), 7.25
(m, 1H), 7.26 (m, 2H), 7.27 (d, J ) 2.4 Hz, 1H), 7.35 (d, J ) 7.8
Hz, 2H), 7.57 (d, J ) 7.8 Hz, 2H); 13C NMR (100 MHz, (CD3)2CO)
δ 81.1, 81.3, 115.4, 115.5, 115.6, 117.9, 120.6, 120.9, 127.1 (2C),
127.9, 128.1 (2C), 128.8 (2C), 128.9 (2C), 129.2, 129.4, 129.5 (2C),
132.1, 137.7, 138.6, 144.8, 145.2, 145.6, 146.2; HRMS (ESI) for
C28H22O4 m/z calcd for [M + H]+ 423.1591, found 423.1597, error
) 1.4 ppm.
1
Dimer of resveratrol: H NMR (400 MHz, (CD3)2CO) δ 4.62
(d, J ) 8.0 Hz, 1H), 5.48 (d, J ) 8.0 Hz, 1H), 6.19 (d, J ) 2.0 Hz,
2H), 6.25 (d, J ) 2.0 Hz, 2H), 6.53 (d, J ) 2.0 Hz, 2H), 6.84 (d,
J ) 8.0 Hz, 2H), 6.88 (d, J ) 8.0 Hz, 1H), 6.92, 7.08 (AB system,
d, J ) 16.0 Hz, 2H), 7.24 (d, J ) 8.0 Hz, 2H), 7.25 (d, J ) 8.0
Hz, 1H), 7.45 (d, J ) 2.0 Hz, 1H); 13C NMR (100 MHz, (CD3)2CO)
δ 57.7, 94.2, 102.4, 102.7, 105.7 (2C), 107.4 (2C), 110.1, 116.1
(2C), 123.9, 127.2, 128.5 (3C), 129.1, 131.7, 132.1, 132.5, 140.7,
145.1, 158.4, 159.5 (2C), 159.7 (2C), 160.5; HRMS (ESI) for
C28H22O6 m/z calcd for [M + H]+ 455.1489, found 455.1481, error
) 1.8 ppm.
4-HS ( 98 mg, 0.5 mmol) and 424 mg of GO• (1.0 mmol) were
mixed in 40 mL of ethanol and the solution was stirred for 8 h at
room temperature. The products were purified by silica gel chroma-
tography and eluted with petroleum ether-acetone (4:1/v:v) to afford
the dimer (41 mg) and recover unreacted 4-HS (52 mg) with the
conversion and yield of 85% and 42%, respectively.
Dimer of 4-HS: 1H NMR (400 MHz, (CD3)2CO) δ 4.63 (d, J )
8.5 Hz, 1H), 5.49 (d, J ) 8.5 Hz, 1H), 6.83 (d, J ) 8.0 Hz, 2H),
6.88 (d, J ) 8.4 Hz, 1H), 6.98, 7.13 (AB system, d, J ) 16.4 Hz,
2H), 7.14 (t, d, J ) 8.0, 2.0 Hz, 1H), 7.16 (d, J ) 2.0 Hz, 1H),
7.19 (d, J ) 8.0 Hz, 2H), 7.19 (d, J ) 8.3 Hz, 2H), 7.21 (t, J ) 8.0
Hz, 1H), 7.21 (t, d, J ) 8.0, 1.5 Hz, 1H), 7.29 (t, d, J ) 8.0, 2.0
Hz, 2H), 7.34 (t, d, J ) 8.0, 1.5 Hz, 1H), 7.46 (d, J ) 8.4 Hz, 1H),
7.49 (t, d, J ) 8.0, 2.0 Hz, 2H); 13C NMR (100 MHz, (CD3)2CO)
δ 57.1, 94.2, 110.2, 116.1 (2C), 123.6, 126.9 (2C), 127.6, 128.0,
128.4 (2C), 128.5, 129.0 (2C), 129.2, 129.3 (2C), 129.6 (2C), 131.6,
Acknowledgment. This work was supported by the National
Natural Science Foundation of China (Grant Nos. 20502010
and 20621091), the 111 Project, and Program for New Century
Excellent Talents in University (NCET-06-0906).
Supporting Information Available: 1D and 2D NMR and
MS spectral data for all compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO9007095
J. Org. Chem. Vol. 74, No. 14, 2009 5031