Synthesis and antioxidant activity of hydroxylated phenanthrenes as cis-restricted resveratrol analogues

Article abstract of DOI:10.1016/j.foodchem.2012.05.074

Five hydroxylated phenanthrenes as "cis-configuration-fixed" resveratrol analogues differing in the number and position of the hydroxyl groups were designed and synthesized. Their antioxidant activity was studied by ferric reducing antioxidant power, 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging, and DNA strand breakage-inhibiting assays, corresponding to their electron-donating, hydrogen-transfer and DNA-protecting abilities, respectively. In the above assays, their activity depends significantly on the number and position of the hydroxyl groups, and most of them are more effective than resveratrol. Noticeably, compound 9b (2,4,6-trihydroxyl phenanthrene) with the same hydroxyl group substitutions as resveratrol, is superior to the reference compound, highlighting the importance of extension of the conjugation over multiple aromatic-rings. Similar activity sequences were obtained in different experimental models, but the appreciable differences could contribute detailed insights into antioxidant mechanisms. Based on these results, the hydroxylated phenanthrenes may be considered as a novel type of resveratrol-directed antioxidants.

Full text of DOI:10.1016/j.foodchem.2012.05.074

Food Chemistry 135 (2012) 1011–1019
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Synthesis and antioxidant activity of hydroxylated phenanthrenes as cis-restricted
resveratrol analogues
De-Jun Ding, Xiao-Yan Cao, Fang Dai, Xiu-Zhuang Li, Guo-Yun Liu, Dong Lin, Xing Fu, Xiao-Ling Jin,
⇑
Bo Zhou
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Five hydroxylated phenanthrenes as ‘‘cis-configuration-fixed’’ resveratrol analogues differing in the num-
ber and position of the hydroxyl groups were designed and synthesized. Their antioxidant activity was
studied by ferric reducing antioxidant power, 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging,
and DNA strand breakage-inhibiting assays, corresponding to their electron-donating, hydrogen-transfer
and DNA-protecting abilities, respectively. In the above assays, their activity depends significantly on the
number and position of the hydroxyl groups, and most of them are more effective than resveratrol.
Noticeably, compound 9b (2,4,6-trihydroxyl phenanthrene) with the same hydroxyl group substitutions
as resveratrol, is superior to the reference compound, highlighting the importance of extension of the
conjugation over multiple aromatic-rings. Similar activity sequences were obtained in different experi-
mental models, but the appreciable differences could contribute detailed insights into antioxidant mech-
anisms. Based on these results, the hydroxylated phenanthrenes may be considered as a novel type of
resveratrol-directed antioxidants.
Received 24 January 2012
Received in revised form 10 April 2012
Accepted 10 May 2012
Available online 26 May 2012
Keywords:
Phenanthrenes
Resveratrol
Antioxidant
Mechanism
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
and have established that the 40-OH in the stilbene scaffold is
responsible for the activity (Cao et al., 2003; Caruso et al., 2004;
´
Leopoldini, Russo, & Toscano, 2011; Queiroz et al., 2009; Stojanovic
Converging evidence indicates excessive production of reactive
oxygen species (ROS) have been considered responsible for a vari-
ety of diseases and pathophysiological events, including inflamma-
tion, cancer and neurodegenerative disorders (Ames, Shigenaga, &
Hagen, 1993; Reuter, Gupta, Chaturvedi, & Aggarwal, 2010). Conse-
quently, there is considerable potential for the applications of anti-
oxidant in prevention or control of these conditions. Efforts to
discover potent antioxidants from the vast reserves of native com-
ponents as useful drug candidates are continuously going on.
Among these naturally-occurring antioxidants, resveratrol (3,5,40-
trihydroxy-stilbene), a triphenolic phytoalexin found in grapes
and other food products (Burns, Yokota, Ashihara, Lean, & Crozier,
2002), has received widespread attention. This molecule has been
proposed to be active in cancer prevention, cardiovascular protec-
tion, anti-ageing, and so on (de la Lastra & Villegas, 2005; Howitz
et al., 2003; Jang et al., 1997; Smoliga, Baur, & Hausenblas, 2011).
Considerable efforts have devoted to study antioxidant activity
of resveratrol (Amorati et al., 2004; Cao et al., 2003; Caruso, Tanski,
Villegas-Estrada, & Rossi, 2004; Fukuhara et al., 2008; Murias et al.,
2005; Queiroz, Gomes, Moraes, & Borges, 2009; Stojanovic´ & Brede,
´
& Brede, 2002; Stojanovic et al., 2001; Wang et al., 1999). The
structural simplicity of this molecule has prompted interest in
designing novel analogues with improved antioxidant potency
(Amorati et al., 2004; Fukuhara et al., 2008; Murias et al., 2005).
In this connection, we have also demonstrated that the introduc-
tion of electron-donating groups at positions ortho and para to
the 4-OH or 40-OH group in the stilbene scaffold (Fang & Zhou,
2008; Shang et al., 2009), construction of the hydroxylated stil-
bene-chroman hybrids (Yang et al., 2010), the insertion of addi-
tional double bonds between two aromatic rings (Tang et al.,
2011) and the substitution of 4-SH for 4-OH in the stilbene scaffold
(Cao et al., 2012) could effectively improve antioxidant activity of
this parent molecule.
Resveratrol presents two isomeric forms (trans and cis).
Although cis-resveratrol was proved to be less efficient as an anti-
oxidant than trans-resveratrol (Belguendouz, Fremont, & Linard,
1997; Mikulski, Górniak, & Molski, 2010), many other beneficial
properties of the former and the related cis-stilbenes were also re-
ported (Leiro et al., 2005; Pettit et al., 2002; Siemann, Chaplin, &
Walicke, 2009). For example, Leiro’s studies (Leiro et al., 2005)
showed cis-resveratrol has anti-inflammatory properties through
´
2002; Stojanovic, Sprinz, & Brede, 2001; Wang, Jin, & Ho, 1999),
a significant modulatory effect on the nuclear factor
genes. In a structure-activity relationship study of the potential
jB related
⇑
Corresponding author. Fax: +86 931 8915557.
E-mail address: bozhou@lzu.edu.cn (B. Zhou).
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.05.074

1012
D.-J. Ding et al. / Food Chemistry 135 (2012) 1011–1019
antineoplastic activity of various stilbene derivatives, Pettit et al.
(2002) likewise reported that the cis-stilbenes exhibit significant
inhibitory effects on a number of cancer cell lines. Of the cis-stilb-
enes, combretastatin A4 stands out as the molecule with the most
potential in cancer therapy, and has now entered clinical trials for
both solid and liquid tumours (Siemann et al., 2009).
activity of cis-resveratrol is relatively weak compared with that
of trans-resveratrol (Belguendouz et al., 1997; Mikulski et al.,
2010), we used trans-resveratrol as a reference compound in the
following antioxidant activity assays.
2. Materials and methods
Nonetheless, the cis-resveratrol is unstable, and during storage
and on the condition of low pH, it can be easily isomerized into
trans-resveratrol, a sterically more stable form (Trela & Water-
house, 1996). Considering the above remarks, our interest is to
design resveratrol-based derivatives by introducing a single bond
linkage to ‘‘lock’’ the rotation of olefinic bridge and construct a
phenanthrene ring (Scheme 1). This strategy not only maintains
the cis-configuration, but also extends the conjugation over mul-
tiple aromatic-rings, resulting in the high resonance stabilization
of the phenoxyl radical formed in antioxidant reaction of the
hydroxylated phenanthrenes. Therefore, it should be a promising
strategy to improve antioxidant activity of resveratrol analogues.
Moreover, numerous phenanthrene-containing plants have been
used in traditional medicine and phenanthrenes have been iden-
tified as the active constituents from phytochemical–pharmaco-
logical investigations (Kovács, Vasas, & Hohmann, 2008). For
example, moscatin (4-methoxyphenanthrene-2,5-diol) obtained
from Bulbophyllum odoratissimum, was demonstrated to induce
cell death in several cancer cell lines (Chen et al., 2008). How-
ever, research on their antioxidant and structure-activity rela-
tionships (SAR) is still rare, and we believe they merit further
investigation.
Herein, we report the designed synthesis of five hydroxylated
phenanthrene derivatives (Scheme 1) differing in the number
and position of the hydroxyl groups, aiming at finding more effec-
tive antioxidants than resveratrol. Of the compounds, 9a had a
similar hydroxyl position to 4- or 40-OH on the phenyl rings of
the stilbene, and 9b retained the same hydroxyl group substitu-
tions as resveratrol, while 9c, 9d and 8e contained ortho-hydro-
xy-methoxy groups and ortho-dihydroxyl groups based on the
consideration that the introduction of electron-donating groups
such as methoxy and hydroxyl in the ortho position of phenolic
hydroxyl group, helps increase its antioxidant activity (Wright,
Johnson, & DiLabio, 2001). In view of the fact that antioxidant
2.1. General experimental procedures
The melting points were measured with an SGWX-4 binocular
microscope melting-point apparatus (Shanghai Precision & Scien-
tific Instrument Co. Ltd., Shanghai, China) and were not corrected
by the standard sample. 1H NMR spectra and 13C NMR were re-
corded by using a Bruker AV 400 (400 MHz) spectrometer with
CDCl3, CD3OD or CD3COCD3 as a solvent. Chemical shifts (d) are re-
ported in parts per million (ppm) using the solvent peak. Mass
spectra were recorded on a Bruker Daltonics Esquire 6000 spec-
trometer (ESI-MS) or a VG ZAB-HS spectrometer (EI-MS).
2.2. Materials
2,2-Diphenyl-1-picrylhydrazyl free radical (DPPHÅ), pBR322
DNA, 2,20-azobis (2-amidinopropane hydrochloride) (AAPH), were
purchased from Sigma–Aldrich. 2,4,6-Tri(2-pyridyl)-s-triazine
(TPTZ) was from Alfa Aesar. Other chemicals used were of analyt-
ical grade. All anhydrous solvents were dried and purified by stan-
dard techniques just before use. Starting materials 3–6 (Cui &
Wang, 2009) and 7 (Tron, Pagliai, Del Grosso, Genazzani, & Sorba,
2005) were synthesized according to the available procedures.
2.3. Synthesis
2.3.1. General procedure for the synthesis of 8c–8e
An appropriate amount of 6c–6e, (5.56 mmol) was added to
powdered copper (28.8 mmol) in 20 ml quinoline, and the result-
ing mixture was heated at 200 °C for 2 h. Upon cooling, acetic ether
was added, and the copper was filtered off through celite. The fil-
trate was washed with 1 M hydrochloric acid, and the aqueous
layer was separated and extracted with acetic ether. The combined
Scheme 1. Molecular structures of the hydroxylated phenanthrenes and resveratrol.

D.-J. Ding et al. / Food Chemistry 135 (2012) 1011–1019
1013
organic layers were washed in turn with saturated aqueous sodium
carbonate, water, brine, dried over MgSO4. The solvent was evapo-
rated and the resultant solid was purified by silica gel chromatog-
raphy (petroleum/EtOAc, 8:1) to give pure compounds.
2, and then extracted with Et2O (3 ꢀ 150 ml). These organic frac-
tions were dried over MgSO4 and the solvent was evaporated to
produce a red-brown solid. The crude residue was chromato-
graphed with a mixture of petroleum/EtOAc (2:1) as eluent.
2.3.1.1. 2,3-Dimethoxy phenanthrene (8c). A canary yellow solid;
m.p. 129–130 °C; yield: 52%; 1H NMR (400 MHz, CDCl3): d = 8.53
(d, J = 8.0 Hz, 1H, H5), 8.00 (s, 1H, H4), 7.87 (d, J = 8.0 Hz, 1H, H8),
7.61 (t, J = 8.0 Hz, 3H, H6, H7, H9), 7.52 (t, J = 8.0 Hz, 1H, H10),
7.22 (s, 1H, H1), 4.10 (s, 3H, –OCH3), 4.03 (s, 3H, –OCH3); 13C
NMR (100 MHz, CDCl3): d = 149.3, 149.2, 131.3, 129.7, 128.6,
127.1, 126.1, 125.9, 125.5, 125.2, 124.8, 122.1, 108.3, 103.1, 56.0,
55.9; MS (EI): (m/z) = 238 [M+].
2.3.3.1. 3,6-Dihydroxy phenanthrene (9a). A white solid; m.p. 221–
222 °C; yield: 86%; 1H NMR (400 MHz, CD3COCD3): d = 8.77 (bs,
2H, –OH), 7.98 (d, J = 2.4 Hz, 2H, H4, H5), 7.78 (d, J = 8.8 Hz, 2H,
H8, H1), 7.51 (s, 2H, H9, H10), 7.22 (dd, J = 8.8 Hz, 2.4 Hz, 2H, H2,
H7); 13C NMR (100 MHz, CD3COCD3): d = 157.1, 132.4, 131.1,
127.6, 124.7, 118.3, 107.6; MS (ESI): (m/z)=211 [M+H].
2.3.3.2. 2,4,6-Trihydroxy phenanthrene (9b). A white solid; m.p.
>300 °C; yield: 70%; 1H NMR (400 MHz, CD3OD): d = 8.99 (d,
J = 2.4 Hz, 1H, H5), 7.51 (d, J = 8.4 Hz, 1H, H9), 7.38 (d, J = 8.8 Hz,
1H, H8), 7. 18 (d, J = 8.4 Hz, 1H, H10), 6.87 (dd, J = 8.8 Hz, 2.4 Hz
1H, H7), 6.59 (d, J = 2.4 Hz, 1H, H1), 6.50 (d, J = 2.4 Hz, 1H, H3);
13C NMR (100 MHz, CD3OD): d = 159.2, 157.1, 156.7, 137.8, 133.9,
130.1, 128.6, 126.8, 124.7, 115.2, 114.3, 113.2, 104.7, 103.1; MS
(ESI): (m/z) = 227 [M+H].
2.3.1.2. 2,3,6,7-Tetramethoxy phenanthrene (8d). A white solid; m.p.
179–181 °C; yield: 65%; 1H NMR (400 MHz, CDCl3): d = 7.76 (s, 2H,
H4, H5), 7.53 (s, 2H, H9, H10), 7.19 (s, 2H, H1, H8), 4.10 (s, 6H, –
OCH3), 4.02 (s, 6H, –OCH3); 13C NMR (100 MHz, CDCl3): d = 149.1,
148.7, 126.3, 124.3, 124.2, 108.3, 102.7, 56.0, 55.8; MS (EI): (m/
z) = 298 [M+].
2.3.1.3. 2-Methoxy-3-hydroxy phenanthrene (8e). A canary yellow
solid; m.p. 145–146 °C; yield: 45%; 1H NMR (400 MHz, CD3COCD3):
d = 8.60 (d, J = 8.0 Hz, 1H, H5), 8.14 (s, 1H, H4), 8.08 (s, 1H, OH),
7.89 (d, J = 8.0 Hz, 1H, H8), 7.71 (d, J = 8.0 Hz, 1H, H9), 7.63 (d,
J = 8.0 Hz, 1H, H10), 7.60 (t, J = 8.0 Hz, 1H, H6), 7.52 (t, J = 8.0 Hz,
1H, H7), 7.44 (s, 1H, H1), 4.02 (s, 3H, OCH3); 13C NMR (100 MHz,
CDCl3): d = 149.4, 149.2, 132.5, 130.8, 129.4, 127.8, 127.3, 127.1,
126.5, 125.3, 123.5, 109.4, 107.9, 56.4; MS (EI): (m/z) = 224 [M+].
2.3.3.3. 2,3-Dihydroxy phenanthrene (9c). A white solid; m.p. 158–
159 °C; yield: 50%; 1H NMR (400 MHz, CD3COCD3): d = 8.59 (s,
1H, –OH), 8.51–8.54 (m, 2H, –OH, H5), 8.16 (s, 1H, H4), 7.84–
7.86 (m, 1H, H8), 7.50–7.63 (m, 3H, H6, H9, H10), 7.47–7.51 (m,
1H, H7), 7.25 (s, 1H, H1); 13C NMR (100 MHz, CD3COCD3):
d = 147.3, 147.0, 132.2, 130.8, 129.3, 128.1, 127.0, 126.9, 126.2,
125.8, 125.1, 123.1, 122.9, 108.0; MS (EI): (m/z) = 210 [M+].
2.3.2. General procedure for the synthesis of 8a, 8b
Anhydrous N2 was passed through a solution containing stil-
bene 7a, 7b (0.16 mmol), iodine (0.02 g, 0.16 mmol), propylene
oxide (6.3 ml, 90 mmol) and cyclohexane (500 ml) for 20 min.
The solution was irradiated using a 500 W Hg lamp for approxi-
mately 3 d until the reaction was completed. The solution was
washed with 15% aqueous Na2S2O3 (50 ml), H2O (50 ml) and brine
(50 ml). The organic extract was dried (MgSO4) and evaporated.
Silica chromatography (petroleum/EtOAc, 10:1) provided the de-
sired products.
2.3.3.4. 2,3,6,7-Tetrahydroxy phenanthrene (9d). A white solid; m.p.
>300 °C; yield: 65%; 1H NMR (400 MHz, CD3COCD3): d = 8.41 (bs,
4H, –OH), 7.87 (s, 2H, H4, H5), 7.36 (s, 2H, H9, H10), 7.23 (s, 2H,
H1, H8); 13C NMR (100 MHz, CD3COCD3): d = 149. 8, 146.0, 127.1,
125.5, 124.3, 112.7, 107.4; MS (ESI): (m/z) = 241 [MꢁH].
2.4. Assay for ferric reducing/antioxidant power (FRAP)
FRAP assay (Benzie & Strain, 1996) was used to evaluate the
reducing capacity of hydroxylated phenanthrenes and the related
procedure was described in our previous work (Tang et al., 2011).
2.3.2.1. 3,6-Dimethoxy phenanthrene (8a). A white solid; m.p. 104–
105 °C; yield: 46%; 1H NMR (400 MHz, CDCl3): d = 7.95 (d,
J = 2.4 Hz, 2H, H4, H5), 7.80 (d, J = 8.8 Hz, 2H, H8, H1), 7.55 (s, 2H,
H9, H10), 7.25 (dd, J = 8.8 Hz, 2.4 Hz, 2H, H2, H7), 4.01 (s, 6H, –
OCH3); 13C NMR (100 MHz, CDCl3): d = 158.2, 131.0, 130.0, 127.1,
124.2, 116.5, 104.4, 55.5; MS (EI): (m/z) = 238 [M+].
2.5. Assay for DPPHÅ-scavenging activity
The EC50 value of hydroxylated phenanthrenes in the scaveng-
ing of DPPHÅ was determined by monitoring the absorbance of
2.3.2.2. 2,4,6-Trimethoxy phenanthrene (8b). A white solid; m.p.
109–110 °C; yield: 40%; 1H NMR (400 MHz, CDCl3): d = 9.09 (d,
J = 2.4 Hz, 1H, H5), 7.78 (d, J = 8.8 Hz, 1H, H9), 7.67 (d, J = 8.8 Hz,
1H, H8), 7.51 (d, J = 8.8 Hz, 1H, H10), 7.20 (dd, J = 8.8 Hz, 2.8 Hz
1H, H7), 6.90 (d, J = 2.8 Hz, 1H, H1), 6.77 (d, J = 2.8 Hz, 1H, H3),
4.08 (s, 3H, –OCH3), 3.98 (s, 3H, –OCH3), 3.93 (s, 3H, –OCH3); 13C
NMR (100 MHz, CDCl3): d = 160.0, 158.2, 158.1, 136.0, 131.6,
129.3, 128.1, 126.5, 124.5, 115.4, 114.6, 109.6, 101.3, 99.0, 55.8,
55.3, 55.2; MS (EI): (m/z) = 268 [M+].
DPPHÅ (60
lmol) at 517 nm in methanol during a 120 min observa-
tion. The stoichiometry n was calculated by the equation, n = 1/
(EC50 ꢀ 2) (Villaño, Fernández-Pachón, Moyá, Troncoso, & García-
Parrilla, 2007).
The rates of hydroxylated phenanthrenes with DPPHÅ in metha-
nol were determined by monitoring the absorbance change at
517 nm, using a Varian Cary 300 Spectrophotometer equipped
with a quartz cell (optical path length, 1 cm) and using the sec-
ond-order kinetics with the ratio of [hydroxylated phenan-
threnes]/[DPPHÅ] being 1/1. The rates of 9b in the presence of
acetic acid were determined in the same manner. Temperature in
the cell was kept at 25 °C by means of a thermostatted bath. The
concentrations of DPPHÅ in methanol were measured from its mo-
2.3.3. General procedure for the synthesis of 9a–9d
To a solution of 8a–8d (0.84 mmol) in CH2Cl2 (5 ml) was added
BBr3 (1.0 M solution in CH2Cl2 1.6 ml, 1.6 mmol). The deep purple
solution obtained was stirred for 24 h and then poured into ice-
water (250 ml). The suspended solid formed was extracted into
Et2O (3 ꢀ 150 ml), and then the combined organic fractions were
re-extracted with aqueous NaOH (2 M, 2 ꢀ 150 ml). The basic solu-
tion was acidified by dropwise addition of concentrated HCl to pH
lar extinction coefficient value,
e
, of 1.023 ꢀ 104 Mꢁ1 cmꢁ1. As the
DPPHÅ-scavenging rates of 9c and 9d in methanol were very fast,
their rates were measured using the stopped-flow technique with
a SFA-20 accessory.

1014
D.-J. Ding et al. / Food Chemistry 135 (2012) 1011–1019
Scheme 2. Synthesis of the hydroxylated phenanthrenes. Reagents and conditions: (a) Ac2O/Et3N; (b) CH3OH/conc. H2SO4; (c) FeCl3/CH2Cl2; (d) NaOH/methanol:H2O = 1:1;
(e) Cu/quinoline; (f) I2, propylene oxide/cyclohexane, UV; (g) BBr3/CH2Cl2.
2.6. Assay for oxidative DNA strand breakage induced by AAPH
iron(III) chloride (FeCl3) (Wang, Lü, Wang, & Huang, 2008), is the
most convenient way to construct the phenanthrene ring system.
Thus, a facile synthesis of the phenanthrene unit by using FeCl3
at room temperature was chosen. Interestingly, only the coupling
of ester 4 with ortho-dimethoxy or hydroxyl substituted 2,3-diphe-
nylacrylate (for 4c, 4d and 4e) gave the oxidative coupling product
5 in an excellent yield, whereas no oxidative coupling product was
formed from 4a and 4b. This indicated substituted ortho-dime-
thoxy or ortho-hydroxy-methoxy groups on phenyl is necessary
for this type of intramolecular oxidative coupling. Ester 5 was con-
verted to its corresponding acids 6 with methanol in the presence
of sodium hydroxide. Decarboxylation reactions of 6 were carried
out in the presence of Cu/quinoline at 220 °C under the protection
of N2 to give the methoxyl phenanthrene 8c–8e, followed by
demethylation with BBr3 in dichloromethane to furnish 9c–9d.
For 4a and 4b, we also tried MnO2 as an oxidative coupling reagent.
Unfortunately, we also failed to construct the phenanthrene ring.
We then turned to photochemical cyclisation of stilbenes with io-
dine as a catalyst, which was reported to efficiently construct the
phenanthrene ring system (Scheme 2) (Lawrence et al., 1999).
Decarboxylation of 3a and 3b provided E- and Z-stilbenes 7a and
7b, which were used in the next stage without separation. Photol-
ysis of a diluted solution of stilbene 7 in the presence of iodine
using a 500 W Hg lamp led to the production of phenanthrene 8a
and 8b. Hydroxyl-substituted phenanthrene 9a and 9b were
The inhibition of AAPH-induced DNA strand breakage by ArOH
was assessed by measuring the conversion of the supercoiled
pBR322 plasmid DNA following the procedure described previ-
ously (Qian et al., 2011).
3. Results
3.1. Synthesis of the hydroxylated phenanthrenes
The overall strategy for synthesis of the hydroxylated phenan-
threnes is outlined in Scheme 2. The E- and Z-2,3-diphenylacrylic
acids 3 were easily available by Perkin condensation of the appro-
priate aromatic aldehyde 1 with the corresponding benzeneacetic
acid 2. Esterification of acids 3 with methanol in the presence of
concentrated sulphuric acid gave the corresponding methyl esters
4 in almost quantitative yields. Construction of the phenanthrene
unit is the key step of the overall synthesis. To the best of our
knowledge, metal-based intramolecular oxidative coupling by
using oxidative coupling reagents, such as thallium(III) trifluoro-
acetate (TTFA) (Bringmann, Walter, & Weirich, 1990), vanadium
oxytrifluoride (VOF3) (Halton, Maidment, Officer,
& Warnes,
1984), manganese(IV) dioxides (MnO2) (Wang et al., 2010) and

D.-J. Ding et al. / Food Chemistry 135 (2012) 1011–1019
1015
Fig. 1. Ferric reducing antioxidant power of the hydroxylated phenanthrenes and
resveratrol. Data are the average of three determinations, which were reproducible
with a deviation of less than 10%.
obtained by demethylation with BBr3. All the phenanthrene com-
pounds were characterised with 1H and 13C NMR and MS.
3.2. Ferric reducing antioxidant power of the hydroxylated
phenanthrenes
Reducing power of the hydroxylated phenanthrenes were
investigated by FRAP assay (Benzie & Strain, 1996), a method that
depends on the reduction of a ferric tripyridyltriazine Fe(TPTZ)2(III)
complex to the ferrous tripyridyltriazine Fe(TPTZ)2(II) in the pres-
ence of antioxidants or reductants (ArOHs) (Eq. (1)):
ArOH þ Fe3þ ! ArOHÅþ þ Fe2þ
ð1Þ
The results are expressed as the number of donated electrons
per molecule and illustrated in Fig. 1. It is seen from the figure that
the reducing capacity depends significantly on the degree of
hydroxylation, and decreases in the order of 9d (four hydroxyl
groups) > 9b (three hydroxyl groups) > 9c ꢂ 9a (two hydroxyl
groups) > 8e (one hydroxyl group) ꢂ resveratrol.
3.3. DPPHÅ-scavenging activity of the hydroxylated phenanthrenes
It is well-known that one of the main characteristics responsi-
ble for antioxidant activity of a phenolic compound is its ability
to scavenge free radicals, and the DPPHÅ-scavenging assay is the
most commonly used method. Therefore, the method was em-
ployed to evaluate antioxidant activity of the hydroxylated phe-
nanthrenes in methanol, and their values of EC50 (concentration
able to scavenge 50% of DPPHÅ) and stoichiometric factor (number
of DPPHÅ reduced by one molecule of antioxidant), n, are presented
Fig. 2. Relationship of the number of hydroxyl groups of the hydroxylated
phenanthrenes with their EC50 (A) or n values (B) in the DPPHÅ-scavenging reaction.
(C) Plot of k vs. [CH3COOH] for the reaction of 9b with DPPHÅ in methanol at 298 K.
Table 1
DPPHÅ-scavenging activity of the hydroxylated phenanthrenes and resveratrol.
in Table 1. Based on the EC50 and n values, their DPPHÅ-scavenging
activity follows the order: 9d > 9b > 9c ꢂ 9a > 8e ꢂ resveratrol,
which is in accordance with the result determined by the FRAP
method. Among the compounds tested, compound 9d with two
a
Compounds
EC50
(
l
M)a
nb
k (Mꢁ1 sꢁ1
)
8e
9a
9c
9b
27.90 0.03
14.53 0.18
13.53 0.63
9.28 0.55
5.76 0.16
26.23 2.12
1.79
3.44
3.70
5.38
8.68
1.90
(1.05 0.03) ꢀ 102
(0.99 0.03) ꢀ 102
(1.14 0.01) ꢀ 104c
(1.74 0.11) ꢀ 103
(6.74 0.13) ꢀ 104c
(2.09 0.01) ꢀ 102d
catechol moieties had the lowest EC50 value (5.76 lM) and highest
n value (8.67) and hence possessed the highest DPPHÅ-scavenging
ability. Compound 9b, with the same hydroxyl group substitution
mode as resveratrol, ranks the second place, and shows more than
3-fold DPPHÅ-scavenging efficiency than resveratrol, suggesting the
importance for construction of a phenanthrene ring. Compounds
9a and 9c with two hydroxyl groups are almost equally effective,
9d
trans-Resveratrol
a
b
c
Data are expressed as the mean SD for three determinations.
Calculated by the equation, n = 1/(EC50 ꢀ 2).
Determined by the stopped-flow technique.
Cited from Ref. Cao et al. (2012).
d

1016
D.-J. Ding et al. / Food Chemistry 135 (2012) 1011–1019
and more active than 8e with one hydroxyl group. Furthermore, a
good linear relationship between the number of hydroxyl groups
and the EC50 (Fig. 2A) or n (Fig. 2B) values was also obtained.
However, both the EC50 and n value assays are rather static, and
do not reflect the kinetic details, namely radical-scavenging rate.
Thus, we monitored the reaction rates of the hydroxylated phenan-
threnes with DPPHÅ in methanol at 25 °C by following the second-
order decay of the absorbance at 517 nm due to the radical. For 9c
and 9d, their rates were very high and were therefore measured
using the stopped-flow technique. These kinetic data (second-or-
der rate constants, k) are given in Table 1. By comparing the k val-
ues, DPPHÅ-scavenging activity follows the sequence of
9d > 9c > 9b > resveratrol > 8e ꢂ 9a. Compound 9b is still more ac-
tive than resveratrol and its k value is about 9-fold larger than that
of this reference compound. Noticeably, a clear difference appears
in this assay, that is, compound 9c with a catechol moiety exhibits
significantly higher activity than compound 9b, while the former
has relatively weak activity in comparison with the latter in the
FRAP, EC50 and n values assays.
3.4. Inhibition of AAPH-initiated DNA strand breakage by the
hydroxylated phenanthrenes
DNA is a primary target for free radical-mediated oxidative
damage, and this damage is widely believed to be a causative fac-
tor in cancer, chronic inflammation and ageing (Ames et al., 1993;
Reuter et al., 2010). Consequently, ability of the hydroxylated phe-
nanthrenes to inhibit AAPH-induced oxidative damage of DNA was
also assessed in vitro by using agarose gel electrophoresis analysis
to measure the conversion of supercoiled pBR322 plasmid DNA to
the open circular and linear forms, indicating of single- and dou-
ble-strand breakage, respectively. As shown lane 2 in Fig. 3A, the
supercoiled DNA was completely converted into its circular and
linear forms by addition of 5 mM AAPH. These compounds
(20 lM) except 9d inhibited significantly the DNA strand breakage
(lanes 3–7) with the activity sequence of 9b > 9c ꢂ 9a > resvera-
trol > 8e based on the percentage of intact supercoiled DNA
(Fig. 3B). Interestingly, 9d, the most active compound in the FRAP
and DPPHÅ-scavenging assays, did not provide any protection effect
against the DNA strand breakage (lane 8 in Fig. 3A). Instead, it con-
verted completely the supercoiled DNA into a fragmental form in
the presence AAPH, further intensifying AAPH-induced DNA dam-
age. On the other hand, there was no such breaking effect in the ab-
sence of AAPH (lane 3 in Fig. 3C), whereas in the presence of AAPH,
the degree of the DNA strand breakage increased with increasing
concentrations of 9d (lanes 4–8 in Fig. 3C). This clearly suggests
the involvement of ortho-quinone, an oxidative product of 9d.
The interesting prooxidant phenomenon will be further discussed
in the following section.
To rationalise the reaction mechanism, the effect of adding ace-
tic acid on DPPHÅ-scavenging reaction of 9b in methanol was inves-
tigated. As shown in Fig. 2C, the rate constant (1.74 ꢀ 103 Mꢁ1 sꢁ1
)
decreased remarkably with increasing concentration of acetic acid
to reach a limiting value (1.46 ꢀ 102 Mꢁ1 sꢁ1), unambiguously sup-
porting that the reaction occurs via a sequential proton loss elec-
tron transfer mechanism and the actual electron donor is the
phenolate anion formed by deprotonation of 9b (Litwinienko & In-
gold, 2007) (see below).
4. Discussion
Antioxidants are compounds that, at low concentrations com-
pared to those of oxidizable substrates, including proteins, DNA,
polyunsaturated lipids and so on, can significantly prevent their
oxidative damage (Halliwell & Gutteridge, 2007), by direct scav-
enging of free radicals or ROS (Leopoldini, Chiodo, Russo, & Toscan-
o, 2011), induction of phase 2 enzymes (Dinkova-Kostova &
Talalay, 2008) and chelation of metal ions (Leopoldini, Russo, Chi-
odo, & Toscano, 2006). Free radical-scavenging activity of polyphe-
nolic antioxidants depends significantly on their molecular
structure such as number and position of hydroxyl groups, as well
as conjugation and resonance effects (Leopoldini, Marino, Russo, &
Toscano, 2004; Leopoldini et al., 2011). This work describes a strat-
egy in structural modifications of resveratrol based on extension of
conjugation and alternation of number and position of hydroxyl
groups on the aromatic rings.
As we expected, compound 9b, with the same hydroxyl group
substitutions as resveratrol, displays significantly increased anti-
oxidant activity as compared to this parent molecule in FRAP
(Fig. 1), DPPHÅ-scavenging (Table 1) and DNA strand breakage-
inhibiting (Fig. 3) assays, highlighting that construction of a phen-
anthrene ring is a successful strategy to improve antioxidant activ-
ity of resveratrol. This could be understood because the presence of
the single bound linkage, between the benzene rings and the ole-
finic bridge of 9b, extends delocalization and conjugation of the
p
electrons over the entire molecule and thus increases stability
of neutral radical species. As shown in Scheme 3, only the 40-OH
phenoxyl radical of resveratrol could be completely delocalized
over the whole molecule due to the para position of the olefinic
bridge to the OH, and this contribution is missing in the 3- or 5-
OH radical since the unpaired electron cannot travel across the ole-
finic bridge. However, such a delocalization could be found for all
the possible hydroxyl-derived phenoxyl radical in the phenan-
threne ring.
Fig. 3. Agarose gel electrophoresis pattern of pBR332 DNA (100 ng/25
ll) after
incubation with AAPH (5 mM) and/or the hydroxylated phenanthrenes (20
lM) in
PBS (pH 7.4) at 37 °C for 60 min. (A) Effect of the hydroxylated phenanthrenes on
AAPH-induced DNA strand breakage. Lane 1, control; lane 2, 5 mM AAPH alone;
lanes 3–8, resveratrol, 8e, 9a, 9c, 9b and 9d, respectively. (B) Histogram of
supercoiled DNA percentages relative to native DNA for the hydroxylated phenan-
threnes. (C) Promoting effect of 9d on AAPH-induced DNA strand breakage. Lane 1,
control; lane 2, 5 mM AAPH alone; lane 3, 20
and 2.5, 5, 10, 15, 20 M 9d, respectively.
lM 9d alone; lanes 4–8, 5 mM AAPH
l

D.-J. Ding et al. / Food Chemistry 135 (2012) 1011–1019
1017
Scheme 3. Resonance structures of phenoxyl radicals derived from 9b and trans-resveratrol.
We turn now to a consideration of the three different experi-
mental models that correspond to different mechanisms and tar-
gets. The first experiment, FRAP assay is relevant to electron-
donating ability (reducing capacity) of these compounds and sta-
bility of the corresponding radical cations (Eq. (1)), which are char-
acterised by their oxidative potentials. In general, the lower the
oxidation potential, the stronger the electron-donating ability.
Therefore, our FRAP results indicate that increasing degree of
hydroxylation and conjugation help decrease their oxidative
potentials, and hence increase their electron-donating ability and
stability of the corresponding radical cations.
electron-donating donor, but also the best hydrogen atom donor
among all compounds investigated. The important role of the cat-
echol moiety in antioxidant activity has also been elucidated in the
case of caffeic acid (Leopoldini et al., 2011), catechin and its planar
analogue (Leopoldini, Russo, & Toscano, 2007) and quercetin (Chi-
odo, Leopoldini, Russo, & Toscano, 2010).
Currently, at least four different chemical pathways was used to
describe the formal hydrogen-transfer from a phenol antioxidant
(ArOH) to free radical (XÅ), that is, direct hydrogen atom transfer
(HAT, Eq. (2)), proton-coupled electron transfer (PCET), sequential
proton loss electron transfer (SPLET, Eq. (3)), and electron transfer
then proton transfer (ETPT) (Litwinienko & Ingold, 2007). Although
the four different pathways ultimately result in generation of the
same ArOÅ radical, their occurrence and contribution depend signif-
icantly on the bond dissociation enthalpy (BDE) of the phenolic O–
H bond, the oxidation (ionisation) potential of ArOH and its anion
(ArOꢁ), as well as the acid dissociation constant (pKa) of ArOH.
Generally, in HAT or PCET reaction, the BDE of the O–H bond is
an important parameter in determining the reaction rate; the low-
er the BDE value, the faster the HAT process (Leopoldini et al.,
2004, 2011; Wright et al., 2001). However, in the ETPT reaction,
the oxidation (ionisation) potential of ArOH controls the ration
rate; the lower the oxidation (ionisation) potential value, the easier
the electron abstraction (Leopoldini et al., 2004, 2011; Wright
et al., 2001). For the SPLET reaction, the pKa of ArOH and oxidation
(ionisation) potential of ArOꢁ are the most significant parameters
(Litwinienko & Ingold, 2007). Moreover, occurrence and contribu-
tion of these reactions are also controlled by the nature of the
attacking radical and the solvent used (Litwinienko & Ingold,
2007). In alcoholic solvents that support ionisation, ArOH first loses
a proton to form the corresponding anion (ArOꢁ), which is a much
stronger electron donor than the parent ArOH, followed by rapid
electron transfer to an electron-deficient radical such as DPPHÅ,
an SPLET mechanism well demonstrated by Litwinienko and Ingold
based on acidified-kinetic analysis (Litwinienko & Ingold, 2007).
The second study on DPPHÅ-scavenging activity is utilised to
estimate hydrogen-transfer ability of these compounds. In term
of the static parameters (EC50 or n values), their hydrogen-transfer
ability also relies on the degree of hydroxylation (Table 1, Fig. 2A
and B). Noticeably, the n values of all compounds except resvera-
trol are larger than the available number of hydroxyl groups (Ta-
ble 1). This could be due to the involvement of their oxidative
products in the radical-scavenging reaction (Dangles, Fargeix, &
Dufour, 1999, 2000; Villaño et al., 2007). Moreover, a methanol
molecule (solvent) can regenerate the catechol moiety of phenol
by nucleophilic attack to the corresponding ortho-quinone (Dan-
gles et al., 1999), and hence enlarge the n values. A comparison
of the activity sequences derived from the static and kinetic (k val-
ues) parameters clearly shows an activity reversal between com-
pounds 9c and 9b. This implies that in addition to the number of
the hydroxyl groups, the position of the hydroxyl groups is also
of great importance for improving the hydrogen-transfer ability
of the hydroxylated phenanthrenes. Obviously, the significantly
high k values of 9c with a catechol moiety relative to that of 9b
due to the intramolecular hydrogen-bonding interaction of the
oxidative intermediate, the ortho-hydroxy-phenoxyl radical,
(Leopoldini et al., 2004, 2011; Wright et al., 2001), is easier to
further oxidise to form the ortho-quinone. This also leads to 9d
with two catechol moieties, which is not only the strongest

1018
D.-J. Ding et al. / Food Chemistry 135 (2012) 1011–1019
We have used the same method to find that the DPPHÅ-scavenging
reaction of resveratrol and its hydroxylated analogues in ethanol
proceed mainly via a SPLET mechanism (Shang et al., 2009). Simi-
larly, the DPPHÅ-scavenging rate of 9b in methanol was decreased
approximately 12-fold by the addition of acetic acid (Fig. 2C). This
result supports that this unacidified-kinetic in methanol is mostly
governed by SPLET (Eq. (3)), whereas the addition of acetic acid
suppresses the ionisation of 9b and hence eliminates SPLET to
leave a limiting rate corresponding to HAT (Eq. (2)). Due to the cru-
cial role of the amount and oxidation potential of ArOꢁ in the SPLET
reaction (Litwinienko & Ingold, 2007), the significant high DPPHÅ-
scavenging rates of 9b–9d relative to resveratrol in methanol, also
confirm that construction of a phenanthrene helps to decrease pKa
value of ArOH or oxidation potential of its anion. As a matter of
fact, the existence of the phenanthrene ring could promote the pro-
ton releasing process, due to the resonance stabilization of the cor-
responding proton releasing product (ArOꢁ) by the larger aromatic
system (Zhu, Wang, & Liang, 2010):
(ii) The number and position of the hydroxyl groups exert sig-
nificant influence on antioxidant activity of the hydroxyl-
ated phenanthrenes.
(iii) Although similar activity sequences were obtained in the
FRAP, DPPHÅ-scavenging and DNA strand breakage-inhibit-
ing assays, the appreciable differences could contribute
detailed insights into antioxidant mechanisms.
(iv) Compound 9d has emerged as a prooxidant in AAPH-
induced DNA strand breakage, suggesting a potential appli-
cation as a DNA cleaving or cross-linking agent.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 20972063), the 111 Project, Program for
New Century Excellent Talents in University (NCET-06-0906) and
the Fundamental Research Funds for the Central Universities.
ArOH þ XÅ ! ArOÅ þ XH
ð2Þ
Appendix A. Supplementary data
—Hþ
XÅ
Hþ
ArOH ! ArOꢁ ! ArOÅ þ Xꢁ ! ArOÅ þ XH
ð3Þ
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2012.
05.074.
Finally, we turned to another target, DNA and employed
AAPH-induced plasmid pBR322 DNA strand breakage as a simple
model to evaluate the protecting ability of these compounds
against the oxidative damage. Although a similar activity se-
quence to that of FRAP and DPPHÅ-scavenging experiments were
obtained in this assay, interestingly, 9d with two catechol moie-
ties further intensified AAPH-induced DNA strand breakage as a
prooxidant rather than an antioxidant (Fig. 3). The prooxidant ac-
tion may be accounted for by the following two factors: (1) this
molecule could directly react with molecular oxygen to generate
superoxide, thereby causing DNA strand breakage, and (2) the for-
mation of ortho-quinone oxidation product in the presence of
AAPH may involve in DNA strand breakage by forming covalent
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